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US 20100028334A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0028334 A1 Cottarel et al. (43) Pub. Date: Feb. 4, 2010

(54) COMPOSITIONS AND METHODS TO Publication Classification POTENTIATE ACTIVITY (51) Int. Cl. A638/12 (2006.01) (75) Inventors: Guillaume Cottarel, Mountain AOIN 37/00 (2006.01) View, CA (US); Jamey A 6LX 39/395 (2006.01) Wierzbowski, Stoneham, MA (US) A6IP3L/04 (2006.01) Correspondence Address: CI2O I/68 (2006.01) RONALDI. EISENSTEIN (52) U.S. Cl...... 424/130.1: 514/2: 514/9: 435/6 100 SUMMER STREET, NIXON PEABODY LLP BOSTON, MA 02110 (US) (57) ABSTRACT A pharmaceutical composition comprising an (73) Assignee: TRUSTEES OF BOSTON agent and an enhancer of an antimicrobial agent, wherein the UNIVERSITY, Boston, MA (US) enhancer of an antimicrobial agent is an inhibitor of gene, that by inactivating the gene product potentiates the effectiveness (21) Appl. No.: 12/519,336 of the antimicrobial agent. In some embodiments, the phar maceutical composition further comprises a pharmaceuti (22) PCT Filed: Dec. 13, 2007 cally acceptable carrier. In some embodiments, the antimi crobial agent is an antimicrobial peptide Such as a , (86). PCT No.: PCT/US07/87397 for example but not limited to colistin. In some embodiments of the present invention provides methods to treat and/or S371 (c)(1), prevent infection of a Subject with a microorganism by (2), (4) Date: Jun. 15, 2009 administering a pharmaceutical composition comprising an antimicrobial agent and an enhancer of an antimicrobial Related U.S. Application Data agent. In some embodiments, the present invention provides (60) Provisional application No. 60/875,003, filed on Dec. methods to inhibit growth of a microorganism by administer 15, 2006, provisional application No. 60/963,265, ing a pharmaceutical composition comprising an antimicro filed on Aug. 3, 2007. bial agent and an enhancer of an antimicrobial agent.

Overnight culture in LB from a previous onvernight incubation in presence of Kanamycin

Transfer 30 into 30 us Transfer 30 ul into 30 ul LB Containing Colistin

Stamp onto LB agar plate

e-plates analysis

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COMPOSITIONS AND METHODS TO ions, it destabilizes the wall and can insinuate into it. It per POTENTIATE COLISTIN ACTIVITY forates the , causing distortion of this structure and the release of intracellular constituents in the outside. Colistin FIELD OF THE INVENTION appears to re-emerge as recent clinical findings have been published, focusing on evaluation of efficacy, emerging resis 0001. The present invention relates generally to methods tance and potential toxicities. The use of colistin at current and compositions to treat drug resistant , and more therapeutic effective doses is often associated with neurotox particularly to methods and compositions to potentate the icity and . Therefore it would be desirable to activity of lipopeptides. reduce the toxicity of colistin. 0007. Therefore, there is great need in the art to for an BACKGROUND OF THE INVENTION effective therapy against multi-drug resistant microorgan 0002 Increasing multidrug resistance in Gram-negative isms, in particular gram-negative bacteria and multi-drug bacteria, in particular aeruginosa, Acineto resistant gram-negative bacteria, for example, polymyxin bacter baumannii, and pneumoniae, presents a resistant gram-negative bacteria Such as P. aeruginosa and A. critical problem. Limited therapeutic options have forced baumannii, or to increase the sensitivity of microorganisms to infectious disease clinicians and microbiologists to reap treatment. praise the clinical application of colistin, a polymyxin anti biotic discovered more than 50 years ago. Colistin is now one SUMMARY OF THE INVENTION of the last drug resorts to fight Gram-negative bacteria. 0008. The present invention relates to methods and com 0003. The world is facing an enormous and growing threat positions to potentate the efficacy of antimicrobial agents, for from the emergence of microorganisms that are resistant to a example antimicrobial peptides. The inventors have discov wide range of currently available . For example, ered that by inactivating certain genes, the effectiveness of infections caused by utility drug resistant gram-negative bac antimicrobial agents, such as antimicrobial peptides is teria, particularly and Acineto increased. Accordingly, the present invention relates to bacter baumannii, are becoming a critical challenge in com inhibitors of such genes as enhancers of antimicrobial agents. promised hospital patients (e.g. patients in intensive care In alternative embodiments, the present invention relates to a unite, patients with cystic fibrosis or diffuse panbroncholitis), composition comprising one or more antimicrobial agents and with seemingly trivial infections in sites Such as middle with one or more enhancers of antimicrobial agents. and central ear and eyes. The level of resistance to front line 0009. As disclosed herein, the inventors have discovered a anti-pseudomonal agents is alarmingly high. Of particular method of reducing the toxicity of antimicrobial agents. Such concern are reports on antibiograms of P aeruginosa and A. as for example, Colistin by co-administering an enhancer of baumannii in hospital outbreaks, where colistin, a member of the antimicrobial agent, for example an inhibitor of the gene the polymyxin class of antibiotics, is the only effective anti products as disclosed herein in Table 4 or Table 2. biotic. Unfortunately, polymyxin-resistant P aeruginosa has 0010. Using a functional genomics approach, the inven been isolated from patients with eye infections, ear infec tors discovered that inactivation of specific gene products tions, and particularly in the sputum of patients with cystic render the bacteria more susceptible to colistin, a potent but fibrosis. The appearance of polymyxin-resistant gram-nega toxic . For example, the inventors demonstrate that tive pathogens is of great concern. deletion of the ubiFH and iscS loci render the bacterial cells 0004 Colistin is a polymyxin antibiotic produced by cer more sensitive to colistin. The inventors also demonstrate that tain strains of Bacillus polymyxa. Colistin is effective against that potassium tellurite, an inhibitor of IscS, potentiates colis Gram negative bacilli, except Proteus and Burkholderia tin efficacy. Accordingly, the inventors have discovered dif cepacia and is particularly effective against multi-drug resis ferent combination therapies with colistin, and their use with tantisolates of Pseudomonas aeruginosa, Acinetobacter bau a wide variety of major classes of antibiotics. mannii and Klebsiella pneumoniae. Colistin interacts with 0011. Another aspect of the present invention relates to the bacterial cytoplasmic membrane, changing its permeabil methods to screen for gene products, which when inactivated, ity therefore causing leakage of the cell contents. potentiate the effect of antimicrobial agents, such as antimi 0005. However, despite the ability of colistin to kill multi crobial peptides. Another aspect of the invention relates to drug resistant bacteria, its use in treating patients is highly identification of inhibitors of such genes, and thus identifica limited by the fact that it is highly neurotoxic and nephrotoxic tion of enhancers of antimicrobial agents, such as antimicro and results in adverse renal effects. Colistin must be admin bial peptides. Another aspect of the present invention relates istered parenterally as it is not absorbed by the gastrointesti to a pharmaceutical formulation comprising a composition of nal tract, mucous membranes or intacted or denuded skin. at least one antimicrobial agent, such as an antimicrobial Colistin administration, particularly intravenously, was aban peptide, and at least one enhancer to the antimicrobial agent. doned due to a high incidence of nephrotoxicity and neuro In such an embodiment, the enhancer of the antimicrobial toxicity. In regard of the emergence of bacteria resistant to agent is an inhibitor of a gene product, which by inactivating major classes of antibiotics and of the lack of new classes of the gene product potentiates the effectiveness of the antimi antibiotics, colistin is re-appearing as a valuable antibacterial crobial agent. For example, the enhancer of the antimicrobial therapeutic agent. In fact it is being reintroduced in clinical agent, such as an inhibitor of one of the genes listed in Table practice due to the emergence of multidrug-resistant gram 1 or 4 as disclosed herein potentiates the efficacy of the negative bacteria therefore reflecting the critical situation to antimicrobial agent, e.g. peptides such as colistin, to a greater cure Some bacterial infection. extent than if either was used alone (synergy), and vice versa, 0006 Colistin, also called Colimycin or polymycin E, a the antimicrobial agent potentiates the effect of the enhancer cyclic lipopeptide, penetrates the cell wall of gram negative of antimicrobial peptide, referred to as bi-directional synergy. bacteria by a self induced mechanism by chelating divalent In some embodiments, the enhancer of antimicrobial agent US 2010/0028334 A1 Feb. 4, 2010

may not have any antimicrobial effect when used on its own, prises both an antimicrobial agent, for example an antimicro whereas when Such enhancer of an antimicrobial agent is bial peptide and an enhancer of antimicrobial agents used concurrently with an antimicrobial agent, it may have described herein. antimicrobial activity. 0017. In further embodiments, the enhancers of antimicro 0012. In a further embodiment, the present invention bial agents are any molecule, compound and/or drug which relates to the use of a composition comprising an antimicro inhibits and/or inactivates a gene that results in the potentia bial agent, Such as an antimicrobial peptide, and an enhancer tion of an antimicrobial agent, such as, for example an anti microbial peptide. In some embodiments, such inhibitors to to the antimicrobial agent for the treatmentofa Subject. In one the gene products include antibodies (polyclonal or mono Such embodiment, the composition comprising an antimicro clonal), neutralizing antibodies, antibody fragments, chi bial agent and an enhancer thereof is used to prevent and/or meric antibodies, humanized antibodies, recombinant anti inhibit the growth of a microorganism. In alternative embodi bodies, peptides, proteins, peptide-mimetics, aptamers, ments, the pharmaceutical formulations of the present inven oligonucleotides, hormones, Small molecules, nucleic acids, tion are used for the treatment and/or prophylaxis of an infec analogues, carbohydrates or variants thereofthat tion in a subject. function to inactivate the nucleic acid and/or protein of the 0013. In some embodiments, the antimicrobial agent is a gene products identified herein, and those as yet unidentified. peptide. Such as a lipopeptide, and in another embodiment, Nucleic acids include, for example but not limited to, DNA, the lipopeptide is a cyclic lipopeptide. In some embodiments, RNA, oligonucleotides, peptide nucleic acid (PNA), pseudo the lipopeptide or cyclic lipopeptide is a polymyxin or complementary-PNA (pcPNA), locked nucleic acid (LNA), belongs to the polymyxin class of antibiotics orderivatives or RNAi, microRNAi, siRNA, shRNA etc. The inhibitors can be analogues thereof, which are defined in more detail below. In selected from a group of a chemical, Small molecule, chemi Some embodiments, the polymyxin is polymyxin A, B1, B2, cal entity, nucleic acid sequences, nucleic acid analogues or D1, D2, E1, E2, F, G, M, P S and/or T or any derivative, protein or polypeptide or analogue or fragment thereof. analogue or variant thereof. In some embodiments the poly 0018. In some embodiments, the enhancer of the antimi myxin is a colistin, for example, but not limited to colistin A crobial agent as disclosed herein does not have any effect on and/or colistin B. In some embodiments, the polymyxin is in decreasing cell viability when it is used by itself. In some the form of a salt, for example a methoane sulfonate or sulfate embodiments, an enhancer of the antimicrobial agent is salt or a colistin salt. selected based on its ability to enhance the effect or activity of 0014. Otherantimicrobial agents can be used, for example the antimicrobial agent. In some embodiments therefore, an but not limited to, Small molecules, peptides, peptidomimet enhancer of the antimicrobial agent may not have any anti ics, chemicals, compounds, and any entity that inhibits the pathogenic effects or ability to decrease cell viability when grown and/or kills a microorganism. used itself, and thus will have no antibiotic or no antimicro 0015. In some embodiments, the gene products, which bial activity when used on its own, but when such enhancer of when inactivated potentiate antimicrobial agents effective the antimicrobial agent is used concurrently with an antimi ness are, for example but not limited to the genes as shown in crobial agent, such as, for example with colistin, the enhancer Table 1, for example, agaA, atp A, atpC, atpB, atp), atpE. of the antimicrobial agent functions to enhance the activity of atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, the antimicrobial agent. lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE. rpiA, 0019. In some embodiments, the microorganisms of the sucB, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY. invention are bacterium. In some embodiments, the bacteria yiaY, yidK, yihV, yfhC), yibn and/or homologues, variants are gram positive and gram negative bacteria. In some and fragments thereof. In some embodiments, the gene prod embodiments, the bacteria are multi-drug resistant bacterium. ucts are atpA, atpH, betB, guaA, guaB, lip A, lySA, rpiA, In further embodiments, the bacteria are polymyxin-resistant and/or trXA or homologues, variants and fragments thereof. bacterium. Examples of gram-negative bacteria are for 0016. In some embodiments, the enhancers of antimicro example, but not limited to P. aeruginosa, A. baumannii, bial agents, such as antimicrobial peptides are for example, Salmonella spp., Klebsiella , Shigella spp. and/or compounds that inhibit the gene products which when inac Stenotrophomonas maltophilia. tivated potentiate the antimicrobial agent, for example anti 0020. In some embodiments, the pharmaceutical compo microbial peptides. Examples of such inhibitors are for sitions of the invention are administered in coformulations example but not limited to, mefloquine, Venturicidin A, anti with one or more other antibiotics or therapeutic agents, for mycin, myxothiazol, Stigmatellin, diuron, iodoacetamide, example but not limited to , carbapenemes, potassium tellurite hydrate, all L-vinylglycine, N-ethylmale , , glycoproteins fluroquinolones/ imide, L-allyglycine, diaryduinoline, betaine aldehyde chlo quinolones, oxazolidinones, , , Sul ride, acivein, psicofuraine, buthionine Sulfoximine, diami fonamides and/or . nopemelic acid, 4-phospho-D-erythronhydroxamic acid, 0021. In a further embodiment, the invention provides motexafin gadolinium and/or Xycitrin or modified versions or methods of administration of the compositions and/or phar analogues thereof. In particular embodiments, the enhancer maceutical formulations of the invention and include any of antimicrobial agents is mefloquine or antimycin, means commonly known by persons skilled in the art. In some myxothiazol, Stigmatellin, diuron, iodoacetamide, potassium embodiments, the Subject is any organism, including for tellurite hydrate, all L-vinylglycine, N-ethylmaleimide, L-al example a mammalian, avian or plant. In some embodiments, lyglycine. The present invention also provides methods for the mammalian is a human, a domesticated animal and/or a the modification of the enhancers of antimicrobial peptides commercial animal. described herein, using structure-based design methods. Also 0022. One aspect of the present invention relates to a com encompassed in the present invention is the use of structure position comprising an antimicrobial agent and an enhancer based design methods to design a single molecule that com to the antimicrobial agent, wherein the enhancer to the anti US 2010/0028334 A1 Feb. 4, 2010

microbial agent is an inhibitor of a gene product that by variants thereof, and the inhibitor or enhancer of antimicro inactivating the gene product potentiates the effectiveness of bial agent is diaminopimelic acid or modified versions or the antimicrobial agent. In some embodiments, the antimi analogues thereof. crobial agent is a peptide, for example but not limited to a 0030. In some embodiments, the composition as disclosed lipopeptide Such as a cyclic lipopeptide. In some embodi herein comprises an enhancer of an antimicrobial agent ments, a cyclic lipopeptide is a polymyxin class of antibiotic which inhibits the gene product is rpiA or homologues or or derivative thereof, for example but not limited to a poly variants thereof, and the enhancer is inhibitor or enhancer of myxin is selected from the group of polymyxin A, B1, B2, D1, antimicrobial agent is 4-phospho-D-erythronhydrixamic acid D2, E1 and/or E2, F, G, M, P S and/or T. In some embodi or modified versions or analogues thereof. ments, the polymyxin is selected from polymyZin B1 and/or 0031. In some embodiments, the composition as disclosed herein comprises an enhancer of an antimicrobial agent polymyxin B2 or from colistin A and/or colistin B. which inhibits the gene product is trx A or homologues or 0023. In some embodiments, the composition as disclosed variants thereof, and the inhibitor or enhancer of antimicro herein comprises colistin in the form of a colistin salt, for bial agent is motexafin gadolinium and/or Xycitrin acid or example methane Sulphonate and/or sulfate salt. modified versions or analogues thereof. 0024. In some embodiments, the compositions comprises 0032. In some embodiments, the enhancer of antimicro an enhancer of an antimicrobial agent which is an agent which bial agent inhibitor comprises a small molecule, nucleic acid, inactivates a gene or gene product, for example agene or gene nucleic acid analogue, peptide, , antibody, and vari product is selected from a group consisting of agaA, atpA, ants and fragments thereof. In some embodiments, the atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, nucleic acid is DNA, RNA, DNA/RNA hybrids, a triple helix guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, nucleic acid, an antisense nucleic acid, ribozyme oran RNAi pdxH, phn L. potE. rpiA, such3, trx A, tusB (YheL), tusB. ubiE, or a homologue or fragment thereof. In some embodiments, ubiH, uncA, visB, yeeY, yiaY, yidK, yihV.yfhC), yibN and/or the nucleic acid analogues comprises peptide nucleic acid ynjD or homologues, variants or fragments thereof. In some (PNA), pseudo-complementary PNA (pcPNA), locked embodiments the compositions comprises an enhancer of an nucleic acid (LNA) and variants thereof. antimicrobial agent, which is an agent that inactivates a gene 0033. In some embodiments, the compositions as dis or gene product which is encoded by any of the genes listed in closed herein are useful in the inhibition of growth and/or Table 1 or Table 4. In some embodiments, an enhancer of an decrease in viability of a microorganism, for example a bac antimicrobial agent is an inhibitor of a gene listed in Table 1 terium. In some embodiments, the bacterium is a gram-posi or 4, for example suitable inhibitors useful as enhancers of tive bacterium, and in alternative embodiments, the bacte antimicrobial agents as disclosed herein include, but are not rium is a gram-negative bacterium. In some embodiments, the limited to mefloquine, Venturicidin A, diarycuinoline, betaine microorganism is a multi-drug resistant microorganism, for aldehyde chloride, acivein, psicofuraine, buthionine Sulfox example but not limited to a multi-drug resistant microorgan imine, diaminopemelic acid, 4-phospho-D-erythronhydrox ism is resistant to at least one member of the polymyxin class amic acid, motexafin gadolinium and/or Xycitrin or modified of antibiotics or derivatives or analogues thereof. In some versions or analogues thereof. embodiments, a multi-drug resistant microorganism is poly 0025. In some embodiments, the composition as disclosed myxin resistant Pseudomonas aeruginosa, Acinetobacter herein comprises an enhancer of an antimicrobial agent baumannii, Stenotrophomonas maltophilia, Salmonella spp. which inhibits the gene product is atpA, atpF or atpH or and/or Klebsiella pneumonia and/or Shigella spp. In some homologues, variants or fragments thereof, and the enhancer embodiments, the multi-drug resistant microorganism is of the antimicrobial agent or inhibitor is mefloquine and/or resistant to colistin and/or a colistin salt. Venturicidin A and/or diarycuinoline or modified versions or 0034. In some embodiments, the composition as disclosed analogues thereof. herein, can be administered at different times, for example a antimicrobial agent can be administered prior to, or simulta 0026. In some embodiments, the composition as disclosed neously with, the administration of the enhancer of the anti herein comprises an enhancer of an antimicrobial agent microbial agent or an inhibitor of the gene product. which inhibits the gene product betB or homologues or vari 0035 Another aspect of the present invention relates to a ants thereof, and the inhibitor is betaine aldehyde chloride or pharmaceutical formulation comprising the composition as modified versions or analogues thereof. disclosed herein and a pharmaceutically acceptable carrier. In 0027. In some embodiments, the composition as disclosed Some embodiments, the composition as disclosed herein us herein comprises an enhancer of an antimicrobial agent useful to treat an infection caused by a microorganism. In which inhibits the gene product is guaA or guaB or homo Some embodiments, the use of the composition as disclosed logues or variants thereof, and the inhibitor or enhancer of the herein comprises the amount of the antimicrobial agent is at antimicrobial agent is acivin and/or psicofluranine or modi least 25% less than the same antimicrobial agent in an fied versions or analogues thereof. isogenic cell except for the addition of the enhancer of anti 0028. In some embodiments, the composition as disclosed microbial agent without a reduction of antimicrobial effect. herein comprises an enhancer of an antimicrobial agent 0036) Another aspect of the present invention relates to a which inhibits the gene product is LipA or homologues or method of killing and/or preventing or inhibiting growth of a variants thereof, and the inhibitor or enhancer of antimicro microorganism comprising administering to a subject in need bial agent is buthionine sulfoximine or modified versions or thereof an effective amount of a pharmaceutical formulation analogues thereof. of the composition as disclosed herein. In some embodi 0029. In some embodiments, the composition as disclosed ments, the present invention provides a method of treatment herein comprises an enhancer of an antimicrobial agent and/or prophylaxis of an infection caused by a microorganism which inhibits the gene product is LySA or homologues or comprising steps of administering to a subject in need thereof US 2010/0028334 A1 Feb. 4, 2010

an effective amount of a pharmaceutical formulation of the allow for growth, and (c) assessing the number of cells, composition as disclosed herein. wherein the number of cells is compared to steps (a)-(c) 0037. In some embodiments, the compositions and meth performed on a non-mutated cell, wherein the decrease in ods as disclosed herein are administered to a Subject, for numbers of cells identifies a gene product that when inacti example, where a Subject is mammalian, avian or a plant. In vated potentiates antimicrobial peptide activity. In some Some embodiments the Subject is a mammalian, for example embodiments, the cell used in the method to identify antimi a human. In further embodiments, the mammalian is a domes crobial agents is a bacterium, for example E. Coli. In alterna ticated animal. tive embodiments, the cell is selected from a group consisting 0038. In some embodiments, the compositions and meth of Bacillus cereus, Bacillus anthracis, Bacillus cereus, ods as disclosed herein are administered to a subject to Bacillus anthracis, Clostridium botulinum, Clostridium dif decrease the viability of a microorganism, for example a ficle, Clostridium tetani, Clostridium perfingens, Coryne bacterium, Such as a gram-positive bacterium or a gram bacteria diptheriae, Enterococcus ( D), List negative bacterium. eria monocytogenes, Pneumococcal infections 0039. In some embodiments, the compositions and meth (Streptococcus pneumoniae), Staphylococcal infections and ods as disclosed herein are administered to a Subject to treat Streptococcal infections; Gram-negative bacteria including an infection, for example infections such as, but not limited Bacteroides, Bordetella pertussis, Brucella, Campylobacter to, infection is selected from the group consisting of bacterial infections, enterohaemorrhagic (EHEC/E. wound infections, mucosal infections, enteric infections, sep coli O157:17), enteroinvasive Escherichia coli (EIEC), tic conditions, infectious in airways, cerebrospinal fluid, enterotoxigenic Escherichia coli (ETEC), Haemophilus blood, eyes and skin. In some embodiments, an infection is influenzae, Helicobacter pylori, Klebsiella pneumoniae, caused by gram-negative bacteria. In some embodiments an Legionella spp., Moraxella catarrhalis, Neisseria gonor infection is caused by multi-drug resistant gram negative rhoeae, Neisseria meningitidis, Proteus spp., Pseudomonas bacteria, Such as for example multi-drug resistant microor aeruginosa, Salmonella spp., Shigella spp., Vibrio ganism is resistant to at least one member of the polymyxin and Yersinia, acid fast bacteria including class of antibiotics and synthetic derivatives thereof. In some , Mycobacterium avium-intracellulars, Myco embodiments, the compositions and methods as disclosed bacterium johnei, Mycobacterium leprae, atypical bacteria, herein are administered to a subject that is infected with a , Myoplasma, , Spirochetes, Treponema microorganism, such as, for example, a microorganisms are pallidum, Borrelia recurrentis, Borrelia burgdorfii and Lep selected from a group comprising: Pseudomonas aeruginosa, to spira icterohemorrhagiae, Actinomyces, Nocardia, P , Stenotrophomonas maltophilia, aeruginosa, A. baumannii, Salmonella spp., Klebsiella pneu Salmonella spp and/or Klebsiella pneumonia and/or Shigella monia, Shigella spp. and/or Stenotrophomonas maltophilia spp. and other miscellaneous bacteria. In some embodiments, the 0040 Another aspect of the present invention relates to the antimicrobial agent used in the methods to identify a gene use of an antimicrobial agent and an enhancer to the antimi product that when inactivated potentiates antimicrobial pep crobial agent, wherein the enhancer inhibits a gene product of tide activity is any antimicrobial agent commonly known by the treatment or prophylaxis of an infection caused by a persons of ordinary skill in the art or are disclosed herein. In microorganism, wherein the antimicrobial agent and the Some embodiments, in the methods to identify gene product inhibitor interact synergistically against the microorganism. that when inactivated potentiates antimicrobial peptide activ In some embodiments, the infection is selected from the ity, mutation results in inactivation of the gene or gene prod group consisting of bacterial wound infections, mucosal uct. infections, enteric infections, septic conditions, infectious in airways, cerebrospinal fluid, blood, eyes and skin. BRIEF DESCRIPTION OF FIGURES 0041 Another aspect of the present invention relates to the 0044 FIG. 1 shows a schematic drawing of the screen use of an antimicrobial agent and an enhancer of an antimi shown in a 96 well plate format for simplicity, the screen was crobial agent, or an inhibitor to a gene product in the manu done in a 384 well plate format. The arrow points to the facture of a medicament for inhibiting or preventing produc expected result. Example of digital image analysis with the tion of at least one pathogenic factor by a microorganism. In iscS and ubiH mutants as example. Some embodiments, the use of an antimicrobial agent and an 0045 FIG. 2 shows a flow chart of the genetic and chemi inhibitor to a gene product in the manufacture of a medica cal screen leading to a combination therapy between colistin ment for inhibiting or preventing production of at least one and potassium tellurite. Mutants are listed in table 1 in the pathogenic factor by a microorganism in a Subject being Supplement section. treated with a medicament comprising a antimicrobial agent 0046 FIG. 3 shows a heat map of gene homology of the and the inhibitor. identified geneloci in different bacteria. The conserved genes 0042 Another aspect of the present invention relates to a loci are dark, and less conserved gene loci are light in color. pharmaceutical formulation comprising the composition as 0047 FIG. 4 shows an example of colistin and mefloquine disclosed herein, wherein the enhancer of the antimicrobial synergy in a minimum inhibitory concentration (MIC) assay agent enhances the anti-pathogenic activity of the antimicro in E. coli. As the concentration of mefloquine increases, the bial agent. minimum inhibitory concentration of colistin decreases. 0043. Another aspect of the present invention relates to a 0048 FIG. 5 shows a phenotypic response to colistin. FIG. method for identifying gene products, wherein inactivation of 5A shows the log change in colony forming units per ml the gene products potentiates antimicrobial agent activity, the (CFU/mil) of wild-type, BW25113 and ubiHandiscS mutant method comprising the steps of: (a) mutating one or more E. coli cells (meants.d.) in presence or absence of colistin. genes in a cell, (b) contacting the cell with the antimicrobial The cells were incubated with colistinata final concentration agent, incubating the cell for a sufficient amount of time to of 1.25 ug/ml. At regular time points (3 and 6 hours), aliquots US 2010/0028334 A1 Feb. 4, 2010

were taken and serially diluted into PBS. 10 ul of cells were peptides. In particular embodiments, the antimicrobial agents plated onto LBagar no colistin plates. Number of colonies are antimicrobial peptides, and in Some embodiments, the were counted after overnight incubation at 37 C. Colony antimicrobial peptides are lipopeptides, in particular cyclic count was normalized to get the same starting amount of cells lipopeptides. Enhancers to antimicrobial agents, for example for all strains. FIG. 5B shows the phenotypic response to enhancers of antimicrobial peptides can include nucleic colistin and potassium tellurite. Log change in colony form acids, peptide, nucleic acid analogues, phage, phagemids, ing units per ml (CFU/ml) of wild-type, BW25113 E. coli polypeptides, peptidomimetics, antibodies, Small or large cells (meants.d.) in presence or absence of colistin and potas organic or inorganic molecules or any combination of the sium tellurite. Cell were grown in the absence or presence of above. The enhancers to the antimicrobial agents can be natu colistin (1.25 ug/ml), potassium tellurite (1.6 ug/ml) alone rally occurring or non-naturally occurring (e.g., recombinant) and combined. At regular time points (3 and 6 hours)) aliquots and are sometimes isolated and/or purified. were taken and serially diluted into PBS. 10 ul of cells were 0051. In some embodiments, the enhancers of antimicro plated onto LBagar no colistin plates. Number of colonies bial agents such as antimicrobial peptides are for example, were counted after overnight incubation at 37 C. Colony compounds that inhibit the gene products which when inac count was normalized to get the same starting amount of cells tivated potentiate the antimicrobial agents. Examples of Such for all strains. inhibitors are for example but not limited to, mefloquine, Venturicidin A, diaryduinoline, betaine aldehyde chloride, DETAILED DESCRIPTION OF THE INVENTION acivein, psicofuraine, buthionine Sulfoximine, diaminope 0049. The present invention relates to methods and com melic acid, 4-phospho-D-erythronhydroxamic acid, motexa positions to potentate the efficacy of antimicrobial agents fin gadolinium and/or xycitrin or modified versions or ana such as antimicrobial peptides. The inventors have discovered logues thereof. In particular embodiments, an enhancer of that by inactivating certain genes, the effectiveness of an antimicrobial agents is mefloquine antimicrobial agent, Such as an antimicrobial peptide is 0052. In some embodiments the enhancers to antimicro increased. Accordingly, the present invention relates to bial agents, for example antimicrobial peptides inhibit a gene inhibitors of such genes as enhancers of antimicrobial agents, or gene product. Examples of Such gene products are shown for example antimicrobial peptides. In alternative embodi in Table 1, and include for example, but are not limited to ments, the present invention relates to compositions compris agaA, atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, ing one or more antimicrobial agents, such as antimicrobial cSdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, mnmA, peptides, with one or more enhancers thereof. nuvC, papa, pdxH, phnL, potE. rpiA, such3, trXA, tusB 0050. The present invention relates to methods and com (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, positions comprising antimicrobial agents, such as peptides yihV.yfhO, yibn and/or homologues, variants and fragments and enhancers thereof. Such as enhancers of antimicrobial thereof.

TABLE 1. List of example genes that can be inhibited. SEQ GeneBank ID NO: Gene BLAT ID ID Accession ID DESCRIPTION 1 agaA b313S 23671.98 AAC76169 putative N-acetylgalactosamine-6-phosphate deacetylase 2 atp A b3734 790172 AAC76757 membrane-bound ATP synthase, F1 sector, alpha-subunit 3 atpB b3738 7901.76 AAC76761 membrane-bound ATP synthase, FO sector, subunita 4 atpC b3731 790169 AAC76754 membrane-bound ATP synthase, F1 sector, epsilon-subunit 5 aptl) b3732 7901.70 AAC767SS Membrane-bound ATP synthase, F1 sector, beta-subunit 6 atpE b3737 7901.75 AAC76760 membrane-bound ATP synthase, FO sector, subunit c 7 atpC b3733 790171 AAC76756 membrane-bound ATP synthase, F1 sector, gamma-Subunit 8 atpH b3735 790173 AAC76758 membrane-bound ATP synthase, F1 sector, delta-subunit 9 betB bO312 786SO4 AAC7341S NAD+-dependent betaine aldehyde dehydrogenase 10 cscA b2810 789175 AAC75852 orf, hypothetical protein 11 cscA b3162 7895.53 AAC761.96 inducible ATP-independent RNA helicase 12 cscB b1680 787970 AAC747SO orf, hypothetical protein 13 epC bOS88 786803 AAC73689 ATP-binding component of ferric enterobactin transport 14 guaA b2507 788.854 AAC75560 GMP synthetase (glutamine-hydrolyzing) 15 guaB b2SO8 788.855 AAC7SS61 MP dehydrogenase 16 iscS b2S30 788879 AAC75583 putative aminotransferase 17 kdgK b3526 7899.45 AAC76SS1 ketodeoxgluconokinase 18 lip A bO628 786846 AAC73729 ipoate synthesis, Sulfur insertion? 19 lysA b2838 7892O3 AAC75877 diaminopimelate decarboxylase 20 minima 87081837 AAC74217.2 RNA (5-methylaminomethyl-2-thiouridylate)- methyltransferase 21 nuvC b2S30 48994898 AAT48142 cysteine desulfurase monomer 22 papA b3734 790172 AAC76757 membrane-bound ATP synthase, F1 sector, alpha-subunit 23 pdhik 787926 AAC74710 pyridoxine 5'-phosphate oxidase, 24 phnL b4096 790534 AACTTO57 ATP-binding component of phosphonate transport 25 pot bO692 786908 AAC73786 putrescine transport protein 26 rpiA b2914 78928O AAC75951 ribosephosphate isomerase, constitutive 27 SucB bO727 786946 AAC73821 2-oxoglutarate dehydrogenase (dihydrolipoyltransSuccinase E2 component) US 2010/0028334 A1 Feb. 4, 2010

TABLE 1-continued List of example genes that can be inhibited. SEQ GeneBank ID NO: Gene BLAT ID ID Accession ID DESCRIPTION 28 trix A b3781 790215 AACT6786 thioredoxin 1 29 tuSB b3343 789741 ACC76368 tRNA 2-thiouridine synthesizing protein 30 tuSE bO969 87081811 ACC74OSS tRNA 2-thiouridine synthesizing protein 31 ubiF b3833 2367307 AACT6836 2-octaprenyl-6-methoxy-1,4-benzoquinone --> 2-octaprenyl 3-methyl-6-methoxy-1,4-benzoquinone 32 ubi b2907 7892.74 AACTS945 2-octaprenyl-6-methoxyphenol--> 2-octaprenyl-6-methoxy-1, 4-benzoquinone 33 uncA b3734 710172 AACT6757 membrane-bound ATP synthase, F1 sector, alpha-subunit 34 wisB b2907 7892.74 AACTS945 2-octaprenyl-6-methoxyphenol--> 2-octaprenyl-6-methoxy-1, 4-benzoquinone 35 yeeY b2O15 788326 AACTSO76 putative transcriptional regulator LYSR-type 36 yiaY b3589 79001S AACT6613 putative oxidoreductase 37 yid K b3679 7901 13 AACT6702 putative cotransporter 38 yihV b3883 790316 AAD13445 putative kinase 39 yfhO b2S30 788879 AACTSS83 putative aminotransferase 40 ybN b4049 790483 AACF7019 orf, hypothetical protein 41 ynjD b1756 788053 AACT4826 YnjD is an ATP-binding component of a predicted metabolite uptake ABC transporter

0053 Definitions those that are resistant to three or more antibiotics in a single 0054 For the purpose of this specification it will be clearly antibiotic class. This also includes microorganisms that are understood that the word “comprising means “including but resistant to a wider range of antibiotics, i.e. microorganisms not limited to’, and that the word “comprises have a corre that are resistant to one or more classes of antibiotics. sponding meaning. 0059. The term “microorganism’ includes any micro 0055 As used in this specification the singular forms “a”, Scopic organism or taxonomically related macroscopic “an and the include the plural references unless the context organism within the categories algae, bacteria, fungi, yeast clearly dictate otherwise, for example, reference to “an anti and or the like. The microorganisms targeted in the microbial peptide' or “an antimicrobial agent” includes mix first aspect of the present invention are multi-drug resistant tures of antimicrobial peptides or agents respectively, refer microorganisms. Preferably, gram-negative microorganisms ence to “a antimicrobial agent' includes mixtures of two or are targeted more such components, and the like. 0060. The term “antimicrobial agent” as used herein refers 0056. The term “microorganism’ includes any micro to any entity with antimicrobial activity, i.e. the ability to scopic organism or taxonomically related macroscopic inhibit the growth and/or kill bacterium, for example gram organism within the categories algae, bacteria, fungi, yeast positive- and gram negative bacteria. An antimicrobial agent and protozoa or the like. It includes Susceptible and resistant includes any chemical, peptide, peptidomimetic, entity or microorganisms, as well as recombinant microorganisms. moiety, or analogues of hybrids thereof, including without Examples of infections produced by Such microorganisms are limitation synthetic and naturally occurring non-proteina provided herein. In one aspect of the invention, the antimi ceous entities. In some embodiments, the antimicrobial agent crobial agents and enhancers thereofare used to target micro is a small molecule having a chemical moiety. For example, organisms in order to prevent and/or inhibit their growth, chemical moieties include unsubstituted or substituted alkyl, and/or for their use in the treatment and/or prophylaxis of an aromatic or heterocyclyl moieties including macrollides, lep infection caused by the microorganism, for example multi tomycins and related natural products or analogues thereof. drug resistant microorganisms. In some embodiments, gram Antimicrobial agents can be any entity known to have a negative microorganisms are also targeted. desired activity and/or property, or can be selected from a 0057 The anti-pathogenic aspects of the invention target library of diverse compounds. The term "agent” as used the broader class of “microorganism' as defined herein. How herein and throughout the application is intended to refer to ever, given that a multi-drug resistant microorganism is so any means such as an organic or inorganic molecule, includ difficult to treat, the antimicrobial agent and an enhancer of ing modified and unmodified nucleic acids such as antisense antimicrobial agent in the context of the anti-pathogenic nucleic acids, RNAi, such as siRNA or shRNA, peptides, aspect of the invention is Suited to treating all microorgan peptidomimetics, receptors, ligands, and antibodies, aptam isms, including for example multi-drug resistant microorgan ers, polypeptides, nucleic acid analogues or variants thereof. isms. 0061 The term “antimicrobial peptide' as used herein 0058. Unless stated otherwise, in the context of this speci refers to any peptides with antimicrobial activity, i.e. the fication, the use of the term “microorganism” alone is not ability to inhibit the growth and/or kill bacterium, for example limited to “multi-drug resistant organism’, and encompasses gram positive- and gram negative bacteria. The term antimi both drug-susceptible and drug-resistant microorganisms. crobial peptides encompasses all peptides that have antimi The term “multi-drug resistant microorganism” refers to crobial activity, and are typically, for example but not limited those organisms that are, at the very least, resistant to more to, short proteins, generally between 12 and 50 amino acids than two antibiotics in different antibiotic classes. This long, however larger proteins with Such as, for example includes those microorganisms that have more resistance than are also encompassed as antimicrobial peptides in US 2010/0028334 A1 Feb. 4, 2010

the present invention. Also includes in the term antimicrobial tide colistin, and the enhancer of such antimicrobial peptide is peptide are antimicrobial peptidomimetics, and analogues or mefloquin, then bidirectional synergy means mefloquin fragments thereof. The term “antimicrobial peptide' also enhances the activity of the antimicrobial peptide colistin and includes all cyclic and non-cyclic antimicrobial peptides, or Vice versa, the antimicrobial peptide colistin can be used to derivatives or variants thereof, including tautomers, see Liet enhance the activity of mefloquin. al. JACS, 2006, 128: 5776-85 and http://aps.unmc.edu/AP/ 0068. In the present invention, the antimicrobial agent and main.php for examples, which are incorporated herein in their an enhancer of antimicrobial agent show at least synergistic entirety by reference. In some embodiments, the antimicro anti-pathogenic activity. The term “bidirectional Synergy’ bial peptide is a lipopeptide, and in some embodiments the refers to the increase in activity of each antimicrobial com lipopeptide is a cyclic lipopeptide. The lipopeptides include, ponent when used in conjunction with the otherantimicrobial for example but not limited to, the polymyxin class of anti component, and not merely an increase in activity of one of microbial peptides. the antimicrobial components. In the present invention, the 0062. The term “polymyxin' is used in its broadest sense antimicrobial agent and enhancer of the antimicrobial agent to encompass all members of the well known polymyxin class component show at least synergistic, and in some instances of antibiotics and synthetic derivatives thereof. Derivatives bidirectional Synergistic antimicrobial activity. within this class are the non-cyclic derivatives of cyclic poly 0069. The term “anti-pathogenic’ refers to activity inhib myxin, derivatives containing amino acid variations, deriva iting or preventing production of a pathogenic factor released tives containing Substitutes of the fatty acid components with by living microorganisms in a host which leads to destructive other fatty acids or substituents, derivatives with D- and effects of tissues at the site(s) of infection. This includes L-amino acid conversions, and derivatives Substituted with inhibition of the expression of flagellin in P. aeruginosa, any one or more optional substituents identified below. Clas perturbing of cytokine production and altering action of poly sic include, but are not limited to, polymyxin A, morphonuclear cell functions in vivo and ex vivo, and/or the B1, B2, C, D1, D2, E1 and/or E2, FC, M, P S and T. The inhibition of production of pyocyanin, which is produced by polymyxins are cationic detergents and are relatively simple microorganisms such as P. aeruginosa and is blue pigment basic peptides with molecular masses of about 1000-1200 which disrupts human ciliary beating in vitro, inhibits epider daltons. mal cell growth and also impedes lymphocyte proliferation. 0063. In this embodiment, the term “polymyxin resistant” 0070 The term “analog as used herein refers to a com refers to those microorganisms that are resistant to the mem position that retains the same structure or function (e.g., bind ber of the polymyxin class of lipopeptides being used in one ing to a receptor) as a polypeptide or nucleic acid herein. embodiment. Examples of analogs include peptidomimetics, peptide 0064. The term “prevent or inhibit growth of a microor nucleic acids, Small and large organic or inorganic com ganism, for example a multi-drug-resistant microorganism pounds, as well as derivatives and variants of a polypeptide or refers to the interference with growth or replication of the nucleic acid herein. The term “analog as used herein refers to microorganism, which can include but does not necessarily a composition that retains the same structure or function (e.g., extend to killing of the microorganism. binding to a receptor) as a polypeptide or nucleic acid herein. 0065. The term “treatment and/prophylaxis' refers gener (0071. The term “derivative' or “variant as used herein ally to afflicting a subject, tissue or cell to obtain a desired refers to a peptide, chemical or nucleic acid that differs from pharmacologic arid/or physiologic effect. The effect may be the naturally occurring polypeptide or nucleic acid by one or prophylactic in terms of completely or partially preventing a more amino acid or nucleic acid deletions, additions, Substi disease or sign or symptom thereof, and/or may be therapeu tutions or side-chain modifications. Amino acid substitutions tic in terms of a partial or complete cure of a disease. include alterations in which an amino acid is replaced with a 0066. The term “synergy' or “synergistically are used different naturally-occurring or a non-conventional amino interchangeably herein refers to the total increase in activity acid residue. Such substitutions may be classified as “conser of the antimicrobial agent and the enhancer of antimicrobial Vative', in which case an amino acid residue contained in a agent components of the invention, over their single and/or polypeptide is replaced with another naturally occurring additive antimicrobial activity. It includes the increase in amino acid of similar character either in relation to polarity, activity of only one of the antimicrobial components. In the side chain functionality or size. present invention, the antimicrobial agent and enhancer of 0072 Substitutions encompassed by the present invention antimicrobial agent component show at least synergistic anti may also be “non conservative', in which an amino acid pathogenic activity. residue which is present in a peptide is substituted with an 0067. The term “bidirectional synergy” refers to the amino acid having different properties. Such as naturally increase in activity of each antimicrobial component (i.e. the occurring amino acid from a different group (e.g., Substitut antimicrobial agent and/or antimicrobial peptide and ing a charged or hydrophobic amino; acid with alanine), or enhancer of the agent or enhancer of antimicrobial peptide) alternatively, in which a naturally-occurring amino acid is when used in conjunction with the other antimicrobial com Substituted with a non-conventional amino acid. In some ponent, and not merely an increase in activity of one of the embodiments amino acid Substitutions are conservative. antimicrobial components. In the present invention, the anti (0073. The term “amino acid” within the scope of the microbial agent and enhancer of antimicrobial agent compo present invention is used in its broadest sense and is meant to nent show at least synergistic antimicrobial activity. Advan include naturally occurring L. C.-amino acids or residues, but tageously, for example, the antimicrobial agent (for example is not necessarily restricted to the naturally occurring amino antimicrobial peptide) and enhancer of the antimicrobial acids. agent (for example antimicrobial peptide) show bidirectional 0074 By “pharmaceutically acceptable derivative' is synergistic antimicrobial activity. Stated in other words, for meant any pharmaceutically acceptable salt, hydrate or any example, if an antimicrobial agent is the antimicrobial pep other compound which, upon administration to the Subject, is US 2010/0028334 A1 Feb. 4, 2010 capable of providing (directly or indirectly) an antimicrobial example but not limited to, agaA, atpA, atpC, atpB, atp). peptide and/or enhancer of antimicrobial component or resi atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, due thereof. kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE, 0075. The term “pro-drug is used herein in its broadest rpiA, such3, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, sense to include those compounds and entities which are yeeY, yiaY, yidK, yihV, yfhC), yibN and etc., and any converted in vivo to active antimicrobial agents and/or active homologs, analogs or fragments thereof. enhancers of antimicrobial agents of the present invention. I0083. The term “gene product(s)' as used herein refers to 0076. The term “tautomer' is used herein in its broadest include RNA transcribed from a gene, or a polypeptide sense to include antimicrobial agents and/or or enhancers of encoded by a gene or translated from RNA. antimicrobial agents which are capable of existing in a state of I0084. The term “inhibition” or “inhibit” when referring to equilibrium between two isometric forms. Such compounds the activity of an antimicrobial agent and/or enhancer of may differ in the bond connecting two atoms or groups and antimicrobial agent refers to prevention of, or reduction in the the position of these atoms or groups in the compound. rate of growth. Inhibition and or inhibit when referring to the 0077. The term “isomer' is used herein in its broadest activity of an enhancer of antimicrobial agent refers to the sense and includes structural, geometric and stereo isomers. prevention or reduction of activity of a gene or gene product, As the antimicrobial agents and/or or enhancers of antimicro that when inactivated potentiates the activity of an antimicro bial agents may have one or more chiral centers, they are bial agent. capable of existing in enantiomeric forms. I0085. The term “homolog’ or “homologous' as used 0078. The term “infection' or “microbial infection' which herein refers to homology with respect to structure and/or are used interchangeably herein refers to in its broadest sense, function. With respect to sequence homology, sequences are any infection caused by a microorganism and includes bac homologs if they are at least 50%, preferably at least 60%, terial infections, fungal infections, yeast infections and pro more preferably at least 70%, more preferably at least 80%, toZomal infections. more preferably at least 90%, more preferably at least 95% 0079 Suitable mammals include members of the orders identical, more preferably at least 97% identical, or more Primates, Rodentla, Lagomorpha, Cetacea, Homo sapiens, preferably at least 99% identical. The term “substantially Carnivora, Perissodactyla and Artiodactyla. Members of the homologous' refers to sequences that are at least 90%, more orders Perissodactyla and Artiodactyla are included in the preferably at least 95% identical, more preferably at least invention because of their similar biology and economic 97% identical, or more preferably at least 99% identical. importance, for example but not limited to many of the eco Homologous sequences can be the same functional gene in nomically important and commercially important animals different species. Such as goats, sheep, cattle and pigs have very similar biology I0086. The term “hybridize” refers to interaction of a and share high degrees of genomic homology. nucleotide sequence with a second nucleotide sequence. Such 0080. As used herein, the term “effective amount” is interaction can be, e.g., in Solution or on a solid Support. Such meant an amount of antimicrobial agent and/or enhancers of as cellulose or nitrocellulose. If a nucleic acid sequence binds antimicrobial agent components of the present invention to a second nucleotide sequence with high affinity, it is said to effective to yield a desired antibiotic activity. The term “effec “hybridize' to the second nucleotide sequence. The strength tive amount as used herein refers to that amount of compo of the interaction between the two sequences can be assessed sition necessary to achieve the indicated effect. The specific by varying the stringency of the hybridization conditions. “effective amount” will, obviously, vary with such factors as Under highly stringent hybridization conditions only highly the particular condition being treated, the physical condition complementary nucleotide sequences hybridize. of the subject, the type of subject being treated, the duration of I0087. The term “organism' as used herein includes all the treatment, the route of administration, the type of antimi living cells including microorganisms (e.g., viruses, bacteria, crobial agent and/or enhancer of antimicrobial agent, the protozoa), plants, and animals (e.g., humans, birds, reptiles, nature of concurrent therapy (if any), and the specific formu amphibians, fish, and domesticated animals, such as cows, lations employed, the ratio of the antimicrobial agent and/or chicken, pigs, dogs, and goats). enhancers antimicrobial agent components to each other, the I0088. The term “peptide' or “polypeptide' are used inter structure of each of these components or their derivatives. changeably herein, refers to any composition that includes 0081. As used herein, a “pharmaceutical carrier' is a phar two or more amino acids joined to each other by a peptide maceutically acceptable solvent, Suspending agent or vehicle bond or peptidomimetic thereof. The term includes both short for delivering the combination of antimicrobial agent and/or chains, which are also commonly referred to in the art as enhancers of antimicrobial agent components to the Subject. peptides, oligopeptides and oligomers, for example, and to The carrier may be liquid or solid and is selected with the longer chains, which generally are referred to in the art as planned manner of administration in mind. Each carrier must proteins. The term "peptide' includes all peptides as be pharmaceutically “acceptable' in the sense of being com described below. It will be appreciated that peptides often patible with other ingredients of the composition and non contain amino acids other than the 20 amino acids commonly injurious to the Subject. referred to as the 20 naturally occurring amino acids, and that 0082. The terms “gene(s) refers to a nucleic acid many amino acids, including the terminal amino acids, can be sequence (DNA, RNA, or analogs and/or combinations modified in a given polypeptide, either by natural processes thereof) that encodes through its template or messenger RNA Such as glycosylation and other post-translational modifica a sequence of amino acids characteristic of a specific peptide. tions, or by chemical modification techniques which are well The term “gene can includes intervening, non-coding known in the art. Known modifications which can be present regions, as well as regulatory regions, and can include 5' and in peptides of the present invention include, but are not lim 3' ends. Examples of genes associated with, when inactivated, ited to, acetylation, acylation, ADP-ribosylation, amidation, potentiation the effect of antimicrobial agents are, for covalent attachment of flavin, covalent attachment of a heme US 2010/0028334 A1 Feb. 4, 2010 moiety, covalent attachment of a polynucleotide or poly also applicable to veterinary treatment, including treatment of nucleotide derivative, covalent attachment of a lipid or lipid companion animals such as dogs and cats, and domestic derivative, covalent attachment of phosphotidylinositol, animals such as horses, ponies, donkeys, mules, llama, cross-linking, cyclization, disulfide bond formation, dem alpaca, pigs, cattle and sheep, or Zoo animals such as pri ethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formulation, gamma-c mates, felids, canids, bovids, and ungulates arboxylation, glycation, glycosylation, GPI anchor forma 0.095 Antimicrobial Agents tion, hydroxylation, iodination, methylation, myristoylation, 0096. One aspect of the invention relates to antimicrobial oxidation, proteolytic processing, phosphorylation, prenyla agents. In some embodiments, the antimicrobial agent is anti tion, racemization, selenoylation, Sulfation, transfer-RNA microbial peptide. The term “antimicrobial peptide' refers to mediated addition of amino acids to proteins such as arginy peptides which have antimicrobial activity. In some embodi lation, and ubiquitination. ments, the antimicrobial agent may be any peptide with anti 0089. The term "peptidomimetic' as used herein refers to microbial activity, for example cyclic and non-cyclic pep molecules which mimic an aspect of a polypeptide structure. The term “mimetic” as used herein is any entity, molecule, tides, lipopeptides, and peptides (or proteins) with long chemical, Small or large molecule, organic or inorganic, Syn chains of amino acids, for example, lyoSymes etc. and modi thetic or natural, that mimics the mechanism of the molecule fied versions thereof. In some embodiments, the antimicro of which it is a mimetic. bial peptide is a lipopeptide. The lipopeptides include, for 0090 The term “purified’ refers to a material (e.g., com example, the polymyxin class of antimicrobial peptides. The pound, molecule, or structure of interest) that is relatively free term “polymyxin' is used in its broadest sense to encompass of other materials that it normally is associated with and is all members of the well known polymyxin class of antibiotics preferably at least 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, and synthetic derivatives thereof. Derivatives within this class 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, are the non-cyclic derivatives of cyclic polymyxin, deriva 97%, 98%, 99% or 100% of total weight of the material. tives containing amino acid variations, derivatives containing 0091. The term “recombinant as used herein refers with substitutes of the fatty acid components with other fatty acids reference to material (e.g., a cell, a nucleic acid, a protein, or a vector) indicates that such material has been modified by the or substituents, derivatives with D- and L-amino acid conver introduction of a heterologous material (e.g., a cell, a nucleic sions, and derivatives Substituted with any one or more acid, a protein, or a vector). Thus, for example, recombinant optional substituents identified below. Classic polymyxins microorganisms or cells express genes that are not found include polymyxin A, B1, B2, C, D1, D2, E1 and/or E2, FC, within the native (non-recombinant) form of the microorgan M. P. S and T. The polymyxins are cationic detergents and are ism or cell or express native genes that are otherwise abnor relatively simple basic peptides with molecular masses of mally expressed, under expressed or not expressed at all. For about 1000-1200 daltons. example, a recombinant antibody is an antibody which is not 0097 Examples of members of the polymyxin class are normally found in native (non-recombinant) antibodies form. (Band B) and polymyxin E (colistin A and B). 0092. The term “treatment” or “treating” as used herein refers to reducing or alleviating symptoms in a Subject, pre Both these members include a cyclic heptapeptide ring with a venting symptoms from worsening or progressing, or inhibi tripeptide side chain. It is envisaged that declyclisation of the tion, elimination, or prevention of the infection, disorder or ring may resultina peptide with effective antimicrobial activ symptoms in a subject who is free therefrom. “Treating as ity. Accordingly, cyclic and non-cyclic derivatives and vari used herein covers any treatment of, or prevention of an ants of the polymyxins and similar peptides are encompassed infection or disease in a vertebrate, a mammal, for example, a within the term “antimicrobial peptide'. human, and includes: (a) preventing the infection and/or dis 0098. Also included within the scope of “antimicrobial ease from occurring in a subject that may be predisposed to peptide' are all the components of polymyxin B and poly the infection and/or disease, but has not yet been diagnosed as having it; (b) inhibiting a infection and/or disease, i.e., arrest myxin B, as well as synthetic derivatives thereof. ing its development; or (c) relieving or ameliorating the 0099. It is envisaged that one or more amino acid sequence effects of the infection and/or disease, i.e., cause regression of (s) of the peptides above can be varied without significant the effects of the infection or disease. effect on the structure or function of the peptide. Thus, the 0093. The term “unit dose when used in reference to a invention further includes variations of the peptide which therapeutic composition of the present invention refers to show antimicrobial activity Such variations or mutants physically discrete units Suitable as unitary dosage for the include amino acid deletions, insertions, inversions, repeats Subject, each unit containing a predetermined quantity of and type substitutions. active material calculated to produce the desired therapeutic effect in association with the required physiologically accept 0100. The structural formula of polymyxin B (B1 and B2) able diluents, i.e., carrier, or vehicle. is as follows: 0094. The term “subject' as used herein refers to any animal having a disease or condition which requires treat ment with a pharmaceutically active agent. The Subject may R -> (a) L-Dab -> L-Thr -> (a) L-Dab -> be a mammal, for example a human, or may be a domestic or commercial or companion animal. While in one embodiment of the invention it is contemplated that the antimicrobial agents and/or enhancers of antimicrobial agents of the inven tion are Suitable for use in medical treatment of humans, it is US 2010/0028334 A1 Feb. 4, 2010

-continued Y-NH2 -continued (C) L-Dab -- D-Phe -- L-Leu Y-NH2 (C., Y) L-Dab | (C) L-Dab -- D-Leu -- L-Leu L-Thr -- (C) p -- (C) p (C., Y) L-Dab Y-NH2 Y-NH2 L-Thr -- (C) L-Dab -- (C) p Y-NH2 Y-NH2 0101 Polymyxin B1: R-(+)-6-methyloctanyl 0102 Polymyxin B2: R-(+)-6-methylheptanyl 0107 Colistin A: R-methyloctanoic acid 0103 Phe: Phenylalanine, Thr: Threonine, Leu: Leucine, Dab=C.Y-diaminobutyric acid, wherein C. and Y indicate the 0.108 Colistin B: R=6-methylheptanoic acid respective —NH involved in the peptide linkage. 0109 Thr: Threonine, Leu: Leucifle, Dab=C.Y-diami nobutyric acid, wherein C. and Y indicate the respective 0104 Colistin —NH involved in the peptide linkage. 0105 Colistin is a multicomponent , 0110 Shown below in (A) are structures of Colistin A and comprised mainly of colistin A and B, that became available Colistin B. Shown in (B) are the structures of colistimethane for clinical use in the 1960s, but was replaced in the 1970s by A and B, fatty acid: 6-methyloctanoic acid for colistin A and antibiotics considered less toxic. There are two forms of 6-methylheptanoic acid for colistin B: Thr-thereonine, Leu colistin commercially available: colistin sulfate for oral and leucine, Dab-C.Y-diaminobutyric acid, C. and Y indicate topical use, and colistimethate Sodium (sodium colistin meth respective amino groups involved in peptide linkage. ane Sulphonate, colistin Sulfomethate sodium) for parenteral use (shown herein); both can be delivered by inhalation. Although there have been a substantial number of clinical reports on the Successful use of colistin or polymyxin B Fatty acid -- (a) L-Dab -- L-Thr -- (which differs by only one amino acid from colistin) against infections caused by multidrug-resistant P aeruginosa, A Y-NH2 baumannii, and Kpneumoniae, there is a dearth of informa (a) L-Dab -- tion on the clinical pharmacokinetics, pharmacodynamics, and toxicodynamics of colistin. Such data are essential for Y-NH2 establishing optimal dosing regimens. Y-NH2 0106 Polymyxin B (colistin) has many different compo nents. Its basic structure is a cyclic heptapeptide ring and a -1 (C) L-Dab -- D-Leu -- L-Leu tripeptide side chain covalently bound to a fatty acid at the N-terminus via an acyl group. At least 30 components have (C., Y) L-Dab been isolated and thirteen identified. They differ from each other by the composition of amino acids and fatty acids. The L-Thr -- (C) L-Dab -- (C) L-Dab two major components are colistin A and colistin B. The structural formula is as follows: Fatty acid -- (a) L-Dab -- L-Thr --

Fatty acid -- (a) L-Dab -- L-Thr -> CH SOH

(a) L-Dab -- (a) L-Dab --

CH SOH US 2010/0028334 A1 Feb. 4, 2010 11

vivo data. With respect to antibacterial activity against P -continued aeruginosa, recent studies have indicated that colistimethate SOH is a non-active prodrug of colistin. A recent review provides more details on the considerable differences between colis CH2 timethate and colistin in their chemistry, pharmacokinetics, and pharmacodynamics. 0114. Another schematic showing the chemical structure of colistin is shown below: -1 (C) L-Dab -- D-Leu -- L-Leu (C., Y) L-Dab Hosseo L-Thr -- (C) L-Dab -- (C) L-Dab lose HN1) O hi, s r H H R2 lot lot 0111 Amino acid substitutions are typically of single resi dues, but may be of multiple residues, either clustered or dispersed. Additions encompass the addition of one or more naturally occurring or non-conventional amino acid residues. He Deletion encompasses the deletion of one or more amino acid H residues. O NH N Ot 0112 Minor components of colistin include the poly O ls S=O myxin E3 and E4, norvaline-polymyxin E1, Valine-poly -SnI NH CH O myxin E1, and valine-polymyxin E2, isoleucine-polymyxin HO O N O OH E1, isoleucine-polymyxin E2, polymyxin E7 and isoleucine H polymyxin E0. In some embodiments, the antimicrobial pep O NH tide comprises any one or more components of colistin, and in some embodiments, colistin A and/or colistin B. The propor tion of colistin A and colistin B in commercial material varies S NH between pharmaceutical Suppliers and batches, but it is gen Ho1 No1\ R erally between 4.5:1 to 0.9:1. Colistin is available commer N cially in two forms, colistin Sulphate and sodium colistin H methaneSulphonate. Sodium colistin methanesulphonate colimycin hydrolyses in aqueous media and forms a complex mixture of HN partially sulphomethylated derivatives plus colistin. One or more of the above forms of colistin or its derivatives are encompassed within the scope of the invention. In some H 2 embodiments, antimicrobial peptide comprises salts of colis HN N N tin, for example salts of pharmaceutically acceptable cations NH Such as sodium, potassium, lithium and the like, acid addition O salts of pharmaceutically acceptable inorganic acids Such as HN O O CH3 hydrochloric orthophosphoric, sulphuric and the like, and/or O salts of pharmaceutically acceptable organic acids such as H3C O O NH CH3 acetic, propionic, methanesulfonic and the like. O 0113 Although widely used in the literature, the terms HO colistin and colistimethate are not interchangeable. Colistin HN (usually used as the Sulphate salt) is a polycation, whereas N colistimethate (used as the Sodium salt) is a polyanion at H physiological pH. Colistimethate is prepared from colistin by reaction of the free y-amino groups of the five C.Y-diaminobu NH2 tyric acid residues with formaldehyde followed by sodium bisulphite. Colistimethate is not stable in vitro or in vivo, and is hydrolysed to a series of methanesulphonated derivatives plus colistin. Colistin is more stable than colistimethate in human plasma. The differences in chemistry between colis timethate and colistin also translate into differences in phar macokinetics and pharmacodynamics. Whereas colistime thate is eliminated mainly by the kidney and the urinary excretion involves renal tubular secretion, colistin is elimi nated predominantly by the non-renal route because, at least in part, the compound undergoes very extensive renal tubular reabsorption. After intravenous administration of colistime thate (sodium), the plasma half-lives of colistimethate are colistin (free base) approximately half of those of the colistin generated from in US 2010/0028334 A1 Feb. 4, 2010 12

0115. Where R1 and R2 are as follows:

component I aest CH ca CH3 CH CH3 component III CH3 CH3 O 2 CH3 as

CH3 component IV an CH CH ca CH CH3

0116 Methods to produce pure biologically active colis perforates the cell wall, causing distortion of this structure tin, or a pharmaceutically acceptable salt thereof, and meth and the release of intracellular constituents in the outside. ods for its use for treatment for Pseudmonomia aeruginosa, I0120 Enhancers of Antimicrobial Agents Stenotrophomonas maltophilia and other are disclosed in I0121 One aspect of the invention relates to enhancers of U.S. Pat. No. 5,767,068 and International Application WO antimicrobial agents. In some embodiments, enhancers of 98/20839 which are incorporated herein in its entirety by antimicrobial agents are inhibitors of the gene products listed reference. In some embodiments, the colistin can be admin in Table 1 or Table 4 as disclosed herein. The inactivation of istered with additional agents, as disclosed in International a gene product by inhibition is considered to potentiate the Patent Number WO2006/045156, which is incorporated effectiveness of the antimicrobial agent if the amount of anti herein by reference. In particular, International Patent Num microbial agent used after inactivation is reduced by at least ber WO2006/045156 discloses using colistin with a mac 10% without adversely affecting the result, for example, rolide component, such as , , without adversely effecting the level of antimicrobial activity. and other components with a lactone ring, In another embodiment, the criteria used to select an enhanc where the combination of the macrollide and colistinenhances ers that potentiate the activity of an antimicrobial agent is a the anti-pathogenic effects of the macrollide component and reduction of at least ... 10%, ... 15%, ... 20%, ... 25%, .. the colisitin as compared to their effects alone. .35%, ... 50%, ... 60%, ...90% and all amounts in-between 0117. In one embodiment, the antimicrobial peptide com of the antimicrobial agent without adversely effecting the prises colistin methanesulphonate and/or colistin Sulphate. In antimicrobial effect when compared to the similar cell with another embodiment, the antimicrobial peptide comprises out the addition of an enhancer of the antimicrobial agent. colistin Sulphate. I0122. In some embodiments, example of such enhancers 0118. It is envisaged that variation of these components, of antimicrobial agents can include nucleic acids, peptides, for example, by Substituting a D-amino acid residue for the nucleic acid analogues, phage, phagemids, polypeptides, same or different L-amino acid residue or vice versa, varying peptidomimetics, antibodies, Small or large organic or inor the R substituents and/or conservative amino acid substitu ganic molecules or any combination of the above. The tions, while maintaining the synergistic is antimicrobial enhancers of antimicrobial agents can also be naturally occur activity with the peptide enhancer component of the inven ring or non-naturally occurring (e.g., recombinant) and are tion, is encompassed within the scope of the invention. Sometimes isolated and/or purified. 0119 Colisitin is associated with and neph (0123. In some embodiments, where the antimicrobial rotoxicity. The inventors have discovered a dosage regimen agent is an antimicrobial peptide, the enhancer is an enhancer and combination of colistin with enhancers of antimicrobial to an antimicrobial peptide. In Such embodiments, these agents as disclosed herein, colistin can be used at reduced enhancers are, for example, but not limited to inhibitors of doses for the same effect and thereby the inventors have gene products. In some embodiments, the gene products are, discovered a method whereby colistin can be administered to for example but not limited to, agaA, atp A, atpC, atpB, atp). reduce toxicity effects. Colistin, a cyclic lipopeptide, pen atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, etrates the cell wall of G-bacteria by a self induced mecha kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE, nism by chelating divalent ions, it destabilizes the wall and rpiA, such3, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, can insinuate into it. Without being bound by theory, colistin yeeY, yiaY, yidK, yihV. y?hC), yibn and/or ynjD or homo US 2010/0028334 A1 Feb. 4, 2010 logues thereof. In some embodiments, the gene products are, F1 consists of five subunits (alpha=atpA, beta-atpl), for example but not limited to, those genes listed in Table 4. In gamma atpG, delta atpH, epsilon atpC). Some embodiments, the gene products are homologues, vari I0127 ATP synthesis is the fundamental process to provide ants, fragments or Substantially homologous to the following cell energy and is generated by oxidative phosphorylation. genes; agaA, atp A, atpC, atpB, atp), atpE, atpG, atpH, betB, The Source of energy is an electrochemical gradient of pro cSdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, tons issued from electron transfer across the membranes (Se mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trx A, nior et al., 2002). Theatp operon of the E. coli ATP syntheses tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK. consists of nine genes arranged in the order atp, atpB, atpE. yihV. y?hO, yibn and/orynjD or alternatively those listed in atpF, atpH, atpA, atpG, atp), and atpC; similar gene organi Table 4. In other embodiments, the gene products are, for Zation have been characterized in other bacteria. Theses gene example but not limited to, agaA, atp A, atpC, atpB, atp). products assembled into two components: F1 and F0. F 1 is on atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, the inner Surface of the membrane and is the catalytic center; kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE, F1 consists of nine polypeptide chain subunits of five differ rpiA, such3, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, ent types. Both the atpA and atpF mutants were primarily yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD. The identi identified in the primary screen using the mutant library. The fication of gene products, which when inactivated enhance atpF mutant was not retained for further studies. The atp A antimicrobial peptides is discussed in more detail below (see Sub-unit is conserved among bacterial species and mamma "Screening to Identify Gene Products that suppress the activ lians. The level of identity is over 80% for Salmonella sp. ity of antimicrobial peptides’). Similarly, identification and Klebsiella pneumo, Yersina, Haemophilus influenza, Yersinia screening of inhibitors of such gene products is also described and Vibrio cholera. in more detail below (see sections titled “Screening For Small I0128. Inhibitors of ATP synthases: In some embodiments Molecules That Inhibit the Gene Product and “Structure where the gene product is an ATP synthase, for example atpA, Based Design Methods to Create Small Molecule enhancers atpH and/or atpH, the inhibitor to the gene product, is an of antimicrobial peptide'). inhibitor to ATP synthases. In some embodiments, the inhibi 0124. In some embodiments, enhancers of antimicrobial tor of ATP synthases is mefloquine or analogues, mimetics or agents are inhibitors to the gene products listed in table 1, and derivatives thereof. Examples of Mefloquine are the orally such inhibitors can be the small molecules as disclosed in administered antimalarial drug used as a prophylaxis against Table 2. In some embodiments, an enhancer of the antimicro and treatment for , known by the trade name Lariam R. bial agent as disclosed herein does not include macrollides, (manufactured by Roche Pharmaceuticals) and chemical Such as for example erthyromycin, clarithromycin, name mefloquine hydrochloride (formulated with HCI). azithromyxin, , azithromycin and ketolides Such Mefloquine was developed in the 1970s at the Walter Reed as tellithromycin. In some embodiments, the enhancer of the Army Institute of Research in the U.S. as a chemical synthetic antimicrobial agent as disclosed herein does not include the similar to quinine. In some embodiments, the ATP synthase following ; , , ampi inhibitor is Mefloquine hydrochloride, and in some embodi cillin- ciprofloZacin, poperacillin-, ments it is a 4-quinolinemethanol derivative. While Meflo , , and ciproflxicin. While quine is known to have some side effects, its co-administra Some of these agents may have been used in combination with tion with an antimicrobial agent, for example an colistin, their ability to decrease the amount of colistin to be antimicrobial peptide may enable its use at lower therapeuti used without decreasing colistin antimicrobial effect as com cally effective doses, and Such, reduce the occurrence of side pared to the use of colistin alone cannot be determined effects. because of the limitations in the study design. 0129. In another embodiment, the inhibitor of an ATP 012.5 ATP synthases: In one embodiment, the gene prod synthase can be selected from; venturicidin A, diaryduino uct, which when inactivated potentiates the activity of anti line, Betaine, Acivin, Psicofuranine or derivatives, mimectics microbial agents, is for example an ATP synthase. In some or analogues thereof. In alternative embodiments, the inhibi embodiments, the ATP synthase is for example atpA. In alter tor of ATP synthases is venturicidin or oligomycin or ossa native embodiments, the gene product is atp A, atpH and/or mycin or derivatives, mimetics or analogues thereof. The atpH. ATP synthases is a general term for an enzyme that can antibiotics venturicidin, oligomycin and ossamycin were synthesize adenosine triphosphate (ATP) from adenosine investigated as potential inhibitors of the Escherichia coli diphosphate (ADP) and inorganic phosphate by utilizing H+-ATP synthase. It was found that venturicidin strongly Some form of energy. inhibited ATP-driven proton transport and ATP hydrolysis, 0126 An ATP synthase (EC 3.6.3.14) is a general term for while oligomycin weakly inhibited these functions. Inhibi an enzyme that can synthesize adenosine triphosphate (ATP) tion of the H+-ATP synthase by venturicidin and oligomycin from adenosine diphosphate (ADP) and inorganic phosphate was correlated with inhibition of FO-mediate proton trans by utilizing some form of energy. ATP syntheses are impor port. Venturicidin had been shown to be an antifungal of tant in energy metabolism, for example ATP-proton motive Potential Use in Agriculture. force interconversion in the synthesis of energy for use by the 0.130. Any inhibitor of ATP synthase is also encompassed cell and/or organism. ATP is formed by proton-conducting, for use in the present invention, including those described membrane-bound ATP synthase. ATP synthase is a multi herein, as well as those yet unidentified. component enzyme complex consisting of two main compo I0131 Inhibitors of BetB. In another embodiment, the gene nents: F1 and FO. F1 is on the inner surface of the membrane product, which when inactivated potentiates the activity of and is the catalytic center; F1 consists of nine polypeptide antimicrobial agents, is for example is a Betaine aldehyde chain subunits of five different types. FO is embedded within dehydrogenase. In some embodiments, the gene product is, the membrane and forms the membrane proton channel; in E. for example, a NAD-dependent Betaine aldehyde dehydro coli FO consists of three subunits (A=atpB, BatpF, C-atpE), genase. In some embodiments, the Betaine aldehyde dehy US 2010/0028334 A1 Feb. 4, 2010

drogenase is, for example betB. Bet genes confer protection sitions as disclosed herein is Psicofuranine, which was shown against osmotic stress by making the osmoprotectant glycine to have some antibacterial activities. (Hanka, 1959). betaine from choline. The bet genes are induced by choline, 0.137 Inhibitors of LipA: In another embodiment, the oxygen, and osmotic stress. gene product, which when inactivated potentiates the activity 0.132. In some embodiments, an inhibitor of an ATP syn of antimicrobial agents, is for example a Lipoyl synthase, or thases is Betaine. One such inhibitor is, for example but not a homologue or variant thereof. An example of a lipoyl Syn limited to Cystadane(R) (betaine anhydrous for oral solution), thase is LipA, which is an iron-sulfur protein with SAM which is currently used as an agent for the treatment of dependent chemistry. Lipoyl synthase is involved in lipoic homocystinuria. In another embodiment, an inhibitor of an acid biosynthesis, where the pathway of lipoic acid biosyn thesis consists solely of two steps in which two sulfur atoms ATP synthase which can be used in the methods and compo are introduced into the carbon skeletonat C-8 and C-6. White sitions as disclosed herein is Acivin, which is an irreversible showed that the sulfur atom in lipoic acid is derived from inhibitor of gamma-glutamyl transpeptidase (ID50-0.54 cysteine. As with the incorporation of the sulfur atom into mM). Inhibits the enzymatic conversion of LTC4 to LTD4. biotin, the insertion of sulfur in lipoid acid occurs without the Acivin is currently used as a potent anti-tumor and anti formation of either unsaturated or hydroxylated intermedi leishmania agent. ates. Two different sulfur atoms are inserted in lipoic acid 0133. In some embodiments where the gene product is a biosynthesis, whereas a single Sulfur atom inserts into both Betaine aldehyde dehydrogenase, for example betB, the carbon atoms in biotin biosynthesis. There is evidence to inhibitor to the gene product, is an inhibitor to Betaine alde indicate that a in each case, a single enzyme is responsible for hyde dehydrogenase. In some embodiments, the inhibitor of the sulfur insertions in each molecule, for example the bioB Betaine aldehyde dehydrogenase is Betaine or anhydrous gene product in biotin biosynthesis and the lip A gene product betaine or betaine aldehyde chloride oran analogue orderiva in lipoic acid biosynthesis. Accordingly, in one embodiment, tive or mimetic thereof. Examples of Betaine are Cystadane(R) the enhancer of antimicrobial agentis, for example an inhibi (betaine anhydrous for oral Solution), which is typically used tor to Lipoyl synthase, or a homologue or variant thereof. In in the treatment of homocystinuria. Any inhibitor of Betaine one embodiment, the inhibitor of Lipoyl synthase, is for aldehyde dehydrogenase is also encompassed for use in the example, buthionine sulfoximine or derivatives or modified present invention, including those described herein, as well as versions, mimetics or analogues thereof. Any inhibitor of those yet unidentified. Lipoyl synthase, for example lip A is also encompassed for 0134. Inhibitors of GuaA and GuaB: In another embodi use in the present invention, including those described herein, ment, the gene product, which when inactivated potentiates as well as those as yet unidentified. the activity of antimicrobial agents, is for example is a gua 0.138. Inhibitors of Lys A: In another embodiment, the nine-monophosphate (GMP) synthase or an -5'- gene product, which when inactivated potentiates the activity monophosphate (IMP) dehydrogenase or homologues or of antimicrobial agents, is for example a diaminopimelate variants thereof. In some embodiments, the GMP synthase is, decarboxylase or a homologue or variant thereof. An example for example, guaA and IMP dehydrogenase is, for example, of a diaminopimelate decarboxylase is lySA. The expression guaB or homologues or variants thereof. GMP synthases and of diaminopimelate decarboxylase depends on the intracel IMP dehydrogenases important in the conversion of IMP to lular concentration of both diaminopimelate, which acts as an AMP and GMP. These are also used for de novo synthesis as inducer, and , which acts as a corepressor. This double well as in the salvage of purine bases. In some embodiments, regulation reflects the special situation of the diaminopime the gene products are for example also purA and purB, which late decarboxylase, as a catabolic enzyme for diaminopime are required for the reactions to of IMP to AMP, whereas in late and an anabolic enzyme for lysine. Moreover, ppGpp other embodiments, the gene products are guaB and guaA, must play a role inlySA expression, which is modified in relA which are required for synthesis of GMP. mutants. Accordingly, in one embodiment, the enhancer of 0135) In another embodiment, the gene product, which antimicrobial agent is an inhibitor to diaminopimelate decar when inactivated potentiates the activity of antimicrobial boxylase, or homologues or mimetics or variants thereof. In agents, is for example is a 2-octaprenyl-6-methylphonel one embodiment, the inhibitor is for example, diami hydroxylase, producing 2-octaprenl-6-methyoxy-1,4-benzo nopimelic acid and/or lysine or analogues, derivatives or quinone and is involved in the electron transport pathway. homologues thereof. Any inhibitor of a diaminopimelate 0136. In some embodiments where the gene product is a decarboxylase, for example lySA, is also encompassed foruse guanine-monophosphate (GMP) synthase, for example guaA in the present invention, including those described herein, as or an inosine-5'-monophosphate (IMP) dehydrogenase, for well as those as yet unidentified. example guaB, the enhancer of the antimicrobial agent is for 0.139. Inhibitors of RpiA: In another embodiment, the example Acivin and/or Psicofurine. Acivin is an irreversible gene product, which when inactivated potentiates the activity inhibitor of gamma-glutamyl transpeptidase (IDso 0.54 of antimicrobial agents, is for example a ribose-5-phosphate mM) and inhibits the enzymatic conversion of LTC4 to LTD4. isomerase or a homologue or variant thereof. In some Typically it is used as a potent anti-tumor and anti-leishmania embodiments, the ribose-5-phosphate isomerase is ribose-5- agent. Psicofuranine was shown to have some antibacterial phosphate isomerase A. In some embodiments, the ribose-5- activities. (Hanka, 1959). Any inhibitor of a guanine-mono phosphate isomerase A is alkali-inducible. An example of phosphate (GMP) synthase, for example guaA or an inosine ribose-5-phosphate isomerase A is rpiA. Ribose 5-phosphate 5'-monophosphate (IMP) dehydrogenase, for example guaB isomerases interconvert ribose 5-phosphate and ribulose is also encompassed for use in the present invention, includ 5-phosphate. This reaction allows the synthesis of ribose ing those described herein, as well as those as yet unidenti from the pentose phosphate pathway and represents a means fied. Accordingly, in another embodiment, an inhibitor of an for the salvage of carbohydrates after nucleotide breakdown. ATP synthase which can be used in the methods and compo However, such a dual role for a metabolic pathway is unusual US 2010/0028334 A1 Feb. 4, 2010 and presents problems for effective metabolic regulation. of endoplasmic reticulum, peroxisomes, lysosomes, Vesicles Two unrelated types of enzymes can catalyze the reaction. and notably the inner membrane of the mitochondrion where RpiA is highly conserved and present in almost all organisms. it is an important part of the electron transport chain; there it In E. coli and Salmonella, the enzyme is constitutively passes reducing equivalents to acceptors such as Coenzyme expressed. The second type of ribose isomerase, RpiB, is Q: cytochrome c-oxidoreductase. ubiH gene product is con Sometimes referred to as AIsB because it can also take part in served among bacteria showing over 75% identity with the the metabolism of the rare Sugar allose. E. coli strains defec ubiH homologs in Shigella, salmonella and Klebsiella. The tive in rpiA are ribose auxotrophs, despite the presence of level of identity with Pseudomonas is lower at 40%. wild-typerpiB. Ribose prototrophs of an rpiA genetic back 0144. Inhibitors of ubiH: ubiH gene which is in the ground arise spontaneously. RpiA exhibits a a/131(a13)/13/a ubiquinone synthesis pathway and the atpA which is a com fold, some portions of which are similar to proteins of the ponent of the ATP synthase indirectly or directly involved in alcoholdehydrogenase family. The two subunits of the dimer the electron transfer chain. Ubiquinone is part of a cascade of in the asymmetric unit have different conformations, repre electron transfer leading to the production of ATP by the ATP senting the opening/closing of a cleft. The enzyme presum synthase. The electron transport chain is the major consumer ably acts by an acid-base catalysis mechanism. of oxygen in the cell. The cascade of redox reactions allows 0140. Accordingly, in one embodiment, the enhancerofan the phosphorylation of ADP, forming ATP utilizing the antimicrobial agent is an inhibitor to ribose-5-phosphate energy derived from various Substrates through the central isomerase, for example an inhibitor to ribose-5-phosphate metabolism (glycolysis, TCA cycle) while reducing NADU isomerase A, or a homologue or variant thereof. In one or FADH2. The reasons for these genes once inactivated embodiment, the inhibitor is for example, 4-phospho-D-er genetically or chemically to potentiate colistin are not clear. It thronhydrixamic acid or analogues, derivatives or homo is possible that the membrane potential chain is required to logues or mimetics thereof. Any inhibitor of ribose-5-phos maintain an intact plasma membrane, as the gene products phate isomerases, for example rpiA are also encompassed for ubiH and atpA are necessary but not essential tough for this use in the present invention, including those described herein, process. as well as those as yet unidentified. 0145. It is also encompassed within the present invention 0141 Inhibitors of TrXA. In another embodiment, the gene that an antimicrobial agent disclosed herein can be combined product, which when inactivated potentiates the activity of with any of the inhibitors of the gene products mentioned antimicrobial agents, is for example is a Thioredoxin or a above, thereby enhancing the activity of antimicrobial agent. homologue or variant thereof. An example of a Thioredoxin is AS Such, inhibitors to the gene products; agaA, atp A, atpC, trXA or trxB, which has general chaperone activity, and func atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, tions as a processitiviy factor for phage T7 gene 5 DNA guaB, iscS, kdgK, lip A, lySA, mnmA. nuvC, papa, pdxH. polymerase. Accordingly, in one embodiment, an enhancer of phnL, potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE, ubiH, antimicrobial agent is an inhibitor to thioredoxin or a variant uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD or homologue thereof. In one embodiment, the inhibitor of or homologues or variants thereofare referred to as "enhanc thioredoxin is, for example, motexafin gadolinium and/or ers of antimicrobial agents’ or "enhancers of antimicrobial Xycistrin or derivatives, analogues or variants or mimetics peptides' herein. In some embodiments, an inhibitor of a thereof. Any inhibitor of thioredoxin, for example an inhibitor gene product is any inhibitor or agent which inhibits the of trXA, is also encompassed for use in the present invention, function of a gene listed in Table 4. Such enhancers of anti including those described herein, as well as those as yet microbial agents are for example, but not limited to, meflo unidentified. quine, Venturicidin A, antimycin A, myxothiazol, Stigma 0142. In some embodiments, the gene product is involved tellin, diuron, idoacetamide, potassium tellurite hydrate, in RNA modifications. Biosynthesis of thionucleoside is a aDL-vinylglycine, N-Ethylmaleimide, L-Allyglycine, dia complex process associated with Sulfur metabolism and ryduinoline, betaine aldehyde chloride, acivein, psicofuraine, involving numerous proteins including, but not limited to, buthionine Sulfoximine, diaminopemelic acid, 4-phospho-D- iscS, tuSA, tusB and mnmA. erythronhydroxamic acid, motexafin gadolinium and/or 0143. In another embodiment, the gene product, which Xycitrin and are Summarized in Table 2. In some embodi when inactivated potentiates the activity of antimicrobial ments, one or more enhancer of antimicrobial agent can be agents, is for example is a ubi or a homologue or variant used to enhance or potentiate the activity of antimicrobial thereof. An example of a ubigenes is ubiH, which is involved agents described herein. Any combination of antimicrobial in the ubiquinone synthesis pathway. Ubiquinone is also agent enhancers can be used, in any amount, in any form and referred to as coenzyme Q. ubiH is a 2-octaprenyl-6-methox by any route of administration. In some embodiments, the yphenol hydroxylase and produces 2-octaprenyl-6-methoxy enhancers of the antimicrobial agent are administered at the 1,4-benzoquinone. Coenzyme Q is found in the membranes same time or sequentially in any order. TABLE 2 Examples of enhancers of antimicrobial agents (referred to as “inhibitors') which inhibit the genes (or gene products) which, when inactivated, potentiate the effect of antimicrobial agents. SEQ ID GeneBank Accession NO: Gene ID ID Inhibitor 44 ace. 2-oxoglutarate or 3-hydroxybutyrate 2 atp A 1790172 AACT6757 mefloquine, venturicidin A 9 betB 1786SO4 AACT341S betaine aldehyde chloride 45 fo , Phosphanilic acid US 2010/0028334 A1 Feb. 4, 2010 16

TABLE 2-continued Examples of enhancers of antimicrobial agents (referred to as “inhibitors') which inhibit the genes (or gene products) which, when inactivated, potentiate the effect of antimicrobial agents. SEQID GeneBank Accession NO: Gene ID ID Inhibitor 14 guaA 1788.854 AAC75560 acivicin, psicofuranine 15 guaB 1788.855 AAC75561 Tiazofurin, mycophenolic acid 19 lipA 1786846 AAC73729 buthionine Sulfxoximine 20 lySA 1789203 AAC75877 diaminopimelic acid 46 nuo menaquinone oxidoreductase 27 rpiA 1789280 AAC75951 4-phospho-D-erythronohydroxamic acid 47 shC iethylpyrocarbonate, Methylmalonic acid, Myeloperoxidase 29 trix A 1790215 AAC76786 motexafin gadolinuim, Xyctrin

0146 Inhibitors of Gene Products for example, by chemical means, linkers and the like. In some 0147 In some embodiments, the inhibitors to the gene embodiments, the conjugation may be by protein fusion, the products which when inactivated, potentiate the effect of methods of which are well know in the art. antimicrobial agents, include for example antibodies (poly 0149. In some embodiments, the pharmaceutical compo clonal or monoclonal), neutralizing antibodies, antibody sition can comprise a pharmaceutically acceptable carrier and fragments, peptides, proteins, peptide-mimetics, aptamers, pharmaceutically effective amounts of a microbial agent and oligonucleotides, hormones, Small molecules, nucleic acids, an enhancer of an antimicrobial agent which are polypeptide nucleic acid analogues, carbohydrates or variants thereofthat and proteins. In one embodiment, the antimicrobial agent is a function to inactivate the nucleic acid and/or protein of the colistin, or a homologue or analogue thereof, and in another gene products identified herein, and those as yet unidentified. embodiment, the enhancer of an antimicrobial agent is an Nucleic acids include, for example but not limited to, DNA, inhibitory antibody or polypeptide inhibitor of one of the RNA, oligonucleotides, peptide nucleic acid (PNA), pseudo genes listed in Table 1 or Table 4. In one embodiment, the complementary-PNA (pcPNA), locked nucleic acid (LNA). antimicrobial agent and an enhancer of an antimicrobial agent RNAi, microRNAi, siRNA, shRNA etc. The inhibitors can be can be conjugated together, using such methods of protein or selected from a group of a chemical, Small molecule, chemi polypeptide conjugation which are well known in the art. One cal entity, nucleic acid sequences, nucleic acid analogues or can use any method for conjugation of molecules known by protein or polypeptide or analogue or fragment thereof. In persons of ordinary skill in the art, for example, conjugation some embodiments, the nucleic acid is DNA or RNA, and by chemical means, covalent bonds, linkers and the like. In nucleic acid analogues, for example can be PNA, pcPNA and Some embodiments, the conjugation may be protein fusion, LNA. A nucleic acid may be single or double stranded, and the methods of which are well known in the art. For example, can be selected from a group comprising: nucleic acid encod BioVertis of Vienna has a dual action compound called ing a protein of interest, oligonucleotides, PNA, etc. Such Oxaquin, which combines the therapeutic moieties of two nucleic acid sequences include, for example, but not limited different antibiotic compounds into one molecule. to, nucleic acid sequence encoding proteins that act as tran 0150. In some embodiments, multi-binding agents are Scriptional repressors, antisense molecules, ribozymes, Small useful in the methods and compositions as disclosed herein, inhibitory nucleic acid sequences, for example but not limited for example multi-binding agents which comprise an antimi to RNAi, shRNAi, siRNA, microRNAi (mRNAi), antisense crobial agents such as colistin and an enhancer of Such anti oligonucleotides etc. A protein and/or peptide inhibitor or microbial agent, such as those inhibiting at least one gene fragment thereof, can be, for example, but not limited to listed in Table 2 or Table 4. Multivalent binding interactions mutated proteins; therapeutic proteins and recombinant pro are characterized by the concurrent interaction of multiple teins. Proteins and peptides inhibitors can also include for ligands with multiple ligand binding sites on one or more example; mutated proteins, genetically modified proteins, cellular receptors. Multivalent interactions differ from collec peptides, synthetic peptides, recombinant proteins, chimeric tions of individual monovalent interactions by imparting proteins, antibodies, humanized proteins, humanized anti enhanced biological and/or therapeutic effect. Just as multi bodies, chimericantibodies, modified proteins and fragments Valent binding can amplify binding affinities; it can also thereof. amplify differences in binding affinities, resulting in 0148. In some embodiments, an enhancer of an antimicro enhanced binding specificity as well as affinity. An example bial agent, such as, but not limited to those listed under of a multi-binding agent is an avimer, which relates to a inhibitors in Table 2 can be combined with the antimicrobial peptide agent which is capable of binding to one or more sites. agent to form one dual mode of action compound. Such 0151. Antibodies methods are well known by person of ordinary skill in the art 0152. In some embodiments, inhibitors of genes and/or and include chemical conjugation of two molecules or con gene products useful in the methods of the present invention jugation by any means known in art. Methods of conjugation that function as enhancers to antimicrobial peptides include, of two molecules or entities to form a binary conjugate is for example, antibodies, including monoclonal, chimeric described in PCT Patent Application No: WO99/66944, humanized, and recombinant antibodies and fragment which is specifically incorporated herein in its entirety by thereof. In some embodiments, neutralizing antibodies can be reference. In some embodiments, where the enhancer is a used as inhibitors of the gene products identified herein. polypeptide. Such as for example an antibody or peptide Antibodies are readily raised in animals such as rabbits or inhibitor as disclosed below, methods of conjugation can be, mice by immunization with the gene product, which when US 2010/0028334 A1 Feb. 4, 2010

inactivated, potentiate the effect of an antimicrobial agent. 0154) One limitation of scFv molecules is their monova Immunized mice are particularly useful for providing sources lent interaction with target antigen. One of the easiest meth of B cells for the manufacture of hybridomas, which in turn ods of improving the binding of a Schv to its target antigen is are cultured to produce large quantities of monoclonal anti to increase its functional affinity through the creation of a bodies. Chimeric antibodies are immunoglobin molecules multimer. Association of identical scFv molecules to form characterized by two or more segments or portions derived diabodies, triabodies and tetrabodies can comprise a number from different animal species. Generally, the variable region of identical Fv modules. These reagents are therefore multi of the chimeric antibody is derived from a non-human mam valent, but monospecific. The association of two different malian antibody, Such as murine monoclonal antibody, and ScFv molecules, each comprising a VH and VL domain the immunoglobin constant region is derived from a human derived from different parent Ig will form a fully functional immunoglobin molecule. Preferably, both regions and the bispecific diabody. A unique application of bispecific ScFVS is combination have low immunogenicity as routinely deter to bind two sites simultaneously on the same target molecule mined. Humanized antibodies are immunoglobin molecules via two (adjacent) Surface epitopes. These reagents gain a created by genetic engineering techniques in which the significant: avidity advantage over a single Sclv or Fab frag murine constant regions are replaced with human counter ments. A number of multivalent scEv-based structures has parts while retaining the murine antigenbinding regions. The been engineered, including for example, miniantibodies, resulting mouse-human chimeric antibody should have dimeric miniantibodies, minibodies, (sclv)2, diabodies and reduced immunogenicity and improved pharmacokinetics in triabodies. These molecules span a range of Valence (two to humans. Some examples of high affinity monoclonal antibod four binding sites), size (50 to 120 kDa), flexibility and ease ies and chimeric derivatives thereof, useful in the methods of of production. Single chain Fv antibody fragments (scFvs) are predominantly monomeric when the VHandVL domains the present invention, are described in the European Patent are joined by, polypeptide linkers of at least 12 residues. The Application EP 186,833; PCT Patent Application WO monomer schv is thermodynamically stable with: linkers of 92/16553; and U.S. Pat. No. 6,090,923. 12 and 25 amino acids length under all conditions. The non 0153. In one embodiment of this invention, the inhibitor to covalent diabody and triabody molecules are easy to engineer the gene products identified herein can be an antibody mol and are produced by shortening the peptide linker that con ecule or the epitope-binding moiety of an antibody molecule nects the variable heavy and variable light chains of a single and the like. Antibodies provide high binding avidity and schv molecule. The schv dimers are joined by amphipathic unique specificity to a wide range of target antigens and helices that offer a high degree of flexibility and the minian haptens. Monoclonal antibodies useful in the practice of the tibody structure can be modified to create a dimeric bispecific present invention include whole antibody and fragments (DiBi) miniantibody that contains two miniantibodies (four thereof and are generated in accordance with conventional schv molecules) connected via a double helix. Gene-fused or techniques, such as hybridoma Synthesis, recombinant DNA disulfide bonded scFv dimers provide an intermediate degree techniques and protein synthesis. Useful monoclonal anti of flexibility and are generated by straightforward cloning bodies and fragments may be derived from any species (in techniques adding a C-terminal Gly4CyS sequence. Scv cluding humans) or may be formed as chimeric proteins CH3 minibodies are comprised of two schv molecules joined which employ sequences from more than one species. Human to an IgG CH3 domain either directly (LD minibody) or via a monoclonal antibodies or “humanized' murine antibody are very flexible hinge region (Flex minibody). With a molecular also used in accordance with the present invention. For weight of approximately 80kDa, these divalent constructs are example, murine monoclonal antibody may be “humanized' capable of significant binding to antigens. The Flex minibody by genetically recombining the nucleotide sequence encod exhibits impressive tumor localization in mice. Bi- and tri ing the murine Fv region (i.e., containing the antigenbinding specific multimers can be formed by association of different sites) or the complementarily determining regions thereof schv molecules. Increase in functional affinity can be reached with the nucleotide sequence encoding a human constant when Fab or single chain Fv antibody fragments (scFv) frag domain region and an Fc region. Humanized targeting moi ments are complexed into dimers, trimers or larger aggre eties are recognized to decrease the immunoreactivity of the gates. The most important advantage of multivalent scvs antibody or polypeptide in the host recipient, permitting an over monovalent scFv and Fab fragments is the gain in func increase in the half-life and a reduction in the possibly of tional binding affinity (avidity) to target antigens. High avid adverse immune reactions in a manner similar to that dis ity requires that Schv multimers are capable of binding simul closed in European Patent Application No. 0.411,893 A2. The taneously to separate target antigens. The gain in functional murine monoclonal antibodies should preferably be affinity for schv diabodies compared to schv monomers is employed in humanized form. Antigen binding activity is significant and is seen primarily in reduced off-rates, which determined by the sequences and conformation of the amino result from multiple binding to two or more target antigens acids of the six complementarily determining regions (CDRS) and to rebinding when one Fv dissociates. When such schv that are located (three each) on the light and heavy chains of molecules associate into multimers, they can be designed the variable portion (Fv) of the antibody. The 25-kDa single with either high avidity to a single target antigen or with chain FV (ScFV) molecule, composed of a variable region multiple specificities to different target antigens. Multiple (VL) of the light chain and a variable region (VH) of the binding to antigens is dependent on correct alignment and heavy chain joined via a short peptide spacer sequence, is the orientation in the Fv modules. For full avidity in multivalent Smallest antibody fragment developed to date. Techniques ScFVs target, the antigen binding sites must point towards the have been developed to display schv molecules on the surface same direction. If multiple binding is not sterically possible of filamentous phage that contain the gene for the ScFv. Sclv then apparent gains in functional affinity are likely to be due molecules with a broad range of antigenic-specificities can be the effect of increased rebinding, which is dependent on dif present in a single large pool of ScFV-phage library. fusion rates and antigen concentration. Antibodies conju US 2010/0028334 A1 Feb. 4, 2010 gated with moieties that improve their properties are also yihV, yfhC), yibn and/or ynjD or homologues or variants contemplated for the instant invention. For example, antibody thereof can be manipulated by a variety of well known tech conjugates with PEG that increases their half-life in vivo can niques for in vitro mutagenesis, among others, to produce be used for the present invention. Immune libraries are pre variants of the naturally occurring human protein or fragment pared by Subjecting the genes encoding variable antibody thereof, herein referred to as muteins, may be used in accor fragments from the B lymphocytes of naive or immunized dance with the invention. animals or patients to PCR amplification. Combinations of 0158. The variation in primary structure of muteins of oligonucleotides which are specific for immunoglobulin agaA, atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, genes or for the immunoglobulin gene families are used. cSdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, mnmA, Immunoglobulin germ line genes can be used to prepare nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, tusB semisynthetic antibody repertoires, with the complementa (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, rily-determining region of the variable fragments being yihV. y?hO, yibn and/orynjDare useful in the invention, for amplified by PCR using degenerate primers. These single-pot instance, may include deletions, additions and Substitutions. libraries have the advantage that antibody fragments againsta The Substitutions may be conservative or non-conservative. large number of antigens can be isolated from one single The differences between the natural protein and the mutein library. The phage-display technique can be used to increase generally conserve desired properties, mitigate or eliminate the affinity of antibody fragments, with new libraries being undesired properties and add desired or new properties. prepared from already existing antibody fragments by ran 0159. Similarly, techniques for making small oligopep dom, codon-based or site-directed mutagenesis, by shuffling tides and polypeptides that inactivate and/or function as the chains of individual domains with those of fragments dominant negative versions (i.e. inactive versions) of larger from naive repertoires or by using bacterial mutator strains. proteins from which they are derived are well known and have 0155 Alternatively, a SCID-humouse, for example the become routine in the art. Thus, peptide analogs of gene model developed by Genpharm, can be used to produce anti products of the invention that inactivate the gene product also bodies, or fragments thereof. In one embodiment, a new type are useful in the invention. of high avidity binding molecule, termed peptabody, created (0160. In some embodiments, RNA interference or by harnessing the effect of multivalent interaction is contem “RNAi can be used as enhancers of antimicrobial agents. In plated. A short peptide ligand was fused via a semirigid hinge Such an embodiment, a RNAi molecule that negatively regu region with the coiled-coil assembly domain of the cartilage lates the expression of the gene products of the invention, for oligomeric matrix protein, resulting in a pentameric multiva example but not limited to agaA, atpA, atpC, atpB, atp), lent binding molecule. In preferred embodiment of this inven atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, tion, ligands and/or chimeric inhibitors can be targeted to kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE, tissue- or tumor-specific targets by using bispecific antibod rpiA, such3, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, ies, for example produced by chemical linkage of an anti yeeY, yiaY, yidK, yihV. y?hC), yibn and/or ynjD or homo ligand antibody (Ab) and an Ab directed toward a specific logues or variants thereof, can be used as enhancers of anti target. To avoid the limitations of chemical conjugates, microbial peptides in the present invention. RNAi is a term molecular conjugates of antibodies can be used for produc initially coined by Fire and co-workers to describe the obser tion of recombinant bispecific single-chain Abs directing vation that double-stranded RNA (dsRNA) can block gene ligands and/or chimeric inhibitors at cell Surface molecules. expression when it is introduced into worms (Fire et al. (1998) Alternatively, two or more active agents and or inhibitors Nature 391, 806-811). dsRNA directs gene-specific, post attached to targeting moieties can be administered, wherein transcriptional silencing in many organisms, including verte each conjugate includes a targeting moiety, for example, a brates, and has provided a new tool for studying gene func different antibody. Each antibody is reactive with a different tion. RNAi involves mRNA degradation of a target gene. target site epitope (associated with the same or a different Results showed that RNAi is ATP-dependent yet uncoupled target site antigen). The different antibodies with the agents from mRNA . That is, protein synthesis is not attached accumulate additively at the desired target site. Anti required for RNAi in vitro. In the RNAi reaction, both strands body-based or non-antibody-based targeting moieties may be (sense and antisense) of the dsRNA are processed to small employed to deliver a ligand or the inhibitor to a target site. RNA fragments or segments of from about 21 to about 23 Preferably, a natural binding agent for an unregulated antigen nucleotides (nt) in length ( with mobility in sequencing is used for this purpose. For example, diseases such as gels that correspond to markers that are 21-23 nt in length, hepatoma or myeloma are generally characterized by unregu optionally referred to as 21-23 nt RNA). Processing of the lated IL-6 receptors for which IL-6 acts as an autocrine or dsRNA to the small RNA fragments does not require the paracrine moiety with respect to rapid proliferation of these targeted mRNA, which demonstrates that the small RNA target cell types. For the treatment of Such ailments, IL-6 may species is generated by processing of the dsRNA and not as a therefore be employed as a targeting moiety in a targeting product of dsRNA-targeted mRNA degradation. The mRNA protocol of the present invention. is cleaved only within the region of identity with the dsRNA. 0156 Nucleic Acid Inhibitors Cleavage occurs at sites 21-23 nucleotides apart, the same 0157. It will be appreciated by those of skill that the genes interval observed for the dsRNA itself, suggesting that the identified herein and those identified by the methods of the 21-23 nucleotide fragments from the dsRNA are guiding present invention can be readily manipulated to alter the mRNA cleavage. Isolated RNA molecules (double-stranded; amino acid sequence of a protein. Genes for example, but not single-stranded) of from about 21 to about 23 nucleotides limited agaA, atp A, atpC, atpB, atp), atpE, atpG, atpH, betB, mediate RNAi. That is, the isolated RNAs mediate degrada cSdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, tion of mRNA of a gene to which the mRNA corresponds mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trx A, (mediate degradation of mRNA that is the transcriptional tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK. product of the gene, which is also referred to as a target gene). US 2010/0028334 A1 Feb. 4, 2010

Isolated RNA molecules specific to G6PD mRNA, which first aspect of the present invention are multi-drug resistant mediate RNAi are antagonists useful in the method of the microorganisms. In some embodiments, gram-negative present invention. Alternative nucleic acid and nucleic acid microorganisms are targeted analogues can be used as enhancers of antimicrobial peptides, (0166 Bacterial infections include, but are not limited to, for example oligonucleotides, antisense nucleic acid con infections caused by Bacillus cereus, Bacillus anthracis, structs, siRNA, microRNA, shRNA etc. Bacillus cereus, Bacillus anthracis, Clostridium botulinum, Clostridium difficle, Clostridium tetani, Clostridium perfrin 0161. In some embodiments of the invention suitable gens, Corynebacteria diptheriae, Enterococcus (Streptococ enhancers of antimicrobial agents may be administered to the cus D), Listeria monocytogenes, Pneumococcal infections Subject in a vector. The vector may be a plasmid vector, a viral (Streptococcus pneumoniae), Staphylococcal infections and vector, or any other suitable vehicle adapted for the insertion Streptococcal infections/Gram-negative bacteria including and foreign sequence and for the introduction into eukaryotic Bacteroides, Bordetella pertussis, Brucella, Campylobacter cells. The vector can be an expression vector capable of infections, enterohaemorrhagic Escherichia coli (EHEC/E. directing the transcription of the DNA sequence of the agonist coli O157:17) enteroinvasive Escherichia coli (EIEC), entero or antagonist nucleic acid molecules into RNA. Viral expres toxigenic Escherichia coli (ETEC), Haemophilus influenzae, sion vectors can be selected from a group comprising, for Helicobacter pylori, Klebsiella pneumoniae, Legionella spp., example, retroviruses, lentiviruses, Epstein Barr virus-, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria bovine papilloma virus, adenovirus- and adeno-associated meningitidis, Proteus spp., Pseudomonas aeruginosa, Sal based vectors or hybrid virus of any of the above. In one monella spp., Shigella spp., Vibrio cholera and Yersinia, acid embodiment, the vector is episomal. The use of a suitable fast bacteria including Mycobacterium tuberculosis, Myco episomal vector provides a means of maintaining the agonist bacterium avium-intracellulars, Mycobacterium johnei, orantagonist nucleic acid molecule in the Subject in high copy Mycobacterium leprae, atypical bacteria, Chlamydia, Myo number extra chromosomal DNA thereby eliminating poten plasma, Rickettsia, Spirochetes, Treponema pallidum, Borre tial effects of chromosomal integration. lia recurrentis, Borrelia burgdorfii and Leptospira icterohe 0162 Another embodiment of the invention, suitable morrhagiae and other miscellaneous bacteria, including enhancers of antimicrobial agents can be achieved by intro Actinomyces and Nocardia. (0167. In some embodiments, the microbial infection is ducing catalytic antisense nucleic acid constructs, such as caused by gram-negative bacterium, for example, P aerugi ribozymes, which are capable of cleaving RNA transcripts nosa, A. baumannii, Salmonella spp., Klebsiella pneumonia, and thereby preventing the production of wildtype protein. Shigella spp. and/or Stenotrophomonas maltophilia. Ribozymes are targeted to and anneal with a particular 0168 Examples of microbial infections include bacterial sequence by virtue of two regions of sequence complemen wound infections, mucosal infections, enteric infections, sep tary to the target flanking the ribozyme catalytic site. After tic conditions, pneumonia, trachoma, onithosis, trichomonia binding the ribozyme cleaves the target in a site specific sis and salmonellosis, especially in Veterinary practice. manner. The design and testing of ribozymes which specifi 0169. Examples of infections caused by P. aeruginosa cally recognize and cleave sequences of the gene products include: A) Nosocomial infections: 1. Respiratory tract infec described herein, for example but not limited to agaA, atpA, tions in cystic fibrosis patients and mechanically-ventilated atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, patients; 2. Bacteraemia and sepsis; 3. Wound infections, guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, particularly in wound patients; 4. Urinary tract infec pdxH, phn L. potE. rpiA, such3, trx A, tusB (YheL), tusB. ubiE, tions: 5. Post-surgery infections on invasive devises 5. ubiH, uncA, visB, yeeY, yiaY, yidK, yihV.yfhC), yibN and/or Endocarditis by intravenous administration of contaminated ynjD or homologues or variants thereof can be achieved by drug Solutions; 7, Infections in patients with acquired immu techniques well known to those in the art (for example Lleber nodeficiency syndrome, , steroid and Strauss, (1995) Mol Cell Biol 15:540.551, the disclosure therapy, hematological malignancies, organ transplantation, of which is incorporated herein by reference). renal replacement therapy, and other situations with severe 0163 Uses of Antimicrobial Agents and Enhancers neutropenia. Thereof 0170 B) Community-acquired infections: 1. Community 0164. Some bacteria, including P. aeruginosa, actively acquired respiratory tract infections; 2. ; 3. Folli form tightly arranged multi-cell structures in vivo known as culitis and infections of the ear canal caused by contaminated biofilm. The production of biofilm is important for the per waters; 4. Malignant otitis externa in the elderly and diabet sistence of infectious processes such as seen in pseudomonal ics; 5. Osteomyelitis of the caleaneus in children: Eye infec lung-infections in patients with cystic fibrosis and diffuse tions commonly associated with contaminated contact lens; panbronchiolitis and many other diseases. Biofilm is resistant 6. Skin infections such as nail infections in people whose to phagocytosis by host immune cells and the effectiveness of hands are frequently exposed to water, 7. Gatrointestinal tract antibiotics at killing bacteria in biofilm structures may be infections; 8. Muscoskeletal system infections. reduced by 10 to 1000 fold. Biofilm production and arrange 0171 Examples of infections caused by A. baumannii ment is governed by quorum sensing systems. The disruption include: A) Nosocomial infections 1. Bacteraemia and sepsis, of the quorum sensing system in bacteria Such as P. aerugi 2. respiratory tract infections in mechanically ventilated nosa is an important anti-pathogenic activity as it disrupts the patients; 3. Post-Surgery infections on invasive devices; 4. biofilm formation and also inhibits alginate production wound infectious, particularly in bum wound patients; 5. 0.165. The term “microorganism’ includes any micro infection in patients with acquired immunodeficiency syn scopic organism or taxonomically related macroscopic drome, cancer chemotherapy, steroid therapy, hematological organism within the categories algae, bacteria, fungi, yeast malignancies, organ transplantation, renal replacement and protozoa or the like. The microorganisms targeted in the therapy, and other situations with severe neutropenia; 6. uri US 2010/0028334 A1 Feb. 4, 2010 20 nary tract infections; 7. Endocarditis by intravenous admin bacillus Oniliformis, Streptococcus agalactiae, Streptococ istration of contaminated drug Solutions; 8. Cellulitis. cus pneumoniae, Salmonella typhi; Salmonella paratyphi; 0172 B) Community-acquired infections; a. community Salmonella schottmulleri, Salmonella hirshieldii; Staphylo acquired pulmonary infections; 2. Meningitis; Cheratitis coccus epidermidis, , Klebsiella associated with contaminated contact lens; 4. War-Zone com pneumoniae, Legionella pneumophila, Helicobacter pylori; munity-acquired infections. pneumonia, Mycobacterium tuberculosis, 0173 C) Atypical infections: 1. Chronic gastritis. Mycobacterium leprae, Yersinia enterocolitica, Yersinia pes 0.174 Examples of infections caused by Stenotrophomo tis, Vibrio cholerae, Vibrio parahaemolyticus, Rickettsia pro nas maltophilia include Bacteremia, pneumonia, meningitis, wOzekii, Rickettsia rickettsii, Rickettsia akari; Clostridium wound infections and urinary tract infections. Some hospital difficile, Clostridium tetani, Clostridium perfingens, breaks are caused by contaminated disinfectant Solutions, Clostridianz novyi, Clostridianz Septicum, Clostridium respiratory devices, monitoring instruments and ice botulinum, Legionella pneumophila, Hemophilus influenzue; machines. Infections usually occur in debilitated patients Hemophilus parainfluenzue, Hemophilus aegyptus, Chlamy with impaired host defense mechanisms. dia psittaci, Chlamydia trachonzatis, Bordetella pertesis, 0175 Examples of infections caused by Klebsiella pneu Shigella spp., Campylobacter jejuni; Proteus spp., Citro moniae include community-acquired primary lobar pneumo bacter spp.; Enterobacter spp., Pseudomonas aeruginosa, nia, particularly in people with compromised pulmonary Propionibacterium spp.; Bacillus anthracis, Pseudomonas function and alcoholics. It also caused wound infections, soft syringae, Spirrillum minus, Neisseria meningitidis, Listeria tissue infections and urinary tract infections. monocytogenes, Neisseria gonorrhoeae, Treponema palli 0176 Examples of infections caused by Salmonella app. dum, Francisella tularensis, Brucella spp.; Borrelia recur are acquired by eating contaminated food products. Infec rentis, Borrelia hermsii; Borrelia turicatue, Borrelia burg tions include enteric fever, and bacteremia. dorferi, Mycobacterium avium, Mycobacterium smegmatis, 0177 Examples of infections caused by Shigella spp. -resistant Staphylococcus aureus, Vanomycin-re include gastroenteristis (shigellosis). sistant enterococcus, and multi-drug resistant bacteria (e.g., 0.178 The antimicrobial agent and/or enhancer of antimi bacteria that are resistant to more than 1, more than 2, more crobial agent components of the invention may also be used in than 3, or more than 4 different drugs). various fields as where treatment or disinfection of materials it required, for example, Surface disinfection. TABLE 3 0179 The antimicrobial agent and enhancers of antimi Examples of bacteria. crobial peptides described herein can be used to treat micro organisms infecting a cell, group of cells, or a multi-cellular Gram positive bacteria organism. Staphylococcusatiretts 0180. In one embodiment, the antimicrobial agent and Bacilius anthracis enhancers of antimicrobial agent described herein can be Bacilius cereus Bacilius subtiis used to reduce the rate of proliferation and/or growth of Streptococci is pneumonia microorganisms. In some embodiments, the microorganism Streptococci is pyogenes are either or both gram-positive or gram-negative bacteria, Cliostridium tetani whether such bacteria are cocci (spherical), rods, vibrio Listeria monocytogenes Mycobacterium tuberculosis (comma shaped), or spiral. Staphylococci is epidermidis 0181. Of the cocci bacteria, micrococcusandstaphylococ Gram negative bacteria cus species are commonly associated with the skin, and Strep tococcus species are commonly associated with tooth enamel Neisseria meningitidis Neisseria gonorrhoeae and contribute to tooth decay. Of the rods family, bacteria Vibrio cholerae Bacillus species produce endospores seen in various stages of Escherichia coli K12 development in the photograph and B. cereus cause a rela Bartoneia henseiae tively mild food poisoning, especially due to reheated fried Haemophilus influenzae Salmonella typhi food. Of the vibrio species, V. cholerae is the most common Shigella dysenteriae bacteria and causes cholera, a severe disease result Yersinia pestis ing from a toxin produced by bacterial growth in the gut. Of Pseudomonas aeruginosa the spiral bacteria, rhodospirillum and Treponema pallidum Helicobacter pylori are the common species to cause infection (e.g., Treponema Legionella pneumophila pallidum causes ). Spiral bacteria typically grow in Others shallow anaerobic conditions and can photosynthesize to Borrelia burgdorferi obtain energy from Sunlight. Ehrlichia chafeensis 0182 Moreover, the present invention relates to antimi Treponema pallidiin crobial agent and enhancers of antimicrobial agent that can be Chlamydia trachomatis used to reduce the rate of growth and/or kill either gram positive, gram negative, or mixed flora bacteria or other 0184. In some embodiments, antimicrobial agent and microorganisms. enhancers of antimicrobial agent described herein is used to 0183 Such bacteria are for example, but are not limited to, treat an already drug resistant bacterial Strain such as Methi listed in Table 3. Further examples of bacteria are, for cillin-resistant Staphylococcus aureus (MRSA) or Vancomy example but not limited to Baciccis Antracis, Enterococcus cin-resistant enterococcus (VRE) of variants thereof. faecalis, Corynebacterium, diphtheriae, Escherichia coli, 0185. The present invention also contemplates the use of Streptococcus coelicolor, Streptococcus pyogenes, Strepto antimicrobial agent and enhancers of antimicrobial agent US 2010/0028334 A1 Feb. 4, 2010

described herein in combinations with other antibiotics to been found to be effective in animals. The effective amount of fight Gram-positive bacteria that cannot maintain resistance an antimicrobial agent and an enhancer to Such an antimicro to certain drugs. bial agent can vary if the antimicrobial agent and an enhancer 0186 AS Such, antimicrobial agents and enhancers of anti of antimicrobial agent is coformulated with another therapeu microbial agents herein may be used to treat infections, for tic agents (for example, antibiotics, antiviral agents, antipro example bacterial infections and other conditions such as toZoan agents). It is contemplated that lower dosages will be urinary tract infections, ear infections, sinus infections, bac needed in Such cases as a result of a synergistic effect of all terial infections of the skin, bacterial infections of the lungs, active ingredients. sexually transmitted diseases, tuberculosis, pneumonia, lyme 0.192 In some embodiments, an effective amount of an disease, and Legionnaire's disease. Thus any of the above active ingredient (e.g., an antimicrobial agent and an conditions and other conditions resulting microorganism enhancer of antimicrobial agent and/or additional therapeutic infections, for example bacterial infections may be prevented agent(s)) is from about 0.0001 mg to about 500 mg active or treated by the compositions of the invention herein. agent per kilogram body weight of a patient, in Some embodi 0187. In another example, an antimicrobial agents and ments from about 0.001 to about 250 mg active agent per enhancers of antimicrobial agents are used to inhibit resis kilogram body weight of the patient, in further embodiments tance to an antiprotozoan agent selected from the group con from about 0.01 mg to about 100 mg active agent per kilo sisting of: Chloroquine; Pyrimethamine; Mefloquine gram body weight of the patient, yet still more embodiments Hydroxychloroquine; ; Atovaquone; Imi from about 0.5 mg to about 50 mg active agent per kilogram docarb; Malarone; Febendazole; Metronidazole; Ivomec; body weight of the patient, and in another embodiment from Iodoquinol; Diloxanide Furoate; and Ronidazole. Examples about 1 mg to about 15 mg active agent per kilogram body of protozoan organisms whose growth is reduced or inhibited weight of the patient. In terms of weight percentage, a phar by antimicrobial agents and enhancers of antimicrobial maceutical formulation of an active agent (e.g., an antimicro agents described herein include but are not limited to, Acan bial agent and an enhancer of antimicrobial agent or addi thameba; Actinophrys. Amoeba, Anisonema; Anthophysa; tional therapeutic agent(s)) can, in some embodiments, Ascaris lumbricoides, Bicosoeca: Blastocystis hominis, comprises of an amount from about 0.0001 wt.% to about 10 Codonella; Coleps: Cothurina; Cryptosporidia Difflugia; wt.%, and in alternative embodiments, from about 0.001 wt. Entamoeba histolytica (a cause of amebiasis and amebic dys % to about 1 wt.%, and in further embodiments from about entery); Entosipilon; Epalxis; Epistylis; Euglypha; Flukes; 0.01 wt.% to about 0.5 wt.%. Giardia lambia, Hookworm Leishmania spp.; Mayorella; (0193 In any of the formulations herein antimicrobial Monosiga: Naegleria Hartmannella; Paruroleptus; Plasmo agents and enhancers of antimicrobial agents can be formu dium spp. (a cause of Malaria) (e.g., Plasmodium falciparum, lated as a salt, a prodrug, or a metabolite. Such formulations Plasmodium malariae, Plasmodium vivax and Plasmodium can also include additional therapeutic agent(s) such as, for ovale); Pneumocystis carinii (a common cause of pneumonia example, antibiotics, antiviral agents, antifungal agents, and/ in immunodeficient persons); microfilariae; Podophya; or antiprotozoan agents. Raphidiophys; Rhynchomonas; Salpingoeca; Schistosoma 0194 Examples of antibiotics that may be coformulated japonicum, Schistosoma haematobium, Schistosoma Man antimicrobial agents and enhancers of antimicrobial agents SOnii Stentor; Strongyloides; Stylonychia; Tapeworms; Tri include, for example, aminoglycosides, , cepha chomonas spp. (e.g., Trichuris trichiuris and Trichomonas losporins, cephems, glycopeptides, fluoroquinolones/quino vaginalis (a cause of vaginal infection)); Tipanosoma spp. lones, oxazolidinones, penicillins, streptogramins, Sulfona and Vorticella. mides, and tetracyclines. 0188 In another example, antimicrobial agents and 0.195 Aminoglycosides are a group of antibiotics found to enhancers of antimicrobial agents used to inhibit antifungal be effective against gram-negative. Aminoglycosides are agent selected from the group consisting of imidazoles (e.g., used to treat complicated urinary tract infections, septicemia, clotrimazole, miconazole; econazole, ketonazole, oxicona peritonitis and other severe intra-abdominal infections, Zole, Sulconazole), ciclopiroZ, butenafine, and allylamines. severe pelvic inflammatory disease, endocarditis, mycobac Examples of fungus infections growth is reduced or inhibited terium infections, neonatal sepsis, and various ocular infec by antimicrobial agents and enhancers of antimicrobial tions. They are also frequently used in combination with agents described herein include but are not limited to, tinea; penicillins and cephalosporins to treat both gram-positive and athlete's foot, jock itch; and candida. gram-negative bacteria. Examples of aminoglycosides 0189 Pharmaceutical Formulations. include amikacin, gentamycin, , netromycin, 0190. The present invention contemplates pharmaceutical , kanamycin, , and . formulations comprising an antimicrobial agent and an 0196. Carbapenems are a class of broad spectrum antibi enhancer to such an antimicrobial agent in an effective otics that are used to fight gram-positive, gram-negative, and amount to achieve a therapeutic or prophylactic effect and a anaerobic microorganisms. Carbapenems are available for pharmaceutically effective carrier. intravenous administration, and as Such are used for serious 0191 The actual effective amount will depend upon the infections which oral drugs are unable to adequately address. condition being treated, the route of administration, the type For example, carbapenems are often used to treat serious and class of the antimicrobial agent and enhancer of the single or mixed bacterial infections, such as lower respiratory antimicrobial agent used to treat the condition, and the medi tract infections, urinary tract infections, intra-abdominal cal history of the patient. Determination of the effective infections, gynecological and postpartum infections, septice amount is well within the capabilities of those skilled in the mia, bone and joint infections, skin and skin structure infec art. The effective amountforuse inhumans can be determined tions, and meningitis. Examples of carbapenems include imi from animal models. For example, a dose for humans can be / Sodium, meropenem, , and formulated to achieve circulating concentrations that have /betamipron. US 2010/0028334 A1 Feb. 4, 2010 22

0.197 Cephalosporins and cephems are broad spectrum of drugs and are currently very expensive. Examples of strep antibiotics used to treat gram-positive, gram-negative, and togramins include quinupristin/dafopristin and pristinamy spirochaetal infections. Cephems are considered the next cin. generation Cephalosporins with newer drugs being stronger 0205 Sulphonamides are broad spectrum antibiotics that against gram negative and older drugs better against gram have had reduced usage due to increase in bacterial resistance positive. Cephalosporins and cephems are commonly Substi to them. Suphonamides are commonly used to treat recurrent tuted for and can be used to treat common attacks of rheumatic fever, urinary tract infections, prevention urinary tract infections and upper respiratory infections (e.g., of infections of the throat and chest, traveler's diarrhea, pharyugitis and tonsillitis). whooping cough, meningococcal disease, sexually transmit 0198 Cephalosporins and cephems are also used to treat ted diseases, toxoplasmosis, and rhinitis. Examples of Sul , Some skin infections, bronchitis, lower respira fonamides include co-trimoxazole, Sulfamethoxazole trime tory infections (pneumonia), and bone infection (certain; thoprim, , Sulfadoxine, and . members), and are a preferred antibiotic for Surgical prophy 0206 Tetracyclines are broad spectrum antibiotics that are laxis. Examples of Cephalosporins include , cefpo often used to treat gram-positive, gram-negative, and/or spi doxime, , , , , , rochaetal infections. Tetracyclines are often used to treat , cephalexin, and cephradineze. Examples of ceph mixed infections, such as chronic bronchitis and peritonitis, ems include , , cefataxidime pentahy urinary tract infections, rickets, chlamydia, , lyme drate, , , ceftazidime, , disease, and periodontal disease. Tetracyclines are an alter , , , , cefuroxime native therapy to penicillin in syphilis treatment and are also axetil, , Sodium, cefazolin, . used to treat and anthrax. Examples of tetracyclines 0199 Fluroquinolones/quinolones are antibiotics used to include , , , and treat gram-negative infections, though some newer agents . have activity against gram-positive bacteria and anaerobes. 0207. Other antibiotics contemplated herein (some of Fluroquinolones/quinolones are often used to treat conditions which may be redundant with the list above) include, but are Such as urinary tract infections, sexually transmitted diseases not limited to; abrifam; acrofloxacin, aptecin, (e.g., gonorrhea, chlamydial urethritisficerviciitis, pelvic plus clavulonic acid; apalcillin, apramycin; astromicin; inflammatory disease), gram-negative gastrointestinal infec arbekacin; aspoxicillin; azidozillin; ; , tions, Soft tissue infections, pphthalmic infections, dermato ; benzathine penicillin; : clarithro logical infections, sinusitis, and respiratory tract infections mycin, carbencillin: cefaclor, cefadroxil, cefalexin; cefaman (e.g., bronchitis, pneumonia, and tuberculosis). Fluroquino dole; cefaparin, , cefazolin, , cef lones/quinolones are used in combination with other antibi capene; cefdinir, , cefepime, , cefixime; otics to treat conditions, such as multi-drug resistant tubercu cefnetazole; cefiminox, , ; cefo losis, neutropenic cancer patients with fever, and potentially taxime; cefotetan, cefotiam, ; ce?pimizole; ce?pira anthrax. Examples of fluoroquinolones/quinolones include mide; ; cefprozil; , , cefsu ciproflaxacin, , and , , nor lodin, ceftazidime; ceftriaxone, cefuroxime; cephalexin; floxacin, , , , spar ; ; ciclacillin; cinoxacin; floxacin, , and paZufloxacin. clemizole penicillin; cleocin, cleocin-T, ; corifam; 0200 Glycopeptides and streptogramins represent antibi ; daptomycin; demeclocycline; descuinolone; otics that are used to treat bacteria that are resistant to other dibekacin; ; dirithromycin; doxycycline; enoxa antibiotics, such as methicillin-resistant staphylococcus cin; ; ; gemifloxacin; fenampicin; aureus (MRSA). They are also be used for patients who are finamicina; fleroxacin; ; flumequine; allergic to penicillin. Examples of glycopeptides include van flurithromycin; ; fosmidomycin; ; comycin, , and daptomycin. gatifloxacin; gemifloxaxin, isepamicin; ; josamy 0201 Carbapenems are used to treatgram-positive, gram cin; kanamycin; kasugamycin; kitasamycin; kairifam, lata negative, and/or anaerobes. moxef levofloxacin, levofloxacing ; ; 0202 Oxazolidinones are commonly administered to treat lomefloxacin; loracarbaf, lymecycline; ; meth gram-positive infections. Oxazolidinones are commonly acycline; methicillin; metronidazole; ; mideca used as an alternative to other antibiotic classes for bacteria mycin; minocycline; miokamycin; moxifloxacin, ; that have developed resistance. Examples of oxazolidinones nafcillin, ; neomycin; ; ; include linezolid. novobiocin, oflaxacin; oleandomycin; , oxolinic 0203 Penicillins are broad spectrum used to treat gram acid; ; paromycin; paZufloxacin; pefloxacin; positive, gram-negative, and spirochaetal infections. Condi penicillin g; penicillin V; phenethicillin; phenoxymethyl tions that are often treated with penicillins include pneumo penicillin; pipemidic acid; and com coccal and meningococcal meningitis, dermatological bination; piromidic acid; procaine penicillin; ; infections, ear infections, respiratory infections, urinary tract pyrimethamine; rifadin: : rifamide; rifampin: rifap infections, acute sinusitis, pneumonia, and . entene; rifomycin; rimactane, rofact, rokitamycin; rollitetra Examples of penicillins include penicillin, amoxicillin, cycline; ; rufloxacin; sitafloxacin; sparfloxa amoxicillin-clavulanate, amplicillin, , piperacillin cin; ; ; Sulfadiazine; Sulfadoxine; taZobactam, , piperacillin, meZocillin, benzathin Sulfamethoxazole; sisomicin; streptomycin; Sulfamethox penicillin G. penicillin V potassium, methicillin, nafcillin, azole; Sulfisoxazole; quinupristan-dalfopristan; teicoplanin; oxacillin, cloxacillin, and dicloxacillin. ; gatifloxacin, tetracycline; tetroXoprim; tellithro 0204 Streptogramins are antibiotics developed in mycin; ; ticarcillin; ; tobramycin; response to bacterial resistance that diminished effectiveness to Sufloxacin; trimethoprin; trimetrexate; trovafloxacin; Van of existing antibiotics. Streptogramins are a very Small class comycin; Verdamicin; azithromycin; and lineZolid. US 2010/0028334 A1 Feb. 4, 2010

0208. A “pharmaceutical acceptable carrier is a pharma pyl-paraben, or chlorobutanol. Suitable pharmaceutical car ceutically acceptable solvent, Suspending agent or vehicle for riers are described in Remington, cited above. delivering the antimicrobial agents and/or an enhancers anti 0215. The present invention additionally contemplates microbial agents of the present invention to an animal or antimicrobial agent and enhancers of antimicrobial agents human. The carrier may be, for example, gaseous, liquid or formulated for veterinary administration by methods conven Solid and is selected with the planned manner of administra tional in the art. tion in mind. 0216. The antimicrobial agents and enhancers of antimi 0209 Examples of pharmaceutically acceptable carriers crobial agents described herein can also be formulated for for oral pharmaceutical formulations include: lactose, industrial applications with, for example, a cleaning product, Sucrose, gelatin, agar and bulk powders. Examples of suitable Such as Soap, laundry detergent, shampoo, dishwashing soap, liquid carriers include water, pharmaceutically acceptable toothpaste, and other house cleaning detergents. fats and oils, alcohols or other organic solvents, including 0217 Selection of Subjects Administered the Composi esters, emulsions, syrups or elixirs, Suspensions, Solutions tions and/or suspensions, and solution and or Suspensions recon 0218. In some embodiments, the subjects administered a stituted from non-effervescent granules and effervescent composition comprising antimicrobial agents and/or enhanc preparations reconstituted from effervescent granules. Such ers of antimicrobial agents are selected based on the desired liquid carriers may contain, for example, Suitable solvents, treatment regime. For instance, preservatives, emulsifying agents, Suspending agents, dilu 0219. Accordingly, in some embodiments, subjects are ents, Sweeteners, thickeners, and melting agents. In some administered the antimicrobial agents, such as colistin, and/or embodiments, the carriers are edible oils, for example, corn or enhancers of antimicrobial agents, such as the inhibitors of canola oils. Polyethylene glycols, e.g. PEG, are also encom the genes as disclosed in Table 1 and Table 4 herein. In some passed in the invention as carriers. embodiments, an enhancer of an antimicrobial agent can be 0210 Examples of pharmaceutically acceptable carriers an antibiotic. In such embodiments, an antibiotic is adminis for topical formulations include: ointments, cream, Suspen tered to the subject for the purpose of its gene inhibiting sions, lotions, powder, Solutions, pastes, gels, spray, aerosol ability, as compared to its normal medical use as an anti or oil. Alternately, a formulation may comprise a transdermal pathogenic or to decrease bacteria viability. Accordingly, the patch or dressing Such as a bandage impregnated with active administration of the antibiotics (with the antimicrobial ingredients (e.g., antimicrobial agents and/or an enhancers of agent) as disclosed herein is different from the normal admin antimicrobial agents) and optionally one or more carriers or istration of antibiotics for medical use. As disclosed herein, diluents. The topical formulations may include a compound the administration of an antibiotic is administered according that enhances absorption or penetration of the active ingredi to a treatment regimen which is determined by the desired ent through the skin or other affected areas. Examples of such duration of the treatment or administration with the antimi dermal penetration enhancers include dimethylsulfoxide and crobial agent. Accordingly, as disclosed herein, the antibiotic related analogues. is administered for its gene inhibitory function as compared to its ability to kill bacteria. For example, in general, the 0211 To be administered in the form of a transdermal medically appropriate administration of an antibiotic is to kill delivery system, the dosage administration can be continuous bacteria, and administration is for a specific amount of time rather than intermittent throughout the dosage regimen. which is determined by its ability to kill all the bacteria to a 0212 Formulations suitable for parenteral administration maximum efficacy but to minimize the development of bac include aqueous and non-aqueous formulations isotonic with terial or viral resistance. As such, antibiotics typically are the blood of the intended recipient; and aqueous and non administered to a subject for a specific period of time, such as, aqueous sterile Suspensions which may include Suspending for example, for 5-7 days if used at a high dose, or 10 to 14 systems designed to target the compound to blood compo days if the antibiotic is used at a lower dose, which results in nents or one or more organs. The formulations may be pre efficient elimination of the bacteria but does not allow devel sented in unit-dose or multi-dose sealed containers, for opment of bacterial resistance. Extended administration of an example, ampoules or vials. Extemporaneous injection solu antibiotic beyond the time the bacteria is killed results in tions and Suspensions may be prepared from sterile powders, deleterious results or undesirable side effects. Accordingly, granules and tablets of the kind previously described. the inventors have discovered that, due to their gene inhibitor Parenteral and intravenous formulation may include minerals functions, antibiotics can be administered to a subjects for a and other materials to make them compatible with the type of prolonged period of time, which is determined by the duration injection or delivery system chosen. of administration of the antimicrobial activity, and impor 0213 Commonly used pharmaceutically acceptable carri tantly, not by the antibiotics ability to eliminate bacteria. As ers for parenteral administration includes, water, a suitable Such, the antibiotic administration as disclosed herein is oil, saline, aqueous dextrose (glucose), or related Sugar Solu counter to the general medical advice on administration of tions and glycols such as propylene glycol or polyethylene antibiotics to eliminate bacterial or other infections. glycols. 0220 Accordingly, in Some embodiments, a Subject is 0214 Solutions for parenteral administration preferably selected for the administration with the compositions as dis contain a water soluble salt of the active ingredient, suitable closed herein by identifying a Subject that needs a specific stabilizing agents and, if necessary, buffer Substances. Anti treatment regimen of antimicrobial agent, and is administered oxidizing agents. Such as sodium bisulfite, Sodium Sulfite, or concurrently an enhancer of a antimicrobial agent such as an ascorbic acid, either alone or combined, are suitable stabiliz antibiotic. As an exemplary example, where the Subject is a ing agents. Citric acid salts and sodium EDTA may also be subject with cystic fibrosis, the subject would be administered used as carriers. In addition, parenteral Solutions may contain a antimicrobial agent to avoid chronic endobronchial infec preservatives, such as , methyl- or pro tions, such as those caused by pseudomonas aeruginosis or US 2010/0028334 A1 Feb. 4, 2010 24 stentrophomonas maltophilia. One Such antimicrobial agent agents selected from the group of Sweetening agents, flavor is colistin. However, administration of colistin at the doses ing agents, coloring agents and preserving agents in order to and the duration required to efficiently prevent such endo produce pharmaceutically elegant and palatable preparations. bronchial infections in Subjects is highly toxic and in some 0227 Parenteral administration may be carried out in any instances fatal. Accordingly, in Some embodiments of the number of ways, but it is preferred that the use of a syringe, present invention, the Subject is selected for a treatment regi catheter, or similar device, be used to effect parenteral admin men of an antimicrobial agent. Such as colistin, and an istration of the formulations described herein. The formula enhancer of an antimicrobial agent, Such as an antibiotic, and tion may be injected systemically such that the active agent the Subject is treated with the compositions as disclosed travels substantially throughout the entire bloodstream. herein for a specific duration of time. Administration of the 0228. In addition, the formulation may also be injected compositions as disclosed herein are not directed by the kill locally to a target site, e.g., injected to a specific portion of the ing of bacteria, but by the need for antimicrobial treatment, body for which inhibition of mutagenesis is desired. An and can be, for example more than one week, more than 2 advantage of local administration via injection is that it limits weeks, more than 3 weeks, a month, 2 months, 3 months, 6 or avoids exposure of the entire body to the active agent(s) months or 12 months or longer. (e.g., antimicrobial peptide and an enhancer of such a peptide 0221) Importantly, as disclosed herein, the enhancer of the and/or other therapeutic agents). It must be noted that in the antimicrobial agent as disclosed herein is not selected for its present context, the term local administration includes effect on decreasing cell viability, it is selected based on its regional administration, e.g., administration of a formulation ability to enhance the effect of the antimicrobial agent. In directed to a portion of the body through delivery to a blood Some embodiments therefore, an enhancer of the antimicro vessel serving that body Zone. Local delivery may be direct, bial agent may not have any anti-pathogenic effects or e.g., intratumoral. Local delivery may also be nearly direct, decrease cell viability when used by themselves, and thus will i.e. intralesional or intraperitoneal, that is, to an area that is have no antibiotic or no antimicrobial activity on their own, Sufficiently close to a site of infection so that the active agent but when used concurrently with an antimicrobial agent as (s) exhibit the desired pharmacological activity. Thus, when disclosed herein, Such as for example with colistin, the local delivery is desired, the pharmaceutical formulations are enhancer of the antimicrobial agent functions to enhance the preferably delivered intralesionally, intratumorally, or intra activity of the antimicrobial agent. peritoneally. 0222 Administration 0229. It is intended that, by local delivery of the presently 0223 The antimicrobial agents and/or enhancers of anti described pharmaceutical formulations, a higher concentra microbial agents components may be administered topically, tion of the active agent may be directed to the target site. including local delivery to the gastrointestinal tract and other There are several advantages to having high concentrations membrane Surfaces including aerosol delivery for adminis delivered directly at the target site. First, since the active agent tration to lungs or nasal cavity, parenterally or orally in dos is more localized, there is less potential for toxicity to the age unit formulations containing conventional non-toxic patient since minimal systemic exposure occurs. Second, pharmaceutically acceptable carriers, adjuvants, and drug efficacy is improved since the target site is exposed to vehicles. The term parenteral as used herein includes subcu higher concentrations of the drug. Third, relatively fast deliv taneous injections, intravenous, intramuscular, intrathecal, ery minimizes solubility and stability liabilities of the active intraventricular, intracranial injection or infusion techniques. agent before reaching its target site. The present invention also provides Suitable topical, 0230 Preferably the pharmaceutical compositions are in parenteral and oral pharmaceutical formulations for use in the unit dosage form. In such form, the composition is divided novel methods of treatment of the present invention. into unit doses containing appropriate quantities of the active 0224. The compositions and pharmaceutical formulation component. The unit dosage form can be a packaged prepa herein can be administered to an organism by any means ration, the package containing discrete quantities of the known in the art. Routes for administering the compositions preparations, for example, packeted tablets, capsules, and and pharmaceutical formulations hereinto an animal. Such as powders in vials or ampoules. The unit dosage form can also a human, include parenterally, intravenously, intramuscu be a capsule, cachet, or tablet, or it can be the appropriate larly, orally, by inhalation, topically, vaginally, rectally, number of any of these packaged forms. nasally, buccally, transdermally, or by an implanted reservoir 0231. Useful pharmaceutical dosage formulations for external pump or catheter. When administered to a plan, Such administration of the compounds of the present invention are means can be by spray or via irrigation. illustrated as follows: 0225. Although any route of administration may be used, 0232 Capsules: A large number of unit capsules are pre parenteral administration, i.e., administration by injection, is pared by filling standard two-piece hard gelatin capsules each preferred. Injectable formulations can be prepared in conven with 1-100 milligrams of powdered active ingredient, milli tional forms, eitheras liquid solutions or Suspensions; as Solid grams of lactose, 50 milligrams of cellulose, and 6 milligrams forms suitable for Solubilization or Suspension in liquid prior magnesium Stearate. to injection; or as emulsions. Preferably, sterile injectable 0233 Soft Gelatin Capsules: A mixture of active ingredi Suspensions are formulated according to techniques known in ent in a digestible oil such as Soybean oil, cottonseed oil or the art using Suitable pharmaceutically acceptable carriers olive oil is prepared and injected by means of a positive and other optional components as discussed above. displacement pump into gelatin to form soft gelatin capsules 0226. The combination of antimicrobial agents and/or containing 1-100 milligrams of the active ingredient. The enhancers of antimicrobial agents components may be capsules are washed and dried. administered orally as tablets, Suspensions, lozenges, tro 0234 Tablets: A large number of tablets are prepared by ches, powders, granules, emulsions, capsules, syrups or elix conventional procedures so that the dosage unit was 1-100 irs. The composition for oral use may contain one or more milligrams of active ingredient, 0.2 milligrams of colloidal US 2010/0028334 A1 Feb. 4, 2010 silicon dioxide, 5-6 milligrams of magnesium Stearate, 275 throughout the particle. Liposomes can be used for controlled milligrams of microcrystalline cellulose, 11 milligrams of release as well as drug targeting of entrapped drug. starch and 98.8 milligrams of lactose. Appropriate coatings 0241 Antimicrobial agents and enhancers of antimicro can be applied to increase palatability or delay absorption. bial agents of the present invention can be administered to any 0235 Injectable: A parenteral composition suitable for organism (eukaryotic or prokaryotic) to prevent or treat drug administration by injection is prepared by stirring 0.5-1.5% resistance. Antimicrobial agent and enhancers of such agents by weight of active ingredient in 10% by volume propylene can also be administered to a first organism in order to target glycol and water. The Solution is made isotonic with sodium a second organism associated with the first organism. For chloride and sterilized. example, an antimicrobial agent and enhancers of the antimi 0236 Suspension: An aqueous Suspension is prepared for crobial agent can be administered to a mammal infected by oral administration so that each 5 ml contains 1-100 mg of bacteria or virus or other pathogen, or for example the com finely divided active ingredient, 200 mg of sodium carboxym positions as disclosed herein can be administered to a plant ethyl cellulose, 5 mg of sodium benzoate, 1 g of sorbitol infected by a fungus or other pathogen. In some embodi solution, U.S.P., and 0.02 ml of vanillin. ments, the methods and compositions as disclosed herein can 0237 Antimicrobial agents and enhancers of antimicro be used to prevent the development of resistance of a micro bial agents of the present invention may be administered in organism to the antimicrobial agent, Such as colistin. Without the form of liposome delivery systems. Such as Small unila being bound by theory, concurrent administration of an mellar vesicles, large unilamellar vesicles, and multilamellar enhancer of the microbial agent and an antimicrobial agent as vesicles. Liposomes can be formed from a variety of phos disclosed in the compositions and methods herein, enables pholipids, such as cholesterol, Stearylamine, orphosphatidyl targeting of at least two different pathways, Such as, for e.g. cholines. parallel or downstream pathways, and thus delays or 0238 Antimicrobial agents and enhancers of antimicro decreases the microorganism’s ability to accumulate sponta bial agents of the present invention may be coupled with neous mutations in the genes involved in the pathways tar soluble polymers as targetable drug carriers. Such polymers geted by the enhancer of antimicrobial agent and/or antimi can include polyvinylpyrrolidone, pyran copolymer, polyhy crobial agent, and thus decreases the ability of the droxylpropylmethacrylamide-, polyhydroxyethylas microorganism to circumvent the ability of the antimicrobial partamidephenol, or polyethyleneoxide-polylysine Substi agent to kill the microorganism by spontaneous mutations. tuted with palmitoyl residues. Furthermore, the compounds 0242 Antimicrobial agents and enhancers of antimicro of the present invention can be coupled to a class of biode bial agents of the present invention can be administered as a gradable polymers useful in achieving controlled release of monotherapy or in combination with additional therapeutic the drug, for example, polylactic acid, polyglycolic acid, agents (e.g., antibiotic, antiviral, antifungal, antiprotozoan copolymers of polylactic and polyglycolic acid, polyepsilon agents etc.) When administered as part of a combination caprolactone, polyhydroxy butyric acid, polyorthoesters, therapy, antimicrobial agents and enhancers of antimicrobial polyacetals, polydibydropyrians, polycyanoacylates, and agents herein can be administered serially or simultaneously crosslinked or amphipathic block copolymers of hydrogels. with the additional agent(s). In some embodiments, antimi 0239. In some embodiment of this invention, antimicro crobial agents and enhancers of antimicrobial agents are bial agents and enhancers of antimicrobial agents can be administered prior to the administration of additional thera incorporated into a biodistribution directing moiety, such as a peutic agent(s). In other embodiments, antimicrobial agents polymer, to direct the biodistribution and/or to allow for con and enhancers of antimicrobial agents are administered after tinuous release of thereof. Alternatively, microparticulate or the administration of additional therapeutic agent(s). For nanoparticulate polymeric bead dosage forms may be example, for prophylactic benefit, antimicrobial agents and employed. In this case, the antimicrobial agents and/or enhancers of antimicrobial agents may be co-administered enhancers of antimicrobial agents will be encapsulated in the (concurrent) to a Subject at risk of developing an infection, for particulate dosage forms. In this manner, the antimicrobial example a bacterial infection. In some embodiments, the agents and/or enhancers of antimicrobial agents are released antimicrobial agents and enhancers of antimicrobial agents over time to provide a sustained therapeutic benefit. These are administered prior to, or after, the administration of the Sustained release dosage forms are also useful with regard to additional therapeutic agents. other active agents useful in the practice of the present inven 0243 The antimicrobial agent and enhancers of antimi tion, Such other therapeutic agents discussed below, for crobial agent components of the present invention may addi example anti-bacterial agents, antibiotics, anti-fungal agents, tionally be combined with other medicaments to provide an anti-protozoan agents etc. Release of the active agents (anti operative combination. It is intended to include any chemi microbial agents and/or enhancers of antimicrobial agents cally compatible combination of pharmacologically-active and/or other therapeutic agents) from the particulate dosage agents, as long as the combination does not eliminate the forms of the present invention can occur as a result of both activity of the antimicrobial peptides and/or macrollide com diffusion and particulate matrix erosion. Biodegradation rate ponents. directly impacts active agent release kinetics. 0244. It will be appreciated that the antimicrobial agents 0240 Controlled release parenteral formulations of the and/or enhancers of antimicrobial agents components of the agonists and/or antagonists of the present invention can be invention and the other medicament may be administered made as implants, oily injections, or as particulate systems. separately, sequentially or simultaneously. Particulate systems include: microspheres, microparticles, 0245. Other medicaments which may be used when treat microcapsules, nanocapsules, nanospheres, and nanopar ing bacterial infections include salbutamol, ipratropium, dor ticles. Microcapsules contain the therapeutic protein as a nase alpha, for example, for use in inhalation for respiratory central core. In microspheres the therapeutic is dispersed infections such as cystic fibrosis. US 2010/0028334 A1 Feb. 4, 2010 26

0246 Screening to Identify Gene Products that when Inac genetic level, the mutants ordinarily are prepared by site tivated Enhance the Activity of Antimicrobial Agents. directed mutagenesis of the DNA encoding the gene. The 0247 The invention relates to a method of enhancing mutants can be substitution mutants, deletion mutants, or function of antimicrobial agents by inhibiting (inactivating) insertion mutants. the function of a gene product. For example, ifa gene product 0253. In some embodiments, the enhancer to the antimi inhibits and/or Suppresses the function of an antimicrobial crobial agent is an inhibitor or a binding agent of the gene agent and/or enhances the activity of a microorganism, its product of the following examples of genes: agaA, atpA, function decreases the effectiveness of the antimicrobial agent at that dose. Inactivation of specific gene products atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, enhances the effectiveness of the antimicrobial agent. guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, Accordingly, inactivation of gene products enables use of pdxH, phn L. potE, rpiA, such3, trx A, tusB (YheL), tusB. ubiE, antimicrobial agents for various implications, for example at ubiH, uncA, visB, yecY, yiaY, yidK, yihV, yfhC), yibN and/or lower doses, thus reducing possible associated toxicity. yn D. In some embodiments, the genes are homologous or 0248. These principles can be used to determine if a given Substantially homologous, analogues or variants thereof of gene product Suppresses antimicrobial agent, and thus an agaA, atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, inhibitor to Such a gene product acts as an enhancer of anti cSdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, mnmA, microbial agent and therefore is a potent candidate in the nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, tusB development of drug to enhance antimicrobial agents. (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, 0249. In one embodiment, a gene product is genetically yihV. y?hO, yibn and/orynjD, in particular the genes atpA. inactivated using known gene disruption techniques. After atpF, atpH, betB, guaA, guaB, lip A, lySA, rpiA and/or trXA. Such a disruption event, the locus that encoded the gene Methods for identifying binding agents are known in the art product would now be unable to produce the gene product and and include yeast two hybrid systems, etc. the cell would lack the function of that gene product. Various 0254 Screening for Small Molecules that Inhibit the Gene known mutability assays are used to assess the effect of the Product gene disruption event on a cell's mutability. See Friedberg, E 0255 Enhancers to antimicrobial agents can be identified C. Walker, GC, Siede, W. DNA Repair and Mutagenesis (ed. by a number of methods including screening libraries of Friedberg, E. C.) American Society of Microbiology, Wash chemical compounds. Combinatorial libraries and methods ington D.C., 1995. For example, an adaptation of the so for searching Such libraries are known in the art and include: called Stressful Lifestyle Associated Mutation (or SLAM) biological libraries, natural products libraries, spatially assay (wherein the evolution of resistance to an antibiotic of addressable parallel solid phase or solution phase libraries, choice is measured) or a forward mutation or reversion assay synthetic library methods requiring deconvolution, the one can be used. See Bull, HJ, Lombardo, MJ, Rosenberg, SM: bead one-compound library method, and synthetic library Stationary-phase mutation in the bacterial chromosome: methods using affinity chromatography selection. The bio recombination protein and DNA polymerase IV dependence. logical library approach is largely limited to polypeptide Bull H.J., Proc. Natl. Acad. Sci. USA (2001) 98: 8334-8341: libraries, while the other four approaches are applicable to Friedberg, E. C. et al. DNA Repair and Mutagenesis (ea. polypeptide, non-peptide oligomer or Small molecule librar Friedberg, E. C.) American Society of Microbiology, Wash ies of compounds. See Lam, K. S. (1997) Anticancer Drug ington D.C., 1995: Crouse, GF: Methods (2000) 22:116-119: DeS. 12:145. Rosenberg, S M Nature. Rev. Genet. (2001) 2:504-515; 0256 In one embodiment, enhancers to antimicrobial Rosche, W. A., Methods (2000) 20:4-17; end roster, PL. agents are screened using Automated Ligand Identification BioEssays (2000) 22:1067-1074. System (referred to herein as “ALIS). See, e.g., U.S. Pat. 0250. In one embodiment, bacterial cells with an inacti Nos. 6,721,665, 6,714,875, 6,694,267, 6,691,046, 6,581,013, vated gene or a wild-type gene are exposed to one or more 6,207,861, and 6,147,344, which are incorporated herein by antibacterial agent. The number of cells that grow and/or reference for all intended purposes. ALIS is a high-through Survive in the presence of the antibacterial agentis quantified put technique for the identification of small molecules that in both cells with the inactive gene and cells with the wild bind to proteins of interest (e.g., agaA, atp A, atpC, atpB. type gene. A decrease in the number of cells that grow in the atpD, atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, inactivated gene group compared with those with the wild iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. type gene Suggests that the inhibitors to the gene being tested pot. rpiA, suc3, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA, are potential enhancers of antimicrobial agent. visB, yeeY, yiaY, yidK, yihV.yfhO,ybN and/orynjD). Small 0251 Numerous techniques are known in the art to inac molecules found to bind tightly to a protein can then be tested tivate genes, many of which could be used to inactivate a test for their ability to inhibit the biochemical activity of that gene of interest. These techniques include the direct inacti protein. Vation of the test gene, for example via mutation of the test 0257 Thus, in some embodiments, a target protein (e.g. gene via homologous recombination. Another useful tech atpA, atpF etc) is mixed with pools of small molecules. Pref nique is the indirect activation of the test gene, for example erably, more than 1,000 pools are used, more preferably more via mutation of a gene whose gene product modulates the than 2,000 pools are used, more preferably more than 3,000 activity of the test gene. pools are used, or more preferably, more than 10,000 pools 0252 Typically, the test gene is inactivated via one or are used. Each pool contains approximately, 1,000 com more mutations such that the resulting protein encoded by the pounds, more preferably approximately 2,500 compounds, or test gene is inactive. Alternatively, the entire gene (or a large more preferably approximately 5,000 compounds that are portion of the gene's open reading frame) is deleted from the mass encoded, meaning that their precise molecular struc genome. Mutation of the test gene may be carried out using ture can be determined using only their mass and knowledge numerous mutagenesis techniques known in the art. At the of the chemical library. US 2010/0028334 A1 Feb. 4, 2010 27

0258. The small molecules and proteins are mixed arraying the reaction mixtures in 96-well plates, where each together and allowed to come to equilibrium (they are incu well also contains a different enhancer of antimicrobial bated together for 30 minutes at room temperature). The agents. mixture is rapidly cooled to trap bound complexes and Subject 0264 Fluorophore labeled nucleoside triphosphates or to rapid size exclusion; chromatography (SEC). Small mol oligonucleotide primers or templates can be used in conjunc ecules that bind tightly to the protein of interest will be co tion with standard plate handling and visualization proce excluded with the protein during SEC. Mass spectroscopic dures to determine which molecules effectively inhibited the analysis is performed to determine the masses of all Small activity of agene product of the invention, for example but not molecules found to bind the protein. Measurement of these limited to agaA, atpA, atpC, atpB, atp), atpE, atpG, atpH, masses allows for the rapid determination of the molecular betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, structures of the small molecules. mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.yiaY, yidK. 0259 Compounds that bind to a target gene product (e.g., yihV, yfhC), yibn and/or ynjD or homologues or variants atpA, atpF etc) in ALIS can then be tested for their ability to thereof. In one embodiment, libraries can be screened in the inhibit atpA function in vitro. Molecules with potent in vitro presence of one or more of the genes identified, for example inhibitory properties can be tested using a modified Stressful agaA, atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, Lifestyle Adaptation and Mutation (referred to herein as cSdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, mnmA, “SLAM) assays, to assess their function as an enhancer of nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, tusB antimicrobial peptide (i.e., the ability to reduce or inhibit the (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, growth and/or survival of E. coli or colistin-resistant E. coli yihV, yfhC), yibn and/or ynjD or homologues or variants grown on colistin, see Examples). Molecules that function to thereof, in order to identify drugs, compounds and/or mol increase the activity of colistin, for example function as an ecules that would most efficiently potentate the effectiveness enhancer of antimicrobial peptide activity in SLAM assays of an antimicrobial agent synergistically, and thus function as can be selected for further tested. enhancers of antimicrobial agents. 0260. In one embodiment, a chemical collection of com 0265 Structure-Based Design Methods to Create Small pounds is screened in a format similar to the SLAM assay to Molecule Enhancers of Antimicrobial Agents: identify molecules that function as an enhancer of antimicro 0266. In some embodiments, the enhancers of the antimi bial peptide. Bacterial cells are exposed to either one test crobial agents identified herein, and those yet not identified compound or a library of compounds and the number of cells can be modified using molecular modeling software tools to create realistic 3-D models of how molecules are shaped. that grown over a period of time in the presence of an anti Such methods include the use of for example, molecular microbial peptide is determined in the presence and absence graphics (i.e., 3D representations) and computational chem of the test compound. A decrease in the number of cells istry (e.g., calculations of the physical and chemical proper indicates increased inhibition of growth and/or decreased ties). Survival of the cells, and thus indicates the test compound or 0267 Using Such molecular modeling, rational drug compound functions as an enhancer the antimicrobial pep design programs can predict which of a collection of different tide. The number of cells is determined both before and after drug like compounds may fit into the active site of an enzyme, bacteria are exposed to the inhibitor, drug and/or compound. and by computationally adjusting their bound conformation, The number of cells is quantified using known assays. See decide which compounds actually might fit the active site Friedberg, E. C. Walker, G C, Siede, W. DNA Repair and well. See William Bains, Biotechnology from A to Z. 2nd Mztagertesis (ea. Friedberg, E. C.) (American Society of edition, Oxford University Press, 1998, at 259. Microbiology, Washington D.C., 1995); Crouse, Methods 0268 For basic information on molecular modeling, see, (2000) 22: 116-119. e.g., M. Schlecht, Molecular Modeling on the PC, 1998, John 0261. In yet another embodiment, the bacterial cells are Wiley & Sons: Gans et al., Fundamental Principals of exposed to an antimicrobial agent and the number of cells Molecular Modeling, 1996, Plenum Pub. Corp.; N. C. Cohen generated is quantified in the presence and absence of the test (editor), Guidebook on Molecular Modeling in Drug Design, compound. 1996, Academic Press; and W. B. Smith, Introduction to 0262. In another example of a method to screen for Theoretical Organic Chemistry and Molecular Modeling, enhancers of antimicrobial agent, purified gene products, for 1996. U.S. Patents which provide detailed information on example agaA, atpA, atpC, atpB, atp), atpE, atpG, atpH, molecular modeling include U.S. Pat. Nos. 6,093,573; 6,080, betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, 576; 5,612,894; 5,583,973; 5,030,103: 4,906,122; and 4,812, mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trx A, 12. tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK. 0269. The present invention permits the use of molecular yihV, yfhC), yibn and/or ynjD or homologues or variants and computer modeling techniques to design, and select com thereofare exposed to test compounds. In the presence of the pounds (e.g., enhancers to antimicrobial agents) that bind and test compounds, inhibitor and/or drug, their function in vitro inhibit agaA, atp A, atpC, atpB, atp), atpE, atpG, atpH, betB, is assessed, and a reduction in activity identifies a potential cSdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, enhancer of antimicrobial agent. The inhibition of different mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, gene products described herein by potential enhancers of tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.yiaY, yidK. antimicrobial agent can be quantified using standard meth yihV.yfhO, yibn and/or other gene products that suppress the ods. activity of antimicrobial peptides. Thus, the invention 0263. Alternatively, high-throughput assays can be used to enables, for example, the use of atomic coordinates deposited screen through large compound libraries to identify potential at the RCSB Protein Data Bank which can be readily identi enhancers of antimicrobial peptides. Such assays rely on fied by persons skilled in the art, to design compounds that US 2010/0028334 A1 Feb. 4, 2010 28 interact with Such gene products (e.g., agaA, atp A, atpC, with molecules composed of a variety of different chemical atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, entities to identify potential sites for interaction of candidate guaB, iscS, kdgK, lip A, lySA, mmm A. nuvC, papa, pdxH. inhibitors. For example, high resolution X-ray diffraction phnL, potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE, ubiH, data collected from crystals saturated with solvent allows the uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD determination of where each type of solvent molecule binds or homologues or variants thereof). For example, this inven the protein. Small molecules that bind tightly to those sites tion enables the design of compounds that act as competitive can then be tested for their ability to inhibit induced mutation inhibitors agaA, atpA, atpC, atpB, atp), atpE, atpG, atpH, (Travis, J., Science (1993) 262: 1374). betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, 0274 Moreover, the present invention enables computa mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trx A, tional screening of Small molecule databases for chemical tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK. entities, agents, or compounds that can bind in whole, or in yihV, yfhC), yibn and/or ynjD or homologues or variants part, to agaA, atp A, atpC, atpB, atp), atpE, atpG, atpH, betB, thereof by binding to, all or a portion of the active site of these cSdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, gene products or the gene products listed in Table 4. mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, 0270. This invention also enables the design of com tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.yiaY, yidK. pounds that act as uncompetitive inhibitors of agaA, atpA, yihV, yfhC), yibn and/or the genes listed in Table 4 and, atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, thereby enhance antimicrobial agent function. guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, 0275. In this screening technique, the quality of fit of such pdxH, phn L. potE. rpiA, such3, trx A, tusB (YheL), tusB. ubiE, entities or compounds to the binding site may be judged either ubiH, uncA, visB, yeeY, yiaY, yidK, yihV.yfhC), yibN and/or by shape complementarily or by estimated interaction energy. ynjD or homologues or variants thereof. These inhibitors may See Meng, E. C. et al. J. Coma. Chem., 13: 505-524 (1992). bind to, all or a portion of the active site of agaA, atpA, atpC, The design of compounds that bind to or inhibit agaA, atpA, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaB, iscS, kdgK, lip A, lySA, mmm A. nuvC, papa, pdxH. guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, phnL, potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE, ubiH, pdxH, phn L. potE, rpiA, such3, trx A, tusB (YheL), tusB. ubiE, uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibN and/or or homologues or variants thereof. Similarly, non-competi the genes listed in Table 4 according to this invention gener tive inhibitors; that bind to either agaA, atp A, atpC, atpB. ally involves consideration of two factors. First, the com atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, pound must be capable of physically associating with agaA, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, csdB. pot. rpiA, suc3, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA, fepC, guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, visB, yeeY, yiaY, yidK, yihV, yfhO, yibn and/or homologues papa, pdxH. phn L. potE. rpiA. Such3, trx A, tusB (YheL), tusB. or variants thereof (whether or not bound to another chemical ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn entity) may be designed using the atomic coordinates of each and/oryn D etc. Inhibition of proteins associated with agaA, gene respectively of this invention. atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, csdB. 0271 Alternatively, the atomic coordinates provided by fepC, guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, the present invention are useful in designing improved ana papa, pdxH. phn L. potE. rpiA. Such3, trx A, tusB (YheL), tusB. logues of known inhibitors of the gene products identified ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn herein; for example, but not limited to (e.g., mefloquine, and/or yn D and/or the genes listed in Table 4 required for Venturicidin A, diayquinoline, betaine aldehyde chloride, their function and/or non-covalent molecular interactions acivein, psicofuraine, buthionine Sulfoximine, diaminope other molecules important in their function, includehydrogen melic acid, 4-phospho-D-erythronhydroxamic acid, motexa bonding, van der Waals and hydrophobic interactions is also fin gadolinium and/or Xycitrin or homologs thereof, and frag encompassed in this invention. Second, the inhibitor must be ments thereof) or to design novel classes of inhibitors. This able to assume a conformation that allows it to associate with provides a novel route for designing potent and selective agaA, atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, inhibitors. cSdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, mnmA, 0272. In alternative embodiments, the present invention nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, tusB also includes the designing of improved analogues of antimi (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, crobial agents, wherein the analogue comprises both the anti yihV, yfhC), yibn and/orynjD etc. or other protein required microbial agent and at least one or more enhancer of antimi for their function. Although certain portions of the inhibitor crobial agents as identified herein. The analogue of the will not directly participate in this association with agaA, antimicrobial agent and enhancer of antimicrobial agents can atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, csdB. be joined by chemical linkage by any means known by per fepC, guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, sons skilled in the art. In additional embodiments, the anti papa, pdxH. phn L. potE. rpiA. Such3, trx A, tusB (YheL), tusB. microbial agent-enhancer of antimicrobial agent molecule ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn can be subjected to molecule modeling as described above for and/orynjD etc. or associated proteins thereof, those portions optimal configuration of the joined molecules. may still influence the overall conformation of the molecule. 0273. The availability of both protein crystals and of This, in turn, may have a significant impact on potency. atomic coordinates determined by X-ray diffraction studies 0276 Such conformational requirements include the over enables soaking experiments with agaA, atpA, atpC, atpB. all three-dimensional structure and orientation of the chemi atpD, atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, cal entity or compound in relation to all or a portion of the iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. active site of agaA, atp A, atpC, atpB, atpD, atpE, atpG, atpH, pot. rpiA, suc3, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA, betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, lipA, lySA, visB, yeeY, yiaY, yidK, yihV, yfhO, yibn and etc. crystals mnmA, nuvC, papa, pdxH. phn L. potE. rpiA. SucB, trXA, US 2010/0028334 A1 Feb. 4, 2010 29 tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK. 0280 Specialized computer programs also assist in the yihV, yfhC), yibn and/orynjD etc. or other protein required process of selecting chemical entities. These include but are for their function or the spacing between functional groups of not limited to GRID (Goodford, P. J. J. Med. Chem. (1985) a compound comprising several chemical entities that 28, 849-857). GRID is available from Oxford University, directly interact with agaA, atpA, atpC, atpB, atp), atpE. Oxford, UK; MCSS (Miranker, A. et al., Structure, Function atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, and Genetics, (1991) Vol. 11, 29-34), MCSS is available from lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE. rpiA, Molecular Simulations, Burlington, Mass. AUTODOCK sucB, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY. (Goodsell, D. S. and A. J. Olsen, “Automated Docking of yiaY, yidK, yihV, yfhC), yibn and/orynjD etc., or other pro Substrates to Proteins by Simulated Annealing Proteins: tein; required their function. Structure, Function, and Genetics, 8, 195-202 (1990)). (0277. The potential inhibitory or binding effect of a (0281 AUTODOCK is available from Scripps Research chemical compound on inhibiting the gene products identi Institute, La Jolla, Calif.; DOCK (Kuntz, I. D. et al., “A fied herein, and other potential gene products that Suppress Geometric Approach to Macromolecule-Ligand Interac the activity of antimicrobial agents may be analyzed prior to tions' J. Mol. Biol., (1982) 161,269-288). DOCK is available its actual synthesis and by the use of computer modeling from University of California, San Francisco, Calif. techniques. If the theoretical structure of the given compound 0282. Once suitable chemical entities, compounds, or precludes any potential association between it and agaA, agents have been selected, they can be assembled into a single atpA, atpC, atpB, atpD, atpE, atpG, atpH, betB, csdA, csdB. compound or inhibitor. Assembly may proceed by visual fepC, guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, inspection of the relationship of the fragments to each other papa, pdxH. phn L. potE. rpiA. Such3, trx A, tusB (YheL), tusB. on the three-dimensional image displayed on a computer ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn screen in relation to the atomic coordinates of agaA, atpA, and/orynjD etc., or other protein required for their function, atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, synthesis and testing of the compound is obviated. guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, 0278 However, if computer modeling Suggests a strong pdxH, phn L. potE, rpiA, such3, trx A, tusB (YheL), tusB. ubiE, interaction is possible, the molecule may then be synthesized ubiH, uncA, visB, yecY, yiaY, yidK, yihV, yfhC), yibN and/or and tested for its ability to interact with a agaA, atp A, atpC, yn D etc., or other protein required for their function. This atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, would be followed by manual model building using software guaB, iscS, kdgK, lip A, lySA, mnmA, nuVC, papa, pdxH. Such as Quanta or Sybyl. Useful programs to aid one of skill phnL, potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE, ubiH, in the art in connecting the individual chemical entities, com uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD pounds, or agents include but are not limited to CAVEAT etc., or other protein required for their function and act as an (Bartlett, P. A. et al., “CAVEAT: A Program to Facilitate the enhancer of antimicrobial agents of the invention. In this Structure-Derived Design of Biologically Active Molecules”. manner, synthesis of inactive compounds may be avoided. In Molecular Recognition in Chemical and Biological Prob lems”. Special Pub., Royal Chem. Soc., 78, pp. 82-196 0279. One skilled in the art may use one of several meth ods to screen chemical entities fragments, compounds, or (1989)). agents for their ability to associate with agaA, atpA, atpC, (0283 CAVEAT is available from the University of Cali atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, fornia, Berkeley, Calif.; 3D Database systems such as guaB, iscS, kdgK, lip A, lySA, mmm A. nuvC, papa, pdxH. MACCS-3D (MDL Information Systems, San Leandro, phnL, potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE, ubiH, Calif.). This area is reviewed in Martin, Y.C., “3D Database uncA, visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/orynjD Searching in Drug Design”. J. Med. Chem., 35, pp. 2145 etc., or other protein required for their function and more 2154 (1992); also HOOK (available from Molecular Simula particularly with the individual binding pockets of such gene tions, Burlington, Mass.). products, or associated proteins required for their function. 0284. Instead of designing an inhibitor of atpA, atpF, This process may begin by visual inspection of, for example, atpH, betB, guaA, guaB, lipA, lySA, rpiA and/or trXA etc., or the active site on the computer screen based on agaA, atpA, other protein required for their in a step-wise fashion one atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, chemical moiety at a time as described above, inhibitors of guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, atpA, atpF, atpH, betB, guaA, guaB, lip A, lySA, rpiA and/or pdxH, phn L. potE. rpiA, such3, trx A, tusB (YheL), tusB. ubiE, trXA etc., or other protein required for their function may be ubiH, uncA, visB, yeeY, yiaY, yidK, yihV.yfhC), yibN and/or designed as a whole or “de novo” using either an empty ynjD etc., or other protein required for their function coordi binding site or optionally including some portion(s) of known nates deposited in the RCSB Protein Data Bank. Selected inhibitor(s). These methods include LUDI (Bohm, H.-J., chemical entities, compounds, or agents may then be posi “The Computer Program LUDI: A New Method for the De tioned in a variety of orientations, or docked, within an indi Novo Design of Enzyme Inhibitors'. J. ComR. Aid. Molec. vidual binding pocket of agaA, atp A, atpC, atpB, atp), atpE. Design, (1992) 6, 61-78). LUDI is available from Biosym atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, Technologies, San Diego, Calif. and LEGEND (Nishibata, Y. lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. potE. rpiA, and A. Itai, Tetrahedron, (1991) 47, p. 8985). LEGEND is sucB, trx A, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY. available from Molecular Simulations, Burlington, Mass. and yiaY, yidK, yihV, yfhC), yibn and/orynjD etc., or other pro LeapFrog (available from Tripos Associates, St. Louis, Mo.). tein required for their function as defined above. Docking 0285) Other molecular modeling techniques may also be may be accomplished using software Such as Quanta and employed in accordance with this invention. See, e.g., Cohen, Sybyl, followed by energy minimization and molecular N. C. et al., “Molecular Modeling Software and; Methods for dynamics with standard molecular mechanics force fields, Medicinal Chemistry,” J. Med. Chem., (1990) 33, 883-894. Such as CHARMM or AMBER. See also, Navia, M. A. and M. A. Murcko, "The Use of US 2010/0028334 A1 Feb. 4, 2010 30

Structural Information in Drug Design. Current Opinions San Diego, Calif., 1994). These programs may be imple Structural Biology, (1992) 2, 202-210. mented, for instance, using a Silicon Graphics workstation, 0286 Once a compound has been designed or selected by IRIS 4D/35 or IBM RISC/6000 workstation model 550. the above methods, the efficiency with which that compound Other hardware systems and software packages will be may bind to agaA, atpA, atpC, atpB, atp), atpE, atpG, atpH, known to those skilled in the art. betB, csdA, csdB, fepC, guaA, guaB, iscS, kdgK, lip A, lySA, 0290. Once an inhibitor of agaA, atpA, atpC, atpB, atpD. mnmA, nuvC, papa, pdxH. phn L. potE. rpiA, such3, trx A, atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, tusB (YheL), tus, ubiE, ubiH, uncA, visB, yeeY.yiaY, yidK, kdgK, lip A, lysA, mnmA. nuvC, papa, pdxH. phn L. potE. yihV, y?hC), yibN and/orynjD etc., or other protein required rpiA, such3, trxA, tusB (YheL), tusB. ubiE., ubiH, uncA, visB, for their function may be tested and optimized by computa yeeY, yiaY, yidK, yihV.yfhO, yibNetc. or other proteins that tional evaluation. An effective antimicrobial agent and/or suppress antimicrobial agents has been optimally selected or enhancer of antimicrobial agent must preferably demonstrate designed, as described above, substitutions may then be made a relatively small difference in energy between its bound and in some of its atoms or side groups to improve or modify its free states (i.e., a small deformation energy of binding). Thus, binding properties. Generally, initial substitutions are conser the most efficient inhibitors of agaA, atpA, atpC, atpB, atpl). vative, e.g., the replacement group will have approximately atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, iscS, the same size, shape, hydrophobicity and charge as the origi kdgK, lip A, lysA, mmm A. nuvC, papa, pdxH. phn L. potE. nal group. It should, of course, be understood that compo rpiA, such3, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, nents known in the art to alter conformation should be yeeY, yiaY, yidK, yihV.yfhO, yibN and/orynjD etc., or other avoided. Such substituted chemical compounds may then be proteins should preferably be designed with deformation analyzed for efficiency of fit into the 3-D structures of agaA, energy of binding of not greater than about 10 kcal/mole, or atpA, atpC, atpB, atplD, atpE, atpG, atpH, betB, csdA, csdB, more preferably, not greater than 7 kcal/mole. fepC, guaA, guaB, iscS, kdgK, lip A. lySA, mnmA. nuvC, 0287. Inhibitors of agaA, atpA, atpC, atpB, atp), atpE. papa, pdxH. phn L. potE. rpiA. Suchs, trx A, tusB (YheL), tusB. atpG. atpH, betB, csdA, csdB, fepC. gua.A. guaB, iscS, kdgK. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, yihV, yfhO, yjbN lip A. lys A, mnmA. nuvC, papa, pdxH. phn L. potE. rpiA. etc. or other proteins by the same computer methods such}, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY. described in detail, above. yiaY, yidK, yihV, yfhO, yibN and/orynjD etc., or other pro 0291. The compounds designed by any of the above meth teins may interact with their target in more than one confor ods are useful for inhibiting genes and/or gene products mation that is similarin overall binding energy. In those cases, which when inactivated potentiate activity of antimicrobial the deformation energy of binding is taken to be the difference agent and thus are useful as therapeutic agents with antimi between the energy of the free compound and the average crobial agents to synergistically inhibit the growth and/or kill energy of the conformations observed when the inhibitor microorganisms. binds to agaA, atpA, atpC, atpB, atpl), atpE, atpG, atpH, betB, csdA, csdB, fepC. guaA, guaB, iscS, kdgK, lip A, lySA, Examples mnmA. nuvC, papa, pdxH. phn L. potE. rpiA, such3, trx A, 0292. The examples presented herein relate to composi tusB (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY.yiaY.yidK, tions comprising antimicrobial agents and enhancers of anti yihV. y?hO, yjbN and/orynjDA etc. microbial agents. In the examples, antimicrobial peptides are 0288 A compound designed or selected, as binding to used as exemplary antimicrobial agents. Further, colistin is agaA, atpA, atpC, atpB, atp), atpE, atpG, atpH, betB, csdA, used as an exemplary antimicrobial agent, in particular as an csdB, fepC, guaA, guaB, iscS, kdgK, lip A, lySA, mnmA, exemplary antimicrobial peptide, although the methods and nuvC, papa, pdxH, phn L. potE. rpiA, such3, trXA, tusB compositions of the invention are applicable to any antimi (YheL), tusB. ubiE, ubiH, uncA, visB, yeeY, yiaY, yidK, crobial agent. Further, mefloquine and potassium tellurite are yihV, yfhO, yjbN and/orynjD etc. can be further computa used as an exemplary enhancer of antimicrobial agents, tionally optimized so that in its bound state it would prefer although the methods and compositions of the invention are ably lack repulsive electrostatic interaction with the target. applicable to any enhancer of the antimicrobial agent, or any Such non-complementary (e.g., electrostatic) interactions molecule which inhibits a gene listed in Table 1 or Table 4. include repulsive charge-charge, dipole-dipole and charged Throughout this application, various publications are refer dipole interactions. Specifically, the sum of all electrostatic enced. The disclosures of all of the publications and those interactions between the inhibitor and the enzyme when the references cited within those publications in their entireties inhibitor is bound to its target (e.g., agaA, atpA, atpC, atpB. are hereby incorporated by reference into this application in atp), atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, order to more fully describe the state of the art to which this iscS, kdgK, lip A, lysA, mnmA, nuvC, papa, pdxH, phn L. invention pertains. The following examples are not intended potE, rpiA, sucB, trx A, tusB (YheL), tusB. ubiE., ubiH, uncA, to limit the scope of the claims to the invention, but are rather visB, yeeY, yiaY, yidK, yihV.yfhO, yibN and/orynjD etc. or intended to be exemplary of certain embodiments. Any varia other protein required for their function), make a neutral or tions in the exemplified methods which occur to the skilled favorable contribution to the enthalpy of binding. artisan are intended to fall within the scope of the present 0289 Specific computer software is available in the art to invention. evaluate compound deformation energy and electrostatic 0293 Methods interaction. Examples of programs designed for such uses (0294 All experiments were performed in Luria-Bertani include: Gaussian 92, revision C (M.J. Frisch, Gaussian, Inc., (LB) medium (Fisher Scientific, Pittsburgh, Pa.). kanamycin Pittsburgh, Pa., 1992); AMBER, version 4.0 (P. A. Kollman, (Fisher Scientific), colistin sulfate (Sigma-Aldrich), and University of California at San Francisco, 1994); QUANTA/ potassium tellurite. Kanamycin (50 mg/ml), colistin (25 CHARMM (Molecular Simulations, Inc., Burlington, Mass. mg/ml), potassium tellurite (10 mg/ml) stocks on water. 1994); and Insight II/Discover (Biosysm Technologies Inc., 0295 Straind US 2010/0028334 A1 Feb. 4, 2010

0296 BW25113 (lacIqrrnBT14 AlaczWJ16 hsdR514 MIC<4 mg/L susceptible, MIC28 mg mg/L resistant and AaraBADAH33 ArhabADLD78) MIC2128 mg/L-highly resistant 0297 Growth of deletion strains ubiH and iscS are con tained in a BW251 13 deletion library (Baba et al., 2006). Example 1 0298 Mutant Deletion Screen 0299. Each plate of knockout strain (11 in total) was first 0306 Over 4,000 single mutant E. coli were initially pre-grown in 50 ug/mL of Kanamycin in LB broth, then screened in a cell based assay for ability to grow and/or re-grown in LB broth without Kanamycin. Survive in the presence of colistin, an antimicrobial peptide 0300 Twelve 384 well plates were filled with 50 uLLB (FIG.1). Approximately 92-95 E. colimutants were unable to broth supplemented with 50 ug/mL of Kanamycin in each grow and Survive in the presence of colistin. The gene loci of well. Using a Beckman Multimek pipetting robot. The plates the mutations were analyzed and identified which are listed in were inoculated from KO library frozen stocks into the newly FIG. 2. The homology of the identified gene loci (herein filled plates using 384 well pin stamper. The stamper was referred to as “gene products”) was analyzed with respect to sterilized between each stamps by allowing the pin stamperto gene homology across different species of bacteria (FIG. 3), sit in 70% Ethanol for 10-15 seconds, briefly placed into and it was found that 24 of 66 gene products had human 100% Ethanol, then placed over a blame to bum off remaining homologues, listed in Table 1. Of these 24 gene products, 7 ethanol. The freshly inoculated plates were incubated for 24 had known identified inhibitor which are listed in Table 2. hours at 37 C. 0307 The efficacy of colistin in inhibiting the growth and/ 0301 A sample of these re-grown cultures was then or Suppressing Survival of E. coli in the presence of one diluted 1/250 and stamped onto an LBagar plate. The remain inhibitor, mefloquine was assessed in a minimum inhibitory ing culture was then treated with colistin 20 ug/mL for 1.5 concentration (MIC) assay. As shown in FIG. 4, in the Hours. After treatment a second sample is then taken and absence of mefloquine, the MIC of colistin is 6.3, whereas in diluted 1/250 insterilized H2O before being stamped onto an the presence of 50, 100 and 200 ug/ml of mefloquine, the MIC agar plate in duplicate. After 24 hours of growth at 37°C. the of colistin required to suppress growth is 6.3, 3.1 and 0.8 plates are photographed and stored for further image analysis. respectively. Therefore, there is a dose-dependent potentia The digital images of treated vs. non treated plates are auto tion of colistin efficacy by mefloquine at the concentrations matically overlapped using a developed image analysis pro above 50 ug/ml or greater. Thus, mefloquine was demon gram to measure size and density of the resulting colonies. strated to act as a potent enhancer to the antimicrobial peptide 0302 Colony Formation Unit Assay colistin. The inventors have also demonstrated that mefloqui 0303 For CFU measurements, 50 ul of stationary phase nine when used concurrently with colistin, potentiates colis culture was inoculated into 5 ml of LB. 240 or 245 ul of the tin ability to decrease gram-positive bacteria (data not freshly diluted cells were placed into wells of a 96 well plate shown). (Costar), 5ul of colistin and or potassium tellurite were added 0308 Accordingly, the inventors have demonstrated that into each well to obtain a final volume of 250 ul. The 96 well mefloquine is an exemplary example of an enhancer of an plates were then incubated at 37° C. At the 0, 3 and 6 hours antimicrobial agent, and was demonstrated to potentiate the time points, 20 ul of culture was collected and then serially activity of an antimicrobial agent Such as colistin. Accord diluted in 180 ul of 1xPBS, pH 7.2 (Fisher). A 10ul portion of ingly, the inventors have discovered that dose and use of each dilution was plated onto LBagar (Fisher)), and the plate mefloquineas an enhancer of the antimicrobial agent is deter was incubated overnight at 37° C. Dilutions that yielded mined by the therapeutic regimen (i.e. duration and adminis between 20 and 100 colonies were counted and CFU were tration) of the antimicrobial agent Such as colistin, as apposed normalized to reflect the same amount of cells for the starting to any ability to decrease cell viability that mefloquine may cultures. have when used by itself. 0304. The resistance or susceptibility of microorganism to an antibiotic is classified by using defined minimum inhibi Example 2 tory concentration (MIC) breakpoints. MIC breakpoints for an antibiotic are determined by the MIC distributions of 0309 The inventors used a systems biology platform to pathogenic in a clinical indication and its pharmacokinetics identify of several genes and therefore pathway, which once and pharmacodynamics in humans. While a microorganism inactivated can increase the killing effect of colistin. This may literally be susceptible to a high concentration of an approach has its origin with the notion of synthetic lethality antibiotic in vitro, the microorganism may in fact be resistant screen in yeast where two genes non essential for viability on to that antibiotic at physiologically realistic concentrations if their own are found to induce a non viable strain when com the concentration of drug required to inhibit growth of or kill, bined and under specific growth condition(s) (Cottarel, the microorganism is greater than the concentration that can 1997). With the access to the yeast deletion knockout library safely be achieved without toxicity Lo the subject, the micro (reviewed in Ooi et al., 2006) major efforts have been initiated organism is considered to be resistant to the antibiotic. To to create genetic mapping of gene interaction (Tong et al., facilitate the identification of antibiotic resistance or suscep 2004). In a further step such reagents were used to link bio tibility using In vitro test results, the National Committee for active compounds to genetic pathways (Parsdons et al., Clinical Laboratory Standards (NCCLS, now known as Clini 2004). Reminiscent to this work, our screen was designed to cal and Laboratory Standards Institute) has formulated stan identify mutants exhibiting hyper sensitivity to antibacterials dards for antibiotic susceptibility that correlate clinical out to (i) map a chemogenomic response pathway and (ii) to come to in vitro determinations of the MIC antibiotics. further identify and/or develop inhibitors which can mimic 0305 Generally, MIC values indicate resistance or sus the genetic observation. Indeed, such gene products can ulti ceptibility of a microorganism. For example, MIC valves of a mately be the target of compounds which can in turn chemi microorganism to colistin/polymyxin B are as follows: cally potentiate Colistin. US 2010/0028334 A1 Feb. 4, 2010 32

0310. The inventors have identified herein using a systems biology platform, several genes and pathways, which once TABLE 4-continued inactivated, can increase the killing effect of colistin. For List of genes identified to exhibit sensitivity example, the inventors have discovered a number of genes, to colistin from the primary screen. such as those listed in Table 1 or Table 4, which normally function in either secondary, parallel or downstream path Gene Function ways to the target action of the antimicrobial agent, and when malG part of maltose permease, inner membrane such pathways are inhibited or inactivated by inhibitors, mbha putative motility protein potentiate the activity of antimicrobial agents, such as for mdoG periplasmic glucans biosynthesis protein nei endonuclease VIII and DNAN-glycosylase with an AP lyase example colistin. The inventors have discovered, using an activity assay or screen designed to identify mutants exhibiting hyper nmpo. outer membrane porin protein; locus of qsr prophage sensitivity to antibacterials to (i) map a chemogenomic nud response pathway and (ii) to further identify and/or develop pdxH pyridoxinephosphate oxidase phnB orf, hypothetical protein inhibitors which can mimic the genetic phenotype. Indeed, phnL ATP-binding component of phosphonate transport Such gene products can ultimately be the target of compounds phnO putative regulator, phn operon which can in turn chemically potentiate colistin. pnuC required for NMN transport potE putrescine transport protein 0311. The Keio collection of E. coli mutants consist of pshM putative general secretion 3,985 single gene knock-out of non essential for viability ptSA PEP-protein phosphotransferase system enzyme 1 genes (Baba et al., 2006). In the goal to identify mutants rhaT rhamnose transport which exhibit increased sensitivity to antibiotics, colistin in rpiA ribosephosphate isomerase, constitutive rseA sigma-E factor, negative regulatory protein that case, the inventors developed a high-throughout assay sbp periplasmic sulfate-binding protein which the screen basic scheme is shown on FIG. 1. The speA biosynthetic arginine decarboxylase collection was re-formatted in a 384 well plate, from a 96 well SucB 2-oxoglutarate dehydrogenase plate format, for ease of manipulation. Each identified can SugE Suppresses groEL, may be chaperone didates found more than once in separated Screens were tdcE probable formate acetyltransferase 3 called positives. 73 Positives were then reformatted into a 96 tolC outer membrane channel well plate for further testing (FIG. 2, Table 4). From these trxA hioredoxin 1 screen we selected the iscS (Lauhon, 2002) and the ubiH ubi 2-octaprenyl-6-methoxy-1,4-benzoquinone --> 2-octaprenyl 3-methyl-6-methoxy-1,4-benzoquinone (Young et al., 1973) mutants for further studies. ubi 2-octaprenyl-6-methoxyphenol-->2-octaprenyl-6-methoxy 0312 Such inhibitors of the gene products as disclosed benzoquinone1.4 herein are termed "enhancers of antimicrobial agents' herein, ubiX 3-octaprenyl-4-hydroxybenzoate carboxy-lyase and are selected based on their ability to inhibit a gene, such Xni as any of those selected from the list of genes in Table 1 or ybb Y putative transport Table 4. In particular, an inhibitor or enhancer of antimicro ycfM orf, hypothetical protein bial agent is selected based on its gene inhibiting function yde orf, hypothetical protein yeeY putative transcriptional regulator LYSR-type rather than any ability it may have to decrease cell viability yfeT orf, hypothetical protein when it is used by itself. ygaA putative 2-component transcriptional regulator ygfZ orf, hypothetical protein TABLE 4 yhdX putative transport system permease protein yheL orf, hypothetical protein List of genes identified to exhibit sensitivity yheM orf, hypothetical protein yiaY putative oxidoreductase to colistin from the primary screen. yid K putative cotransporter Gene Function yihV putative kinase ybN orf, hypothetical protein agaA putative N-acetylgalactosamine-6-phosphate deacetylase yjcR putative membrane protein atpA membrane-bound ATP synthase, F1 sector, alpha-subunit ycZ orf, hypothetical protein atpF membrane-bound ATP synthase, F0 sector, subunit b atpH membrane-bound ATP synthase, F1 sector, delta-subunit ygeC betB NAD+-dependent betaine aldehyde dehydrogenase ygiH putative membrane protein bg|F PTS system beta-glucosides, enzyme II, cryptic yrfA orf, hypothetical protein cysE serine acetyltransferase cysI Sulfite reductase, alpha Subunit The genes identified in bold are the qualified mutants after a secondary fepG ATP-binding component of ferric enterobactin transport screen in a 96 well plate format. fepD ferric enterobactin (enterochelin) transport frvR. putative frv operon regulatory protein 0313 The cells were grown with or without colistin, ali guaa GMP synthetase (glutamine-hydrolyzing) guaB IMP dehydrogenase quots were taken at defined time points and serial dilution of hoff putative general protein secretion protein the cells plated, colony count allowed to quantify the bacte hsdS specificity determinant for hsdM and hsdR ricidal effect of colistin. We noticed that the iscS and ubi iscS putative aminotransferase mutants were slow growers and formed Small size colonies JW5075 compared to the parent strain. JW 5227 0314. The used amount of colistin was chosen to not affect JW 5257 or little the parent strain, no significant decrease in viability UWS360 kdgK ketodeoxygluconokinase was detected when compared to the non treated cells at the 3 lip A lipoate synthesis, Sulfur insertion? and 5 hour time points. To the contrary, the mutants showed a lySA diaminopimelate decarboxylase significant decrease in viability when exposed to colistin, resulting to almost no colony detection at the 5 hour time US 2010/0028334 A1 Feb. 4, 2010

point (FIG. 5A). The mutants have a slower growth rate and bly. The role of these genes to potentiate colistin is likely are characterized as forming Smaller size colonies (data not related to a more global protein functionality aspect. These shown), the reduced growth rate cannot on its own justify the Fe—S cluster groups are important for many cellular pro low colony formation in response to colistin. cesses Such as thNA modification, regulation of enzyme 0315. There are known inhibitors of the iscS gene product activity, Substrate binding and activation as well as regulation such as iodoacetamine (Urbina et al., 2001) potassium tellu of gene expression. The Fe—S clusters main biochemical rite (Rojas and Vasquez, 2005; Tantalean, 2003). The iscS roles are (1) as acceptor and donor of electrons and (2) as mutant was shown to be sensitive to potassium tellurite (Rojas binder of the oxygen nitrogen groups of diverse Substrates and Vasquez, 2005) while ectopic expression of iscS confers (review in Inlay, 2006). Consequently their roles are linked resistance to potassium tellurite (Tantalean et al., 2003). into the process to synthesize ATP 0316 Potassium tellurite is a heavy metal carrier (Te4+) 0320 Such an approach can lead to a combination therapy which is known to be toxic for bacteria. It is in fact a known involving colistin and selected compounds which can ulti antibacterial agent not use as a therapeutic though and served mately reduce the toxic adverse effect of colistin either by as a selective agent in microbiological culture medium (e.g. reducing the colistin dosage and/or by reducing the treatment Perez et al., 2007; Zanaroli et al., 2002). Potassium tellurite length. mediates thiol oxidation in E. coli (Turneret al., 1999). How ever, the inventors chose to continue experiments with Potas REFERENCES sium tellurite based on its ability to inhibit the gene iscS, and 0321. The references cited herein and throughout the not due to any potential anti-bacterial activity it may have. application are incorporated herein by reference. Thus, the inventors demonstrated that inhibition of a gene 0322 Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. listed in Table 4, in particular inhibition of iscS by potassium Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. Mol tellurite potentiated colistin effects was a proof of principal Syst Biol. 2:2006.0008 (2006). that inhibition of the genes listed in table 4 could potentiate 0323 Falagas ME and Kasakiou S K Clin Infect Dis 40: antimicrobial activity, such as potentiation of colistinactivity. 1333-1341 (2005). 0317. The inventors used the CFU assay to observe that the 0324 Imlay J A. Mol Microbiol. 59:1073-82 (2006). amount of colistin used was not affecting the parent strain, no 0325 Lauhon J Bacteriol. 184:6820-6829 (2002). significant decrease in viability was detected when compared 0326 Li J, Nation RL, Turnidge JD, Milne RW, Coulth to the non treated cells at the 3 hour time point (FIG. 5B). A ard K. Rayner C R. Paterson D. L. Lancet Infect Dis. 6:589 decrease in viability was noticed at the 5 hour time point. 601 (2006). Potassium tellurite at 1.6 ug/ml was not or little affecting the 0327 Perez J. M. Calderon L, Arenas FA, Fuentes DE, CFU at the 3 hour time point while it shows a stronger growth Pradenas GA, Fuentes EL, Sandoval JM, Castro ME, Elias inhibition at 6 hours. The inventors observed that the combi AO, Vasquez C C. PLoS ONE. 14:2:e211 (2007). nation of colistin and potassium tellurite resulted in a signifi 0328. Rojas DM, Vasquez C C. Res Microbiol. 156:465 cant decrease in viability closed to a 2 log decrease in average. 71 (2005). 0318. The inventors have therefore designed systems biol 0329 Song JY, Kee SY. Hwang IS, Seo Y B, Jeong H.W. ogy platform that can be used to discover and identify E. coli Kim WJ, Cheong HJ.J Antimicrob Chemother. 60:317-322 mutants which exhibit increased sensitivity to colistin. From (2007). the pool of mutant candidates the inventors qualified the 0330 Tan TY, Ng L S. Tan E. Huang G. J. Antimicrob strains disrupted on the ubiH and iscS loci. These gene prod Chemother. 60:421-423 (2007). ucts are incorporated into two major cellular processes. ubiH 0331 Tantalean J C, Araya MA, Saavedra C P Fuentes D is involved the process to generate ATP while the iscS gene E. Perez J. M. Calderon I L. Youderian P. Vasquez C C. J product is involved the transfer of iron-sulfur clusters. Bacteriol. 185:5831-7 (2003). 0319 Iron-Sulfur Fe—S clusters are prosthetic groups 0332 Turner RJ, Weiner J. H. Taylor D E. Microbiology. which are required for crucial biological processes such as 145:2549-2557 (1999). electron transfer, iron/sulfur storage, gene regulation, tRNA 0333 Urbina HD, Silberg JJ, Hoff KG, Vickery L. E. J. modification and enzyme activity. The iscS gene product, a Biol. Chem., 276:44521-44526 (2001). cysteine desulfurase, acts mostly as a Sulfur donor for other 0334 Young IG, Stroobant P. Macdonald CG, Gibson F. proteins involved in diverse cellular regulatory pathways. J Bacteriol. 114:42-52 (1973). iscS obtains sulfur from cysteine which is then converted to 0335 Zanaroli G, Fedi S, Carnevali M. Fava F. Zannoni D. alanine and serves as Sulfur donor for Fe—S cluster assem Res Microbiol. 153:353–360 (2002).

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 42

<21 Os SEQ ID NO 1 &211s LENGTH: 167 212s. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs SEQUENCE: 1 US 2010/0028334 A1 Feb. 4, 2010 34

- Continued Met His Cys Tyr Asn Gly Met Thr Gly Lieu. His His Arg Glu Pro Gly 1. 5 1O 15 Met Val Gly Ala Gly Lieu. Thir Asp Lys Arg Ala Trp Lieu. Glu Lieu. Ile 2O 25 3O Ala Asp Gly His His Val His Pro Ala Ala Met Ser Lieu. Cys Cys Cys 35 4 O 45 Cys Ala Lys Glu Arg Ile Val Lieu. Ile Thr Asp Ala Met Glin Ala Ala SO 55 6 O Gly Met Pro Asp Gly Arg Tyr Thr Lieu. Cys Gly Glu Glu Val Glin Met 65 70 7s 8O His Gly Gly Val Val Arg Thr Ala Ser Gly Gly Lieu Ala Gly Ser Thr 85 90 95 Lieu. Ser Val Asp Ala Ala Val Arg Asn Met Val Glu Lieu. Thr Gly Val 1OO 105 11 O Thr Pro Ala Glu Ala Ile His Met Ala Ser Lieu. His Pro Ala Arg Met 115 12 O 125 Lieu. Gly Val Asp Gly Val Lieu. Gly Ser Lieu Lys Pro Gly Lys Arg Ala 13 O 135 14 O Arg Val Val Ala Lieu. Asp Ser Gly Lieu. His Val Glin Glin Ile Trp Ile 145 150 155 160 Glin Gly Glin Lieu Ala Ser Phe 1.65

<210s, SEQ ID NO 2 &211s LENGTH: 513 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 2 Met Glin Lieu. Asn. Ser Thr Glu Ile Ser Glu Lieu. Ile Lys Glin Arg Ile 1. 5 1O 15 Ala Glin Phe Asin Val Val Ser Glu Ala His Asn Glu Gly Thr Ile Val 2O 25 3O Ser Val Ser Asp Gly Val Ile Arg Ile His Gly Lieu Ala Asp Cys Met 35 4 O 45 Glin Gly Glu Met Ile Ser Lieu Pro Gly Asn Arg Tyr Ala Ile Ala Lieu. SO 55 6 O Asn Lieu. Glu Arg Asp Ser Val Gly Ala Val Val Met Gly Pro Tyr Ala 65 70 7s 8O Asp Lieu Ala Glu Gly Met Llys Val Lys Cys Thr Gly Arg Ile Lieu. Glu 85 90 95 Val Pro Val Gly Arg Gly Lieu. Lieu. Gly Arg Val Val Asn. Thir Lieu. Gly 1OO 105 11 O Ala Pro Ile Asp Gly Lys Gly Pro Lieu. Asp His Asp Gly Phe Ser Ala 115 12 O 125 Val Glu Ala Ile Ala Pro Gly Val Ile Glu Arg Glin Ser Val Asp Glin 13 O 135 14 O Pro Val Glin Thr Gly Tyr Lys Ala Val Asp Ser Met Ile Pro Ile Gly 145 150 155 160 Arg Gly Glin Arg Glu Lieu. Ile Ile Gly Asp Arg Glin Thr Gly Lys Thr 1.65 17O 17s Ala Lieu Ala Ile Asp Ala Ile Ile Asin Glin Arg Asp Ser Gly Ile Llys 18O 185 19 O US 2010/0028334 A1 Feb. 4, 2010 35

- Continued

Cys Ile Tyr Val Ala Ile Gly Gln Lys Ala Ser Thr Ile Ser Asn Val 195 2OO 2O5 Val Arg Llys Lieu. Glu Glu. His Gly Ala Lieu Ala Asn. Thir Ile Val Val 21 O 215 22O Val Ala Thir Ala Ser Glu Ser Ala Ala Lieu. Glin Tyr Lieu Ala Pro Tyr 225 23 O 235 24 O Ala Gly Cys Ala Met Gly Glu Tyr Phe Arg Asp Arg Gly Glu Asp Ala 245 250 255 Lieu. Ile Ile Tyr Asp Asp Lieu. Ser Lys Glin Ala Val Ala Tyr Arg Glin 26 O 265 27 O Ile Ser Lieu. Lieu. Lieu. Arg Arg Pro Pro Gly Arg Glu Ala Phe Pro Gly 27s 28O 285 Asp Val Phe Tyr Lieu. His Ser Arg Lieu. Lieu. Glu Arg Ala Ala Arg Val 29 O 295 3 OO Asn Ala Glu Tyr Val Glu Ala Phe Thir Lys Gly Glu Wall Lys Gly Lys 3. OS 310 315 32O Thr Gly Ser Lieu. Thir Ala Leu Pro Ile Ile Glu Thr Glin Ala Gly Asp 3.25 330 335 Val Ser Ala Phe Val Pro Thr Asn Val Ile Ser Ile Thr Asp Gly Glin 34 O 345 35. O Ile Phe Lieu. Glu Thir Asn Lieu. Phe Asn Ala Gly Ile Arg Pro Ala Val 355 360 365 Asn Pro Gly Ile Ser Val Ser Arg Val Gly Gly Ala Ala Glin Thir Lys 37 O 375 38O Ile Met Lys Llys Lieu. Ser Gly Gly Ile Arg Thr Ala Lieu Ala Glin Tyr 385 390 395 4 OO Arg Glu Lieu Ala Ala Phe Ser Glin Phe Ala Ser Asp Lieu. Asp Asp Ala 4 OS 41O 415 Thir Arg Lys Glin Lieu. Asp His Gly Glin Llys Val Thr Glu Lieu. Lieu Lys 42O 425 43 O Gln Lys Glin Tyr Ala Pro Met Ser Val Ala Glin Glin Ser Leu Val Lieu. 435 44 O 445 Phe Ala Ala Glu Arg Gly Tyr Lieu Ala Asp Val Glu Lieu. Ser Lys Ile 450 45.5 460 Gly Ser Phe Glu Ala Ala Lieu. Lieu Ala Tyr Val Asp Arg Asp His Ala 465 470 47s 48O Pro Leu Met Glin Glu Ile Asn Gln Thr Gly Gly Tyr Asn Asp Glu Ile 485 490 495 Glu Gly Lys Lieu Lys Gly Ile Lieu. Asp Ser Phe Lys Ala Thr Glin Ser SOO 505 51O Trp

<210s, SEQ ID NO 3 &211s LENGTH: 271 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 3 Met Ala Ser Glu Asn Met Thr Pro Glin Asp Tyr Ile Gly His His Leu 1. 5 1O 15 Asn Asn Lieu. Glin Lieu. Asp Lieu. Arg Thr Phe Ser Lieu Val Asp Pro Glin 2O 25 3O US 2010/0028334 A1 Feb. 4, 2010 36

- Continued

Asn Pro Pro Ala Thr Phe Trp Thir Ile Asin Ile Asp Ser Met Phe Phe 35 4 O 45 Ser Val Val Lieu. Gly Lieu. Lieu. Phe Lieu Val Lieu. Phe Arg Ser Val Ala SO 55 6 O Lys Lys Ala Thr Ser Gly Val Pro Gly Llys Phe Glin Thr Ala Ile Glu 65 70 7s 8O Lieu Val Ile Gly Phe Val Asn Gly Ser Val Lys Asp Met Tyr His Gly 85 90 95 Llys Ser Lys Lieu. Ile Ala Pro Lieu Ala Lieu. Thir Ile Phe Val Trp Val 1OO 105 11 O Phe Lieu Met Asn Lieu Met Asp Lieu. Lieu Pro Ile Asp Lieu. Lieu Pro Tyr 115 12 O 125 Ile Ala Glu. His Val Lieu. Gly Lieu Pro Ala Lieu. Arg Val Val Pro Ser 13 O 135 14 O Ala Asp Val Asn Val Thr Lieu. Ser Met Ala Lieu. Gly Val Phe Ile Lieu 145 150 155 160 Ile Leu Phe Tyr Ser Ile Llys Met Lys Gly Ile Gly Gly Phe Thr Lys 1.65 17O 17s Glu Lieu. Thir Lieu Gln Pro Phe Asn His Trp Ala Phe Ile Pro Val Asn 18O 185 19 O Lieu. Ile Lieu. Glu Gly Val Ser Lieu. Lieu. Ser Llys Pro Val Ser Lieu. Gly 195 2OO 2O5 Lieu. Arg Lieu. Phe Gly Asn Met Tyr Ala Gly Glu Lieu. Ile Phe Ile Lieu. 21 O 215 22O Ile Ala Gly Lieu Lleu Pro Trp Trp Ser Glin Trp Ile Lieu. Asn Val Pro 225 23 O 235 24 O Trp Ala Ile Phe His Ile Lieu. Ile Ile Thr Lieu. Glin Ala Phe Ile Phe 245 250 255 Met Val Lieu. Thir Ile Val Tyr Lieu Ser Met Ala Ser Glu Glu. His 26 O 265 27 O

<210s, SEQ ID NO 4 &21.1 L ENGTH: 139 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 4 Met Ala Met Thr Tyr His Lieu. Asp Val Val Ser Ala Glu Glin Glin Met 1. 5 1O 15 Phe Ser Gly Lieu Val Glu Lys Ile Glin Val Thr Gly Ser Glu Gly Glu 2O 25 3O Lieu. Gly Ile Tyr Pro Gly His Ala Pro Leu Lieu. Thir Ala Ile Llys Pro 35 4 O 45 Gly Met Ile Arg Ile Val Lys Gln His Gly His Glu Glu Phe Ile Tyr SO 55 6 O Lieu. Ser Gly Gly Ile Lieu. Glu Val Glin Pro Gly Asn Val Thr Val Lieu. 65 70 7s 8O Ala Asp Thir Ala Ile Arg Gly Glin Asp Lieu. Asp Glu Ala Arg Ala Met 85 90 95 Glu Ala Lys Arg Lys Ala Glu Glu. His Ile Ser Ser Ser His Gly Asp 1OO 105 11 O Val Asp Tyr Ala Glin Ala Ser Ala Glu Lieu Ala Lys Ala Ile Ala Glin US 2010/0028334 A1 Feb. 4, 2010 37

- Continued

115 12 O 125 Lieu. Arg Val Ile Glu Lieu. Thir Lys Lys Ala Met 13 O 135

<210s, SEQ ID NO 5 &211s LENGTH: 460 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 5 Met Ala Thr Gly Lys Ile Val Glin Val Ile Gly Ala Val Val Asp Wall 1. 5 1O 15 Glu Phe Pro Glin Asp Ala Val Pro Arg Val Tyr Asp Ala Lieu. Glu Val 2O 25 3O Glin Asn Gly Asn. Glu Arg Lieu Val Lieu. Glu Val Glin Glin Glin Lieu. Gly 35 4 O 45 Gly Gly Ile Val Arg Thir Ile Ala Met Gly Ser Ser Asp Gly Lieu. Arg SO 55 6 O Arg Gly Lieu. Asp Wall Lys Asp Lieu. Glu. His Pro Ile Glu Val Pro Val 65 70 7s 8O Gly Lys Ala Thr Lieu. Gly Arg Ile Met Asin Val Lieu. Gly Glu Pro Val 85 90 95 Asp Met Lys Gly Glu Ile Gly Glu Glu Glu Arg Trp Ala Ile His Arg 1OO 105 11 O Ala Ala Pro Ser Tyr Glu Glu Lieu. Ser Asn. Ser Glin Glu Lieu. Lieu. Glu 115 12 O 125 Thr Gly Ile Llys Val Ile Asp Lieu Met Cys Pro Phe Ala Lys Gly Gly 13 O 135 14 O Llys Val Gly Lieu. Phe Gly Gly Ala Gly Val Gly Llys Thr Val Asn Met 145 150 155 160 Met Glu Lieu. Ile Arg Asn. Ile Ala Ile Glu. His Ser Gly Tyr Ser Val 1.65 17O 17s Phe Ala Gly Val Gly Glu Arg Thr Arg Glu Gly Asn Asp Phe Tyr His 18O 185 19 O Glu Met Thr Asp Ser Asn Val Ile Asp Llys Val Ser Lieu Val Tyr Gly 195 2OO 2O5 Glin Met Asn. Glu Pro Pro Gly Asn Arg Lieu. Arg Val Ala Lieu. Thr Gly 21 O 215 22O Lieu. Thir Met Ala Glu Lys Phe Arg Asp Glu Gly Arg Asp Val Lieu. Lieu. 225 23 O 235 24 O Phe Val Asp Asn Ile Tyr Arg Tyr Thr Lieu Ala Gly Thr Glu Val Ser 245 250 255 Ala Lieu. Leu Gly Arg Met Pro Ser Ala Val Gly Tyr Gln Pro Thr Lieu. 26 O 265 27 O Ala Glu Glu Met Gly Val Lieu. Glin Glu Arg Ile Thr Ser Thr Lys Thr 27s 28O 285 Gly Ser Ile Thir Ser Val Glin Ala Val Tyr Val Pro Ala Asp Asp Lieu. 29 O 295 3 OO Thr Asp Pro Ser Pro Ala Thr Thr Phe Ala His Lieu. Asp Ala Thr Val 3. OS 310 315 32O Val Lieu. Ser Arg Glin Ile Ala Ser Lieu. Gly Ile Tyr Pro Ala Val Asp 3.25 330 335 US 2010/0028334 A1 Feb. 4, 2010 38

- Continued Pro Lieu. Asp Ser Thr Ser Arg Glin Lieu. Asp Pro Lieu Val Val Gly Glin 34 O 345 35. O Glu. His Tyr Asp Thr Ala Arg Gly Val Glin Ser Ile Lieu. Glin Arg Tyr 355 360 365 Glin Glu Lieu Lys Asp Ile Ile Ala Ile Lieu. Gly Met Asp Glu Lieu. Ser 37 O 375 38O Glu Glu Asp Llys Lieu Val Val Ala Arg Ala Arg Lys Ile Glin Arg Phe 385 390 395 4 OO Lieu. Ser Glin Pro Phe Phe Val Ala Glu Val Phe Thr Gly Ser Pro Gly 4 OS 41O 415 Llys Tyr Val Ser Lieu Lys Asp Thir Ile Arg Gly Phe Lys Gly Ile Met 42O 425 43 O Glu Gly Glu Tyr Asp His Leu Pro Glu Glin Ala Phe Tyr Met Val Gly 435 44 O 445 Ser Ile Glu Glu Ala Val Glu Lys Ala Lys Llys Lieu. 450 45.5 460

<210s, SEQ ID NO 6 &211s LENGTH: 79 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 6 Met Glu Asn Lieu. ASn Met Asp Lieu. Lieu. Tyr Met Ala Ala Ala Val Met 1. 5 1O 15 Met Gly Lieu Ala Ala Ile Gly Ala Ala Ile Gly Ile Gly Ile Lieu. Gly 2O 25 3O Gly Llys Phe Lieu. Glu Gly Ala Ala Arg Glin Pro Asp Lieu. Ile Pro Lieu. 35 4 O 45 Lieu. Arg Thr Glin Phe Phe Ile Val Met Gly Lieu Val Asp Ala Ile Pro SO 55 6 O Met Ile Ala Val Gly Lieu. Gly Lieu. Tyr Val Met Phe Ala Wall Ala 65 70 7s

<210s, SEQ ID NO 7 &211s LENGTH: 287 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OO > SEQUENCE: 7 Met Ala Gly Ala Lys Glu Ile Arg Ser Lys Ile Ala Ser Val Glin Asn 1. 5 1O 15 Thr Gln Lys Ile Thr Lys Ala Met Glu Met Val Ala Ala Ser Lys Met 2O 25 3O Arg Llys Ser Glin Asp Arg Met Ala Ala Ser Arg Pro Tyr Ala Glu Thr 35 4 O 45 Met Arg Llys Val Ile Gly. His Lieu Ala His Gly Asn Lieu. Glu Tyr Lys SO 55 6 O His Pro Tyr Lieu. Glu Asp Arg Asp Wall Lys Arg Val Gly Tyr Lieu Val 65 70 7s 8O Val Ser Thir Asp Arg Gly Lieu. Cys Gly Gly Lieu. Asn. Ile Asn Lieu. Phe 85 90 95 Llys Llys Lieu. Lieu Ala Glu Met Lys Thir Trp Thr Asp Llys Gly Val Glin 1OO 105 11 O US 2010/0028334 A1 Feb. 4, 2010 39

- Continued Cys Asp Leu Ala Met Ile Gly Ser Lys Gly Val Ser Phe Phe Asin Ser 115 12 O 125 Val Gly Gly Asn Val Val Ala Glin Val Thr Gly Met Gly Asp Asn Pro 13 O 135 14 O Ser Lieu. Ser Glu Lieu. Ile Gly Pro Val Llys Val Met Lieu. Glin Ala Tyr 145 150 155 160 Asp Glu Gly Arg Lieu. Asp Llys Lieu. Tyr Ile Val Ser Asn Llys Phe Ile 1.65 17O 17s

Asn. Thir Met Ser Glin Wall Pro Thir Ile Ser Glin Leu Lleu Pro Leu Pro 18O 185 19 O Ala Ser Asp Asp Asp Asp Lieu Lys His Llys Ser Trp Asp Tyr Lieu. Tyr 195 2OO 2O5 Glu Pro Asp Pro Lys Ala Lieu. Lieu. Asp Thir Lieu. Lieu. Arg Arg Tyr Val 21 O 215 22O Glu Ser Glin Val Tyr Glin Gly Val Val Glu Asn Lieu Ala Ser Glu Glin 225 23 O 235 24 O Ala Ala Arg Met Val Ala Met Lys Ala Ala Thr Asp Asn Gly Gly Ser 245 250 255 Lieu. Ile Lys Glu Lieu. Glin Lieu Val Tyr Asn Lys Ala Arg Glin Ala Ser 26 O 265 27 O Ile Thr Glin Glu Lieu. Thr Glu Ile Val Ser Gly Ala Ala Ala Val 27s 28O 285

<210s, SEQ ID NO 8 &211s LENGTH: 177 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 8 Met Ser Glu Phe Ile Thr Val Ala Arg Pro Tyr Ala Lys Ala Ala Phe 1. 5 1O 15 Asp Phe Ala Val Glu. His Glin Ser Val Glu Arg Trp Glin Asp Met Lieu 2O 25 3O Ala Phe Ala Ala Glu Val Thir Lys Asn. Glu Gln Met Ala Glu Lieu. Lieu 35 4 O 45 Ser Gly Ala Lieu Ala Pro Glu Thir Lieu Ala Glu Ser Phe Ile Ala Val SO 55 6 O Cys Gly Glu Gln Lieu. Asp Glu Asin Gly Glin Asn Lieu. Ile Arg Val Met 65 70 7s 8O Ala Glu Asn Gly Arg Lieu. Asn Ala Lieu Pro Asp Val Lieu. Glu Glin Phe 85 90 95 Ile His Lieu. Arg Ala Val Ser Glu Ala Thr Ala Glu Val Asp Val Ile 1OO 105 11 O Ser Ala Ala Ala Lieu. Ser Glu Glin Glin Lieu Ala Lys Ile Ser Ala Ala 115 12 O 125 Met Glu Lys Arg Lieu. Ser Arg Llys Wall Lys Lieu. Asn. Cys Lys Ile Asp 13 O 135 14 O Llys Ser Wal Met Ala Gly Val Ile Ile Arg Ala Gly Asp Met Val Ile 145 150 155 160 Asp Gly Ser Val Arg Gly Arg Lieu. Glu Arg Lieu Ala Asp Val Lieu. Glin 1.65 17O 17s

Ser US 2010/0028334 A1 Feb. 4, 2010 40

- Continued

<210s, SEQ ID NO 9 &211s LENGTH: 490 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 9 Met Ser Arg Met Ala Glu Gln Gln Leu Tyr Ile His Gly Gly Tyr Thr 1. 5 1O 15 Ser Ala Thr Ser Gly Arg Thr Phe Glu Thir Ile Asn Pro Ala Asn Gly 2O 25 3O Asn Val Lieu Ala Thr Val Glin Ala Ala Gly Arg Glu Asp Val Asp Arg 35 4 O 45 Ala Wall Lys Ser Ala Glin Glin Gly Glin Lys Ile Trp Ala Ser Met Thr SO 55 6 O Ala Met Glu Arg Ser Arg Ile Lieu. Arg Arg Ala Val Asp Ile Lieu. Arg 65 70 7s 8O Glu Arg Asn Asp Glu Lieu Ala Lys Lieu. Glu Thir Lieu. Asp Thr Gly Lys 85 90 95 Ala Tyr Ser Glu Thir Ser Thr Val Asp Ile Val Thr Gly Ala Asp Val 1OO 105 11 O Lieu. Glu Tyr Tyr Ala Gly Lieu. Ile Pro Ala Lieu. Glu Gly Ser Glin Ile 115 12 O 125 Pro Leu Arg Glu Thir Ser Phe Val Tyr Thr Arg Arg Glu Pro Leu Gly 13 O 135 14 O Val Val Ala Gly Ile Gly Ala Trp Asn Tyr Pro Ile Glin Ile Ala Lieu 145 150 155 160 Trp Llys Ser Ala Pro Ala Lieu Ala Ala Gly Asn Ala Met Ile Phe Lys 1.65 17O 17s Pro Ser Glu Val Thr Pro Leu. Thir Ala Leu Lys Lieu Ala Glu Ile Tyr 18O 185 19 O Ser Glu Ala Gly Lieu Pro Asp Gly Val Phe Asin Val Lieu Pro Gly Val 195 2OO 2O5 Gly Ala Glu Thr Gly Glin Tyr Lieu. Thr Glu. His Pro Gly Ile Ala Lys 21 O 215 22O Val Ser Phe Thr Gly Gly Val Ala Ser Gly Lys Llys Val Met Ala Asn 225 23 O 235 24 O Ser Ala Ala Ser Ser Lieu Lys Glu Val Thr Met Glu Lieu. Gly Gly Lys 245 250 255 Ser Pro Lieu. Ile Val Phe Asp Asp Ala Asp Lieu. Asp Lieu Ala Ala Asp 26 O 265 27 O Ile Ala Met Met Ala Asn Phe Phe Ser Ser Gly Glin Val Cys Thr Asn 27s 28O 285 Gly Thr Arg Val Phe Val Pro Ala Lys Cys Lys Ala Ala Phe Glu Glin 29 O 295 3 OO Lys Ile Lieu Ala Arg Val Glu Arg Ile Arg Ala Gly Asp Val Phe Asp 3. OS 310 315 32O Pro Gln Thr Asn Phe Gly Pro Leu Val Ser Phe Pro His Arg Asp Asn 3.25 330 335 Val Lieu. Arg Tyr Ile Ala Lys Gly Lys Glu Glu Gly Ala Arg Val Lieu 34 O 345 35. O Cys Gly Gly Asp Val Lieu Lys Gly Asp Gly Phe Asp Asn Gly Ala Trp 355 360 365 US 2010/0028334 A1 Feb. 4, 2010 41

- Continued

Val Ala Pro Thr Val Phe Thr Asp Cys Ser Asp Asp Met Thr Ile Val 37 O 375 38O Arg Glu Glu Ile Phe Gly Pro Val Met Ser Ile Lieu. Thr Tyr Glu Ser 385 390 395 4 OO Glu Asp Glu Val Ile Arg Arg Ala Asn Asp Thr Asp Tyr Gly Lieu Ala 4 OS 41O 415 Ala Gly Ile Val Thr Ala Asp Lieu. Asn Arg Ala His Arg Val Ile His 42O 425 43 O Gln Leu Glu Ala Gly Ile Cys Trp Ile Asn. Thir Trp Gly Glu Ser Pro 435 44 O 445 Ala Glu Met Pro Val Gly Gly Tyr Lys His Ser Gly Ile Gly Arg Glu 450 45.5 460 Asn Gly Val Met Thr Lieu. Glin Ser Tyr Thr Glin Val Lys Ser Ile Glin 465 470 47s 48O Val Glu Met Ala Lys Phe Glin Ser Ile Phe 485 490

<210s, SEQ ID NO 10 &211s LENGTH: 4 O1 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 10 Met Asin Val Phe Asn Pro Ala Glin Phe Arg Ala Glin Phe Pro Ala Leu 1. 5 1O 15 Glin Asp Ala Gly Val Tyr Lieu. Asp Ser Ala Ala Thr Ala Lieu Lys Pro 2O 25 3O Glu Ala Val Val Glu Ala Thr Glin Glin Phe Tyr Ser Leu Ser Ala Gly 35 4 O 45 Asn. Wal His Arg Ser Glin Phe Ala Glu Ala Glin Arg Lieu. Thir Ala Arg SO 55 6 O Tyr Glu Ala Ala Arg Glu Lys Val Ala Glin Lieu. Lieu. Asn Ala Pro Asp 65 70 7s 8O Asp Llys Thir Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met 85 90 95 Val Ala Glin Cys Tyr Ala Arg Pro Arg Lieu. Glin Pro Gly Asp Glu Ile 1OO 105 11 O Ile Val Ser Val Ala Glu. His His Ala Asn Lieu Val Pro Trp Lieu Met 115 12 O 125 Val Ala Glin Glin Thr Gly Ala Lys Val Val Lys Lieu Pro Lieu. Asn Ala 13 O 135 14 O Glin Arg Lieu Pro Asp Wall Asp Lieu. Lieu Pro Glu Lieu. Ile Thr Pro Arg 145 150 155 160 Ser Arg Ile Lieu Ala Lieu. Gly Glin Met Ser Asn Val Thr Gly Gly Cys 1.65 17O 17s Pro Asp Lieu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val 18O 185 19 O Val Met Val Asp Gly Ala Glin Gly Ala Wal His Phe Pro Ala Asp Wall 195 2OO 2O5 Glin Glin Lieu. Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Llys Lieu. Tyr 21 O 215 22O Gly Pro Thr Gly Ile Gly Val Lieu. Tyr Gly Lys Ser Glu Lieu. Lieu. Glu US 2010/0028334 A1 Feb. 4, 2010 42

- Continued

225 23 O 235 24 O Ala Met Ser Pro Trp Lieu. Gly Gly Gly Lys Met Val His Glu Val Ser 245 250 255 Phe Asp Gly Phe Thr Thr Glin Ser Ala Pro Trp Llys Lieu. Glu Ala Gly 26 O 265 27 O Thr Pro Asn. Wall Ala Gly Val Ile Gly Lieu. Ser Ala Ala Lieu. Glu Trp 27s 28O 285 Lieu Ala Asp Tyr Asp Ile Asn Glin Ala Glu Ser Trp Ser Arg Ser Lieu. 29 O 295 3 OO Ala Thr Lieu Ala Glu Asp Ala Lieu Ala Lys Arg Pro Gly Phe Arg Ser 3. OS 310 315 32O Phe Arg Cys Glin Asp Ser Ser Lieu. Lieu Ala Phe Asp Phe Ala Gly Val 3.25 330 335 His His Ser Asp Met Val Thir Lieu. Lieu Ala Glu Tyr Gly Ile Ala Lieu. 34 O 345 35. O Arg Ala Gly Glin His Cys Ala Glin Pro Lieu. Lieu Ala Glu Lieu. Gly Val 355 360 365 Thr Gly. Thir Lieu. Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp 37 O 375 38O Val Asp Ala Lieu Val Asn Ala Val Asp Arg Ala Lieu. Glu Lieu. Lieu Val 385 390 395 4 OO Asp

<210s, SEQ ID NO 11 &211s LENGTH: 646 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 11 Met Met Ser Tyr Val Asp Trp Pro Pro Leu. Ile Lieu. Arg His Thr Tyr 1. 5 1O 15 Tyr Met Ala Glu Phe Glu Thir Thr Phe Ala Asp Lieu. Gly Lieu Lys Ala 2O 25 3O Pro Ile Lieu. Glu Ala Lieu. Asn Asp Lieu. Gly Tyr Glu, Llys Pro Ser Pro 35 4 O 45 Ile Glin Ala Glu. Cys Ile Pro His Lieu. Lieu. Asn Gly Arg Asp Val Lieu. SO 55 6 O Gly Met Ala Glin Thr Gly Ser Gly Lys Thr Ala Ala Phe Ser Leu Pro 65 70 7s 8O Lieu. Lieu. Glin Asn Lieu. Asp Pro Glu Lieu Lys Ala Pro Glin Ile Lieu Val 85 90 95 Lieu Ala Pro Thir Arg Glu Lieu Ala Val Glin Val Ala Glu Ala Met Thr 1OO 105 11 O Asp Phe Ser Lys His Met Arg Gly Val Asin Val Val Ala Lieu. Tyr Gly 115 12 O 125 Gly Glin Arg Tyr Asp Val Glin Lieu. Arg Ala Lieu. Arg Glin Gly Pro Glin 13 O 135 14 O Ile Val Val Gly Thr Pro Gly Arg Lieu. Lieu. Asp His Lieu Lys Arg Gly 145 150 155 160 Thir Lieu. Asp Lieu. Ser Llys Lieu. Ser Gly Lieu Val Lieu. Asp Glu Ala Asp 1.65 17O 17s Glu Met Leu Arg Met Gly Phe Ile Glu Asp Val Glu Thir Ile Met Ala US 2010/0028334 A1 Feb. 4, 2010 43

- Continued

18O 185 19 O Glin Ile Pro Glu Gly His Glin Thr Ala Leu Phe Ser Ala Thr Met Pro 195 2OO 2O5 Glu Ala Ile Arg Arg Ile Thr Arg Arg Phe Met Lys Glu Pro Glin Glu 21 O 215 22O Val Arg Ile Glin Ser Ser Val Thir Thr Arg Pro Asp Ile Ser Glin Ser 225 23 O 235 24 O Tyr Trp Thr Val Trp Gly Met Arg Lys Asn. Glu Ala Lieu Val Arg Phe 245 250 255 Lieu. Glu Ala Glu Asp Phe Asp Ala Ala Ile Ile Phe Val Arg Thr Lys 26 O 265 27 O Asn Ala Thir Lieu. Glu Val Ala Glu Ala Lieu. Glu Arg Asn Gly Tyr Asn 27s 28O 285 Ser Ala Ala Lieu. Asn Gly Asp Met Asn Glin Ala Lieu. Arg Glu Glin Thr 29 O 295 3 OO

Lieu. Glu Arg Lieu Lys Asp Gly Arg Lieu. Asp I e Lieu. Ile Ala Thr Asp 3. OS 310 3 5 32O Val Ala Ala Arg Gly Lieu. Asp Val Glu Arg Ile Ser Lieu Val Val Asn 3.25 330 335 Tyr Asp Ile Pro Met Asp Ser Glu Ser Tyr Val His Arg Ile Gly Arg 34 O 345 35. O Thr Gly Arg Ala Gly Arg Ala Gly Arg Ala Lieu. Lieu. Phe Val Glu ASn 355 360 365 Arg Glu Arg Arg Lieu. Lieu. Arg Asn. Ile Glu Arg Thr Met Lys Lieu. Thr 37 O 375 38O Ile Pro Glu Val Glu Lieu Pro Asn Ala Glu Lieu. Lieu. Gly Lys Arg Arg 385 390 395 4 OO Lieu. Glu Lys Phe Ala Ala Lys Val Glin Glin Gln Lieu. Glu Ser Ser Asp 4 OS 41O 415 Lieu. Asp Glin Tyr Arg Ala Lieu. Lieu. Ser Lys Ile Glin Pro Thir Ala Glu 42O 425 43 O Gly Glu Glu Lieu. Asp Lieu. Glu Thir Lieu Ala Ala Ala Lieu. Lieu Lys Met 435 44 O 445 Ala Glin Gly Glu Arg Thr Lieu. Ile Val Pro Pro Asp Ala Pro Met Arg 450 45.5 460 Pro Lys Arg Glu Phe Arg Asp Arg Asp Asp Arg Gly Pro Arg Asp Arg 465 470 47s 48O Asn Asp Arg Gly Pro Arg Gly Asp Arg Glu Asp Arg Pro Arg Arg Glu 485 490 495 Arg Arg Asp Val Gly Asp Met Glin Lieu. Tyr Arg Ile Glu Val Gly Arg SOO 505 51O Asp Asp Gly Val Glu Val Arg His Ile Val Gly Ala Ile Ala Asn. Glu 515 52O 525 Gly Asp Ile Ser Ser Arg Tyr Ile Gly Asn. Ile Llys Lieu. Phe Ala Ser 53 O 535 54 O His Ser Thr Ile Glu Lieu Pro Lys Gly Met Pro Gly Glu Val Leu Gln 5.45 550 555 560 His Phe Thr Arg Thr Arg Ile Lieu. Asn Llys Pro Met Asn Met Glin Leu 565 st O sts Lieu. Gly Asp Ala Glin Pro His Thr Gly Gly Glu Arg Arg Gly Gly Gly 58O 585 59 O US 2010/0028334 A1 Feb. 4, 2010 44

- Continued

Arg Gly Phe Gly Gly Glu Arg Arg Glu Gly Gly Arg Asn. Phe Ser Gly 595 6OO 605 Glu Arg Arg Glu Gly Gly Arg Gly Asp Gly Arg Arg Phe Ser Gly Glu 610 615 62O Arg Arg Glu Gly Arg Ala Pro Arg Arg Asp Asp Ser Thr Gly Arg Arg 625 630 635 64 O Arg Phe Gly Gly Asp Ala 645

<210s, SEQ ID NO 12 &211s LENGTH: 406 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 12 Met Ile Phe Ser Val Asp Llys Val Arg Ala Asp Phe Pro Val Lieu. Ser 1. 5 1O 15 Arg Glu Val Asn Gly Lieu Pro Lieu Ala Tyr Lieu. Asp Ser Ala Ala Ser 2O 25 3O Ala Glin Llys Pro Ser Glin Val Ile Asp Ala Glu Ala Glu Phe Tyr Arg 35 4 O 45 His Gly Tyr Ala Ala Wal His Arg Gly Ile His Thr Lieu. Ser Ala Glin SO 55 6 O Ala Thr Glu Lys Met Glu Asn Val Arg Lys Arg Ala Ser Lieu. Phe Ile 65 70 7s 8O Asn Ala Arg Ser Ala Glu Glu Lieu Val Phe Val Arg Gly. Thir Thr Glu 85 90 95 Gly Ile Asn Lieu Val Ala Asn. Ser Trp Gly Asn. Ser Asn Val Arg Ala 1OO 105 11 O Gly Asp Asn. Ile Ile Ile Ser Glin Met Glu. His His Ala Asn. Ile Val 115 12 O 125 Pro Trp Gln Met Lieu. Cys Ala Arg Val Gly Ala Glu Lieu. Arg Val Ile 13 O 135 14 O Pro Leu. Asn Pro Asp Gly Thr Lieu Gln Leu Glu Thir Lieu Pro Thr Lieu 145 150 155 160 Phe Asp Glu Lys Thr Arg Lieu. Lieu Ala Ile Thr His Val Ser Asn. Wall 1.65 17O 17s Lieu. Gly. Thr Glu Asn Pro Lieu Ala Glu Met Ile Thr Lieu Ala His Glin 18O 185 19 O His Gly Ala Lys Val Lieu Val Asp Gly Ala Glin Ala Wal Met His His 195 2OO 2O5 Pro Val Asp Val Glin Ala Lieu. Asp Cys Asp Phe Tyr Val Phe Ser Gly 21 O 215 22O His Llys Lieu. Tyr Gly Pro Thr Gly Ile Gly Ile Lieu. Tyr Val Lys Glu 225 23 O 235 24 O Ala Lieu. Leu Gln Glu Met Pro Pro Trp Glu Gly Gly Gly Ser Met Ile 245 250 255 Ala Thr Val Ser Leu Ser Glu Gly Thr Thr Trp Thr Lys Ala Pro Trp 26 O 265 27 O Arg Phe Glu Ala Gly Thr Pro Asn Thr Gly Gly Ile Ile Gly Lieu. Gly 27s 28O 285 Ala Ala Lieu. Glu Tyr Val Ser Ala Lieu. Gly Lieu. Asn. Asn. Ile Ala Glu US 2010/0028334 A1 Feb. 4, 2010 45

- Continued

29 O 295 3 OO Tyr Glu Glin Asn Lieu Met His Tyr Ala Lieu. Ser Glin Lieu. Glu Ser Val 3. OS 310 315 32O Pro Asp Lieu. Thir Lieu. Tyr Gly Pro Glin Asn Arg Lieu. Gly Val Ile Ala 3.25 330 335 Phe Asn Lieu. Gly Llys His His Ala Tyr Asp Val Gly Ser Phe Lieu. Asp 34 O 345 35. O Asn Tyr Gly Ile Ala Val Arg Thr Gly His His Cys Ala Met Pro Leu 355 360 365 Met Ala Tyr Tyr Asn Val Pro Ala Met Cys Arg Ala Ser Leu Ala Met 37 O 375 38O Tyr Asn. Thir His Glu Glu Val Asp Arg Lieu Val Thr Gly Lieu. Glin Arg 385 390 395 4 OO Ile His Arg Lieu. Lieu. Gly 4 OS

<210s, SEQ ID NO 13 &211s LENGTH: 271 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 13 Met Thr Glu Ser Val Ala Arg Lieu. Arg Gly Glu Glin Lieu. Thir Lieu. Gly 1. 5 1O 15 Tyr Gly Lys Tyr Thr Val Ala Glu Asn Lieu. Thr Val Glu Ile Pro Asp 2O 25 3O Gly His Phe Thr Ala Ile Ile Gly Pro Asn Gly Cys Gly Lys Ser Thr 35 4 O 45 Lieu. Lieu. Arg Thir Lieu. Ser Arg Lieu Met Thr Pro Ala His Gly His Val SO 55 6 O Trp Lieu. Asp Gly Glu. His Ile Glin His Tyr Ala Ser Lys Glu Val Ala 65 70 7s 8O Arg Arg Ile Gly Lieu. Lieu Ala Glin Asn Ala Thir Thr Pro Gly Asp Ile 85 90 95 Thr Val Glin Glu Lieu Val Ala Arg Gly Arg Tyr Pro His Glin Pro Leu 1OO 105 11 O Phe Thr Arg Trp Arg Lys Glu Asp Glu Glu Ala Val Thir Lys Ala Met 115 12 O 125 Glin Ala Thr Gly Ile Thr His Lieu Ala Asp Glin Ser Val Asp Thir Lieu. 13 O 135 14 O Ser Gly Gly Glin Arg Glin Arg Ala Trp Ile Ala Met Val Lieu Ala Glin 145 150 155 160 Glu Thir Ala Ile Met Leu Lleu. Asp Glu Pro Thir Thr Trp Lieu. Asp Ile 1.65 17O 17s Ser His Glin Ile Asp Lieu. Lieu. Glu Lieu. Lieu. Ser Glu Lieu. Asn Arg Glu 18O 185 19 O Lys Gly Tyr Thir Lieu Ala Ala Val Lieu. His Asp Lieu. Asn Glin Ala Cys 195 2OO 2O5 Arg Tyr Ala Ser His Lieu. Ile Ala Lieu. Arg Glu Gly Lys Ile Val Ala 21 O 215 22O Glin Gly Ala Pro Llys Glu Ile Val Thir Ala Glu Lieu. Ile Glu Arg Ile 225 23 O 235 24 O US 2010/0028334 A1 Feb. 4, 2010 46

- Continued Tyr Gly Lieu. Arg Cys Met Ile Ile Asp Asp Pro Val Ala Gly Thr Pro 245 250 255 Lieu Val Val Pro Leu Gly Arg Thr Ala Pro Ser Thr Ala Asn Ser 26 O 265 27 O

<210s, SEQ ID NO 14 &211s LENGTH: 525 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 14 Met Thr Glu Asn. Ile His Llys His Arg Ile Lieu. Ile Lieu. Asp Phe Gly 1. 5 1O 15 Ser Glin Tyr Thr Glin Lieu Val Ala Arg Arg Val Arg Glu Lieu. Gly Val 2O 25 3O Tyr Cys Glu Lieu. Trp Ala Trp Asp Val Thr Glu Ala Glin Ile Arg Asp 35 4 O 45 Phe Asin Pro Ser Gly Ile Ile Leu Ser Gly Gly Pro Glu Ser Thir Thr SO 55 6 O Glu Glu Asn Ser Pro Arg Ala Pro Glin Tyr Val Phe Glu Ala Gly Val 65 70 7s 8O Pro Val Phe Gly Val Cys Tyr Gly Met Glin Thr Met Ala Met Gln Leu 85 90 95 Gly Gly His Val Glu Ala Ser ASn Glu Arg Glu Phe Gly Tyr Ala Glin 1OO 105 11 O Val Glu Val Val Asn Asp Ser Ala Lieu Val Arg Gly Ile Glu Asp Ala 115 12 O 125 Lieu. Thir Ala Asp Gly Llys Pro Lieu. Lieu. Asp Val Trp Met Ser His Gly 13 O 135 14 O Asp Llys Val Thr Ala Ile Pro Ser Asp Phe Ile Thr Val Ala Ser Thr 145 150 155 160 Glu Ser Cys Pro Phe Ala Ile Met Ala Asn Glu Glu Lys Arg Phe Tyr 1.65 17O 17s Gly Val Glin Phe His Pro Glu Val Thr His Thr Arg Glin Gly Met Arg 18O 185 19 O Met Lieu. Glu Arg Phe Val Arg Asp Ile Cys Glin Cys Glu Ala Lieu. Trp 195 2OO 2O5 Thr Pro Ala Lys Ile Ile Asp Asp Ala Val Ala Arg Ile Arg Glu Glin 21 O 215 22O Val Gly Asp Asp Llys Val Ile Lieu. Gly Lieu. Ser Gly Gly Val Asp Ser 225 23 O 235 24 O Ser Val Thir Ala Met Lieu. Lieu. His Arg Ala Ile Gly Lys Asn Lieu. Thir 245 250 255 Cys Val Phe Val Asp Asn Gly Lieu. Lieu. Arg Lieu. Asn. Glu Ala Glu Glin 26 O 265 27 O Val Lieu. Asp Met Phe Gly Asp His Phe Gly Lieu. Asn. Ile Val His Val 27s 28O 285 Pro Ala Glu Asp Arg Phe Lieu. Ser Ala Lieu Ala Gly Glu Asn Asp Pro 29 O 295 3 OO Glu Ala Lys Arg Lys Ile Ile Gly Arg Val Phe Val Glu Val Phe Asp 3. OS 310 315 32O Glu Glu Ala Lieu Lys Lieu. Glu Asp Wall Lys Trp Lieu Ala Glin Gly. Thir 3.25 330 335 US 2010/0028334 A1 Feb. 4, 2010 47

- Continued

Ile Pro Asp Wall Ile Glu Ser Ala Ala Ser Ala Thir Gly Lys Ala 34 O 345 35. O

His Wall Ile Ser His His Asn Wall Gly Gly Lell Pro Lys Glu Met 355 360 365

Met Gly Luell Wall Glu Pro Luell Glu Luell Phe Asp Glu Wall 37 O 375

Arg Ile Gly Lell Glu Lell Gly Luell Pro Tyr Asp Met Luell Tyr Arg 385 390 395 4 OO

His Pro Phe Pro Gly Pro Gly Luell Gly Wall Arg Wall Lell Gly Glu Wall 4 OS 415

Glu Tyr Cys Asp Lell Luell Arg Arg Ala Asp Ala Ile Phe Ile 425 43 O

Glu Glu Luell Arg Ala Asp Luell Tyr Asp Wall Ser Glin Ala Phe 435 44 O 445

Thir Wall Phe Luell Pro Wall Arg Ser Wall Gly Wall Met Gly Asp Gly Arg 450 45.5 460

Lys Asp Trp Wall Wall Ser Luell Arg Ala Wall Glu Thir Ile Asp Phe 465 470

Met Thir Ala His Trp Ala His Luell Pro Tyr Asp Phe Lell Gly Arg Wall 485 490 495

Ser Asn Arg Ile Ile Asn Glu Wall Asn Gly Ile Ser Arg Wall Wall Tyr SOO 505 51O

Asp Ile Ser Gly Pro Pro Ala Thir Ile Glu Trp Glu 515 52O 525

<210s, SEQ ID NO 15 &211s LENGTH: 488 212. TYPE : PRT &213s ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 15

Met Lieu. Arg Ile Ala Glu Ala Luell Thir Phe Asp Asp Wall Luell Luell 1. 5 15

Wall Pro Ala His Ser Thir Wall Luell Pro Asn Thir Ala Asp Luell Ser Thir 25

Glin Luell Thir Thir Ile Arg Luell Asn Ile Pro Met Lell Ser Ala Ala 35 4 O 45

Met Asp Thir Wall Thir Glu Ala Arg Luell Ala Ile Ala Lell Ala Glin Glu SO 55 6 O

Gly Gly Ile Gly Phe Ile His Asn Met Ser Ile Glu Arg Glin Ala 65 70

Glu Glu Wall Arg Arg Wall His Glu Ser Gly Wall Wall Thir Asp 85 90 95

Pro Glin Thir Wall Lell Pro Thir Thir Thir Luell Arg Glu Wall Lys Glu Luell 105 11 O

Thir Glu Arg Asn Gly Phe Ala Gly Tyr Pro Wall Wall Thir Glu Glu Asn 115 12 O 125

Glu Luell Wall Gly Ile Ile Thir Gly Arg Asp Wall Arg Phe Wall Thir Asp 13 O 135 14 O

Lell Asn Glin Pro Wall Ser Wall Met Thir Pro Glu Arg Luell Wall 145 150 155 160

Thir Wall Arg Glu Gly Glu Ala Arg Glu Wall Wall Lell Ala Met His US 2010/0028334 A1 Feb. 4, 2010 48

- Continued

1.65 17O 17s

Glu Lys Arg Val Glu Lys Ala Luell Wall Wall Asp Asp Glu Phe His Luell 18O 185 19 O

Ile Gly Met Ile Thr Val Lys Asp Phe Glin Ala Glu Arg Lys Pro 195

Asn Ala Cys Lys Asp Glu Glin Gly Arg Luell Arg Wall Gly Ala Ala Wall 21 O 215 22O

Gly Ala Gly Ala Gly Asn. Glu Glu Arg Wall Asp Ala Lell Wall Ala 225 23 O 235

Gly Val Asp Val Lieu. Lieu. Ile Asp Ser Ser His Gly His Ser Glu 245 250 255

Val Lieu. Glin Arg Ile Arg Glu Thir Arg Ala Tyr Pro Asp Luell 26 O 265 27 O

Ile Ile Gly Gly Asn Val Ala Thir Ala Ala Gly Ala Arg Ala Luell 27s 28O 285

Glu Ala Gly Cys Ser Ala Wall Wall Gly Ile Gly Pro Gly Ser 29 O 295 3 OO

Cys Thir Thr Arg Ile Val Thr Gly Wall Gly Wall Pro Glin Ile Thir 3. OS 310 315

Val Ala Asp Ala Val Glu Ala Luell Glu Gly Thir Gly Ile Pro Wall 3.25 330 335

Ala Asp Gly Gly Ile Arg Phe Ser Gly Asp Ile Ala Ala Ile 34 O 345 35. O

Ala Gly Ala Ser Ala Wal Met Wall Gly Ser Met Lell Ala Gly Thir 355 360 365

Glu Ser Pro Gly Glu Ile Glu Luell Glin Gly Arg Ser Ser 37 O 375

Tyr Arg Gly Met Gly Ser Lieu. Gly Ala Met Ser Lys Gly Ser Ser Asp 385 390 395 4 OO

Arg Tyr Phe Glin Ser Asp Asn Ala Ala Asp Lell Wall Pro Glu Gly 4 OS 415

Ile Glu Gly Arg Val Ala Tyr Gly Arg Luell Lys Glu Ile Ile His 42O 425 43 O

Glin Gln Met Gly Gly Lieu. Arg Ser Met Gly Lell Thir Gly Gly 435 44 O 445

Thir Ile Asp Glu Lieu. Arg Thr Ala Glu Phe Wall Arg Ile Ser Gly 450 45.5 460

Ala Gly Ile Glin Glu Ser His Wall His Asp Wall Thir Ile Thir Glu 465 470 47s 48O

Ser Pro Asn Tyr Arg Lieu. Gly Ser 485

<210s, SEQ ID NO 16 &211s LENGTH: 412 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 16 Met Met Tyr Gly Val Tyr Arg Ala Met Lys Lieu Pro Ile Tyr Lieu. Asp 1. 5 15 Tyr Ser Ala Thr Thr Pro Val Asp Pro Arg Val Ala Glu Lys Met Met 2O 25 US 2010/0028334 A1 Feb. 4, 2010 49

- Continued Glin Phe Met Thr Met Asp Gly Thr Phe Gly ASn Pro Ala Ser Arg Ser 35 4 O 45 His Arg Phe Gly Trp Glin Ala Glu Glu Ala Val Asp Ile Ala Arg Asn SO 55 6 O Glin Ile Ala Asp Lieu Val Gly Ala Asp Pro Arg Glu Ile Val Phe Thr 65 70 7s 8O Ser Gly Ala Thr Glu Ser Asp Asn Lieu Ala Ile Lys Gly Ala Ala Asn 85 90 95 Phe Tyr Gln Lys Lys Gly Lys His Ile Ile Thr Ser Lys Thr Glu. His 1OO 105 11 O Lys Ala Val Lieu. Asp Thr Cys Arg Glin Lieu. Glu Arg Glu Gly Phe Glu 115 12 O 125 Val Thr Tyr Lieu Ala Pro Glin Arg Asn Gly Ile Ile Asp Lieu Lys Glu 13 O 135 14 O Lieu. Glu Ala Ala Met Arg Asp Asp Thir Ile Lieu Val Ser Ile Met His 145 150 155 160 Val Asn. Asn. Glu Ile Gly Val Val Glin Asp Ile Ala Ala Ile Gly Glu 1.65 17O 17s Met Cys Arg Ala Arg Gly Ile Ile Tyr His Val Asp Ala Thr Glin Ser 18O 185 19 O Val Gly Lys Lieu Pro Ile Asp Lieu. Ser Glin Lieu Lys Val Asp Lieu Met 195 2OO 2O5 Ser Phe Ser Gly His Lys Ile Tyr Gly Pro Lys Gly Ile Gly Ala Leu 21 O 215 22O Tyr Val Arg Arg Llys Pro Arg Val Arg Ile Glu Ala Gln Met His Gly 225 23 O 235 24 O Gly Gly His Glu Arg Gly Met Arg Ser Gly Thr Lieu Pro Val His Glin 245 250 255 Ile Val Gly Met Gly Glu Ala Tyr Arg Ile Ala Lys Glu Glu Met Ala 26 O 265 27 O Thr Glu Met Glu Arg Lieu. Arg Gly Lieu. Arg Asn Arg Lieu. Trp Asn Gly 27s 28O 285 Ile Lys Asp Ile Glu Glu Val Tyr Lieu. Asn Gly Asp Lieu. Glu. His Gly 29 O 295 3 OO Ala Pro Asn. Ile Lieu. Asn Val Ser Phe Asn Tyr Val Glu Gly Glu Ser 3. OS 310 315 32O Lieu. Ile Met Ala Lieu Lys Asp Lieu Ala Val Ser Ser Gly Ser Ala Cys 3.25 330 335 Thir Ser Ala Ser Lieu. Glu Pro Ser Tyr Val Lieu. Arg Ala Lieu. Gly Lieu. 34 O 345 35. O Asn Asp Glu Lieu Ala His Ser Ser Ile Arg Phe Ser Lieu. Gly Arg Phe 355 360 365 Thir Thr Glu Glu Glu Ile Asp Tyr Thr Ile Glu Lieu Val Arg Llys Ser 37 O 375 38O Ile Gly Arg Lieu. Arg Asp Lieu. Ser Pro Lieu. Trp Glu Met Tyr Lys Glin 385 390 395 4 OO Gly Val Asp Lieu. Asn. Ser Ile Glu Trp Ala His His 4 OS 41O

<210s, SEQ ID NO 17 &211s LENGTH: 382 212. TYPE: PRT US 2010/0028334 A1 Feb. 4, 2010 50

- Continued

<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 17 Met Phe Llys Ser Asp Ala Ala Arg Thr Arg Glin Arg Lieu. Ser Ala Arg 1. 5 1O 15 Lys Gly Arg Thr Lieu Pro Pro Gly Arg Gly Llys Phe Pro His Ser Thr 2O 25 3O Thr Glu Ser Phe Arg Asn Thr Phe Trp Leu Lys Lys Ile Met Glu. His 35 4 O 45 Cys Phe Asn Met Val Asp Gln Gln Thr Thr Thr Ala Glin Thr Asn Ala SO 55 6 O Asn Phe Leu Glin Ile Arg Phe Thr Thr Met Ser Lys Lys Ile Ala Val 65 70 7s 8O Ile Gly Glu. Cys Met Ile Glu Lieu. Ser Glu Lys Gly Ala Asp Wall Lys 85 90 95 Arg Gly Phe Gly Gly Asp Thir Lieu. Asn. Thir Ser Val Tyr Ile Ala Arg 1OO 105 11 O Glin Val Asp Pro Ala Ala Lieu. Thr Val His Tyr Val Thr Ala Lieu. Gly 115 12 O 125 Thir Asp Ser Phe Ser Glin Gln Met Lieu. Asp Ala Trp His Gly Glu Asn 13 O 135 14 O Val Asp Thir Ser Lieu. Thr Glin Arg Met Glu Asn Arg Lieu Pro Gly Lieu 145 150 155 160 Tyr Tyr Ile Glu Thr Asp Ser Thr Gly Glu Arg Thr Phe Tyr Tyr Trp 1.65 17O 17s Arg Asn. Glu Ala Ala Ala Lys Phe Trp Lieu. Glu Ser Glu Glin Ser Ala 18O 185 19 O Ala Ile Cys Glu Glu Lieu Ala Asn. Phe Asp Tyr Lieu. Tyr Lieu. Ser Gly 195 2OO 2O5 Ile Ser Lieu Ala Ile Lieu. Ser Pro Thir Ser Arg Glu Lys Lieu. Lieu. Ser 21 O 215 22O Lieu. Lieu. Arg Glu. Cys Arg Ala Asn Gly Gly Llys Val Ile Phe Asp Asn 225 23 O 235 24 O Asn Tyr Arg Pro Arg Lieu. Trp Ala Ser Lys Glu Glu Thr Glin Glin Val 245 250 255 Tyr Glin Gln Met Lieu. Glu. Cys Thr Asp Ile Ala Phe Lieu. Thir Lieu. Asp 26 O 265 27 O Asp Glu Asp Ala Lieu. Trp Gly Glin Glin Pro Val Glu Asp Val Ile Ala 27s 28O 285 Arg Thr His Asn Ala Gly Val Lys Glu Val Val Val Lys Arg Gly Ala 29 O 295 3 OO Asp Ser Cys Lieu Val Ser Ile Ala Gly Glu Gly Lieu Val Asp Val Pro 3. OS 310 315 32O Ala Wall Lys Lieu Pro Llys Glu Lys Val Ile Asp Thir Thr Ala Ala Gly 3.25 330 335 Asp Ser Phe Ser Ala Gly Tyr Lieu Ala Val Arg Lieu. Thr Gly Gly Ser 34 O 345 35. O Ala Glu Asp Ala Ala Lys Arg Gly. His Lieu. Thir Ala Ser Thr Val Ile 355 360 365 Glin Tyr Arg Gly Ala Ile Ile Pro Arg Glu Ala Met Pro Ala 37 O 375 38O US 2010/0028334 A1 Feb. 4, 2010 51

- Continued

<210s, SEQ ID NO 18 &211s LENGTH: 321 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 18 Met Ser Llys Pro Ile Val Met Glu Arg Gly Val Llys Tyr Arg Asp Ala 1. 5 1O 15 Asp Llys Met Ala Lieu. Ile Pro Wall Lys Asn Val Ala Thr Glu Arg Glu 2O 25 3O Ala Lieu. Lieu. Arg Llys Pro Glu Trp Met Lys Ile Llys Lieu Pro Ala Asp 35 4 O 45 Ser Thr Arg Ile Glin Gly Ile Lys Ala Ala Met Arg Lys Asn Gly Lieu. SO 55 6 O His Ser Val Cys Glu Glu Ala Ser Cys Pro Asn Lieu Ala Glu. Cys Phe 65 70 7s 8O Asn His Gly Thr Ala Thr Phe Met Ile Leu Gly Ala Ile Cys Thr Arg 85 90 95 Arg Cys Pro Phe Cys Asp Wall Ala His Gly Arg Pro Wall Ala Pro Asp 1OO 105 11 O Ala Asn. Glu Pro Val Llys Lieu Ala Glin Thir Ile Ala Asp Met Ala Lieu 115 12 O 125 Arg Tyr Val Val Ile Thr Ser Val Asp Arg Asp Asp Lieu. Arg Asp Gly 13 O 135 14 O Gly Ala Gln His Phe Ala Asp Cys Ile Thir Ala Ile Arg Glu Lys Ser 145 150 155 160 Pro Glin Ile Lys Ile Glu Thir Lieu Val Pro Asp Phe Arg Gly Arg Met 1.65 17O 17s Asp Arg Ala Lieu. Asp Ile Lieu. Thir Ala Thr Pro Pro Asp Val Phe Asn 18O 185 19 O His Asn Lieu. Glu Asn Val Pro Arg Ile Tyr Arg Glin Val Arg Pro Gly 195 2OO 2O5 Ala Asp Tyr Asn Trp Ser Lieu Lys Lieu. Lieu. Glu Arg Phe Lys Glu Ala 21 O 215 22O His Pro Glu Ile Pro Thr Lys Ser Gly Lieu Met Val Gly Lieu. Gly Glu 225 23 O 235 24 O Thir Asn. Glu Glu Ile Ile Glu Val Met Arg Asp Lieu. Arg Arg His Gly 245 250 255 Val Thr Met Lieu. Thir Lieu. Gly Glin Tyr Lieu Gln Pro Ser Arg His His 26 O 265 27 O Lieu Pro Val Glin Arg Tyr Val Ser Pro Asp Glu Phe Asp Glu Met Lys 27s 28O 285 Ala Glu Ala Leu Ala Met Gly Phe Thr His Ala Ala Cys Gly Pro Phe 29 O 295 3 OO Val Arg Ser Ser Tyr His Ala Asp Lieu. Glin Ala Lys Gly Met Glu Val 3. OS 310 315 32O Lys

<210s, SEQ ID NO 19 &211s LENGTH: 42O 212. TYPE: PRT <213> ORGANISM: Escherichia coli US 2010/0028334 A1 Feb. 4, 2010 52

- Continued <4 OOs, SEQUENCE: 19 Met Pro His Ser Leu Phe Ser Thr Asp Thr Asp Lieu. Thir Ala Glu Asn 1. 5 1O 15 Lieu. Lieu. Arg Lieu Pro Ala Glu Phe Gly Cys Pro Val Trp Val Tyr Asp 2O 25 3O Ala Glin Ile Ile Arg Arg Glin Ile Ala Ala Lieu Lys Glin Phe Asp Wall 35 4 O 45 Val Arg Phe Ala Gln Lys Ala Cys Ser Asn. Ile His Ile Lieu. Arg Lieu SO 55 6 O Met Arg Glu Glin Gly Val Llys Val Asp Ser Val Ser Lieu. Gly Glu Ile 65 70 7s 8O Glu Arg Ala Lieu Ala Ala Gly Tyr Asn Pro Glin Thr His Pro Asp Asp 85 90 95 Ile Val Phe Thir Ala Asp Val Ile Asp Glin Ala Thr Lieu. Glu Arg Val 1OO 105 11 O Ser Glu Lieu. Glin Ile Pro Val Asn Ala Gly Ser Val Asp Met Lieu. Asp 115 12 O 125 Glin Lieu. Gly Glin Val Ser Pro Gly His Arg Val Trp Lieu. Arg Val Asn 13 O 135 14 O Pro Gly Phe Gly His Gly His Ser Gln Lys Thr Asn Thr Gly Gly Glu 145 150 155 160 ASn Ser Lys His Gly Ile Trp Tyr Thr Asp Lieu. Pro Ala Ala Lieu. Asp 1.65 17O 17s Val Ile Glin Arg His His Leu Gln Leu Val Gly Ile His Met His Ile 18O 185 19 O Gly Ser Gly Val Asp Tyr Ala His Lieu. Glu Glin Val Cys Gly Ala Met 195 2OO 2O5 Val Arg Glin Val Ile Glu Phe Gly Glin Asp Lieu. Glin Ala Ile Ser Ala 21 O 215 22O Gly Gly Gly Lieu. Ser Val Pro Tyr Glin Glin Gly Glu Glu Ala Val Asp 225 23 O 235 24 O Thr Glu. His Tyr Tyr Gly Lieu. Trp Asn Ala Ala Arg Glu Glin Ile Ala 245 250 255 Arg His Lieu. Gly His Pro Wall Lys Lieu. Glu Ile Glu Pro Gly Arg Phe 26 O 265 27 O Lieu Val Ala Glin Ser Gly Val Lieu. Ile Thr Glin Val Arg Ser Val Lys 27s 28O 285 Glin Met Gly Ser Arg His Phe Val Lieu Val Asp Ala Gly Phe Asn Asp 29 O 295 3 OO Lieu Met Arg Pro Ala Met Tyr Gly Ser Tyr His His Ile Ser Ala Leu 3. OS 310 315 32O Ala Ala Asp Gly Arg Ser Lieu. Glu. His Ala Pro Thr Val Glu Thr Val 3.25 330 335 Val Ala Gly Pro Leu. Cys Glu Ser Gly Asp Val Phe Thr Glin Glin Glu 34 O 345 35. O Gly Gly Asn Val Glu Thir Arg Ala Lieu Pro Glu Val Lys Ala Gly Asp 355 360 365 Tyr Lieu Val Lieu. His Asp Thr Gly Ala Tyr Gly Ala Ser Met Ser Ser 37 O 375 38O Asn Tyr Asn. Ser Arg Pro Lieu. Lieu Pro Glu Val Lieu. Phe Asp Asn Gly 385 390 395 4 OO US 2010/0028334 A1 Feb. 4, 2010 53

- Continued

Glin Ala Arg Lieu. Ile Arg Arg Arg Glin Thir Ile Glu Glu Lieu. Lieu Ala 4 OS 41O 415

Lieu. Glu Lieu. Lieu.

<210s, SEQ ID NO 2 O &211s LENGTH: 368 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 2O Met Ser Glu Thir Ala Lys Llys Val Ile Val Gly Met Ser Gly Gly Val 1. 5 1O 15 Asp Ser Ser Val Ser Ala Trp Lieu. Lieu. Glin Glin Glin Gly Tyr Glin Val 2O 25 3O Glu Gly Lieu. Phe Met Lys Asn Trp Glu Glu Asp Asp Gly Glu Glu Tyr 35 4 O 45 Cys Thr Ala Ala Ala Asp Lieu Ala Asp Ala Glin Ala Val Cys Asp Llys SO 55 6 O Lieu. Gly Ile Glu Lieu. His Thr Val Asn. Phe Ala Ala Glu Tyr Trp Asp 65 70 7s 8O Asn Val Phe Glu Lieu. Phe Lieu Ala Glu Tyr Lys Ala Gly Arg Thr Pro 85 90 95 Asn Pro Asp Ile Lieu. Cys Asn Lys Glu Ile Llys Phe Lys Ala Phe Lieu 1OO 105 11 O Glu Phe Ala Ala Glu Asp Lieu. Gly Ala Asp Tyr Ile Ala Thr Gly. His 115 12 O 125 Tyr Val Arg Arg Ala Asp Wall Asp Gly Lys Ser Arg Lieu. Lieu. Arg Gly 13 O 135 14 O Lieu. Asp Ser Asn Lys Asp Glin Ser Tyr Phe Lieu. Tyr Thr Lieu. Ser His 145 150 155 160 Glu Glin Ile Ala Glin Ser Lieu. Phe Pro Val Gly Glu Lieu. Glu Lys Pro 1.65 17O 17s Glin Val Arg Lys Ile Ala Glu Asp Lieu. Gly Lieu Val Thr Ala Lys Llys 18O 185 19 O Lys Asp Ser Thr Gly Ile Cys Phe Ile Gly Glu Arg Llys Phe Arg Glu 195 2OO 2O5 Phe Leu Gly Arg Tyr Lieu Pro Ala Glin Pro Gly Lys Ile Ile Thr Val 21 O 215 22O Asp Gly Asp Glu Ile Gly Glu. His Glin Gly Lieu Met Tyr His Thir Lieu 225 23 O 235 24 O Gly Glin Arg Lys Gly Lieu. Gly Ile Gly Gly. Thir Lys Glu Gly Thr Glu 245 250 255 Glu Pro Trp Tyr Val Val Asp Lys Asp Val Glu Asn. Asn. Ile Lieu Val 26 O 265 27 O Val Ala Glin Gly His Glu. His Pro Arg Lieu Met Ser Val Gly Lieu. Ile 27s 28O 285 Ala Glin Gln Lieu. His Trp Val Asp Arg Glu Pro Phe Thr Gly Thr Met 29 O 295 3 OO Arg Cys Thr Val Lys Thr Arg Tyr Arg Glin Thr Asp Ile Pro Cys Thr 3. OS 310 315 32O Val Lys Ala Lieu. Asp Asp Asp Arg Ile Glu Val Ile Phe Asp Glu Pro US 2010/0028334 A1 Feb. 4, 2010 54

- Continued

3.25 330 335 Val Ala Ala Val Thr Pro Gly Glin Ser Ala Val Phe Tyr Asn Gly Glu 34 O 345 35. O Val Cys Lieu. Gly Gly Gly Ile Ile Glu Glin Arg Lieu Pro Lieu Pro Val 355 360 365

<210s, SEQ ID NO 21 &211s LENGTH: 404 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 2 Met Lys Lieu Pro Ile Tyr Lieu. Asp Tyr Ser Ala Thr Thr Pro Val Asp 1. 5 1O 15 Pro Arg Val Ala Glu Lys Met Met Glin Phe Met Thr Met Asp Gly Thr 2O 25 3O Phe Gly Asn Pro Ala Ser Arg Ser His Arg Phe Gly Trp Glin Ala Glu 35 4 O 45 Glu Ala Val Asp Ile Ala Arg Asn Glin Ile Ala Asp Lieu Val Gly Ala SO 55 6 O Asp Pro Arg Glu Ile Val Phe Thir Ser Gly Ala Thr Glu Ser Asp Asn 65 70 7s 8O Lieu Ala Ile Lys Gly Ala Ala Asn. Phe Tyr Glin Llys Lys Gly Llys His 85 90 95 Ile Ile Thir Ser Llys Thr Glu. His Lys Ala Val Lieu. Asp Thir Cys Arg 1OO 105 11 O Glin Lieu. Glu Arg Glu Gly Phe Glu Val Thir Tyr Lieu Ala Pro Glin Arg 115 12 O 125 Asn Gly Ile Ile Asp Lieu Lys Glu Lieu. Glu Ala Ala Met Arg Asp Asp 13 O 135 14 O Thir Ile Leu Val Ser Ile Met His Val Asn Asn Glu Ile Gly Val Val 145 150 155 160 Glin Asp Ile Ala Ala Ile Gly Glu Met Cys Arg Ala Arg Gly Ile Ile 1.65 17O 17s Tyr His Val Asp Ala Thr Glin Ser Val Gly Lys Lieu Pro Ile Asp Lieu. 18O 185 19 O Ser Gln Leu Lys Val Asp Leu Met Ser Phe Ser Gly His Lys Ile Tyr 195 2OO 2O5 Gly Pro Lys Gly Ile Gly Ala Lieu. Tyr Val Arg Arg Llys Pro Arg Val 21 O 215 22O Arg Ile Glu Ala Gln Met His Gly Gly Gly His Glu Arg Gly Met Arg 225 23 O 235 24 O Ser Gly. Thir Lieu Pro Val His Glin Ile Val Gly Met Gly Glu Ala Tyr 245 250 255 Arg Ile Ala Lys Glu Glu Met Ala Thr Glu Met Glu Arg Lieu. Arg Gly 26 O 265 27 O Lieu. Arg Asn Arg Lieu. Trp Asn Gly Ile Lys Asp Ile Glu Glu Val Tyr 27s 28O 285 Lieu. Asn Gly Asp Lieu. Glu. His Gly Ala Pro Asn. Ile Lieu. Asn Val Ser 29 O 295 3 OO Phe Asn Tyr Val Glu Gly Glu Ser Lieu. Ile Met Ala Lieu Lys Asp Lieu. 3. OS 310 315 32O US 2010/0028334 A1 Feb. 4, 2010 55

- Continued Ala Val Ser Ser Gly Ser Ala Cys Thr Ser Ala Ser Lieu. Glu Pro Ser 3.25 330 335 Tyr Val Lieu. Arg Ala Lieu. Gly Lieu. Asn Asp Glu Lieu Ala His Ser Ser 34 O 345 35. O Ile Arg Phe Ser Lieu. Gly Arg Phe Thr Thr Glu Glu Glu Ile Asp Tyr 355 360 365 Thir Ile Glu Lieu Val Arg Llys Ser Ile Gly Arg Lieu. Arg Asp Lieu. Ser 37 O 375 38O Pro Lieu. Trp Glu Met Tyr Lys Glin Gly Val Asp Lieu. Asn. Ser Ile Glu 385 390 395 4 OO Trp Ala His His

<210s, SEQ ID NO 22 &211s LENGTH: 513 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 22 Met Glin Lieu. Asn. Ser Thr Glu Ile Ser Glu Lieu. Ile Lys Glin Arg Ile 1. 5 1O 15 Ala Glin Phe Asin Val Val Ser Glu Ala His Asn Glu Gly Thr Ile Val 2O 25 3O Ser Val Ser Asp Gly Val Ile Arg Ile His Gly Lieu Ala Asp Cys Met 35 4 O 45 Glin Gly Glu Met Ile Ser Lieu Pro Gly Asn Arg Tyr Ala Ile Ala Lieu. SO 55 6 O Asn Lieu. Glu Arg Asp Ser Val Gly Ala Val Val Met Gly Pro Tyr Ala 65 70 7s 8O Asp Lieu Ala Glu Gly Met Llys Val Lys Cys Thr Gly Arg Ile Lieu. Glu 85 90 95 Val Pro Val Gly Arg Gly Lieu. Lieu. Gly Arg Val Val Asn. Thir Lieu. Gly 1OO 105 11 O Ala Pro Ile Asp Gly Lys Gly Pro Lieu. Asp His Asp Gly Phe Ser Ala 115 12 O 125 Val Glu Ala Ile Ala Pro Gly Val Ile Glu Arg Glin Ser Val Asp Glin 13 O 135 14 O Pro Val Glin Thr Gly Tyr Lys Ala Val Asp Ser Met Ile Pro Ile Gly 145 150 155 160 Arg Gly Glin Arg Glu Lieu. Ile Ile Gly Asp Arg Glin Thr Gly Lys Thr 1.65 17O 17s Ala Lieu Ala Ile Asp Ala Ile Ile Asin Glin Arg Asp Ser Gly Ile Llys 18O 185 19 O Cys Ile Tyr Val Ala Ile Gly Gln Lys Ala Ser Thr Ile Ser Asn Val 195 2OO 2O5 Val Arg Llys Lieu. Glu Glu. His Gly Ala Lieu Ala Asn. Thir Ile Val Val 21 O 215 22O Val Ala Thir Ala Ser Glu Ser Ala Ala Lieu. Glin Tyr Lieu Ala Pro Tyr 225 23 O 235 24 O Ala Gly Cys Ala Met Gly Glu Tyr Phe Arg Asp Arg Gly Glu Asp Ala 245 250 255 Lieu. Ile Ile Tyr Asp Asp Lieu. Ser Lys Glin Ala Val Ala Tyr Arg Glin 26 O 265 27 O US 2010/0028334 A1 Feb. 4, 2010 56

- Continued Ile Ser Lieu. Lieu. Lieu. Arg Arg Pro Pro Gly Arg Glu Ala Phe Pro Gly 27s 28O 285 Asp Val Phe Tyr Lieu. His Ser Arg Lieu. Lieu. Glu Arg Ala Ala Arg Val 29 O 295 3 OO Asn Ala Glu Tyr Val Glu Ala Phe Thir Lys Gly Glu Wall Lys Gly Lys 3. OS 310 315 32O Thr Gly Ser Lieu. Thir Ala Leu Pro Ile Ile Glu Thr Glin Ala Gly Asp 3.25 330 335 Val Ser Ala Phe Val Pro Thr Asn Val Ile Ser Ile Thr Asp Gly Glin 34 O 345 35. O Ile Phe Lieu. Glu Thir Asn Lieu. Phe Asn Ala Gly Ile Arg Pro Ala Val 355 360 365 Asn Pro Gly Ile Ser Val Ser Arg Val Gly Gly Ala Ala Glin Thir Lys 37 O 375 38O Ile Met Lys Llys Lieu. Ser Gly Gly Ile Arg Thr Ala Lieu Ala Glin Tyr 385 390 395 4 OO Arg Glu Lieu Ala Ala Phe Ser Glin Phe Ala Ser Asp Lieu. Asp Asp Ala 4 OS 41O 415 Thir Arg Lys Glin Lieu. Asp His Gly Glin Llys Val Thr Glu Lieu. Lieu Lys 42O 425 43 O Gln Lys Glin Tyr Ala Pro Met Ser Val Ala Glin Glin Ser Leu Val Lieu. 435 44 O 445 Phe Ala Ala Glu Arg Gly Tyr Lieu Ala Asp Val Glu Lieu. Ser Lys Ile 450 45.5 460 Gly Ser Phe Glu Ala Ala Lieu. Lieu Ala Tyr Val Asp Arg Asp His Ala 465 470 47s 48O Pro Leu Met Glin Glu Ile Asn Gln Thr Gly Gly Tyr Asn Asp Glu Ile 485 490 495 Glu Gly Lys Lieu Lys Gly Ile Lieu. Asp Ser Phe Lys Ala Thr Glin Ser SOO 505 51O Trp

<210s, SEQ ID NO 23 &211s LENGTH: 218 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 23 Met Ser Asp Asin Asp Glu Lieu. Glin Glin Ile Ala His Lieu. Arg Arg Glu 1. 5 1O 15 Tyr Thr Lys Gly Gly Lieu. Arg Arg Arg Asp Lieu Pro Ala Asp Pro Lieu. 2O 25 3O Thir Lieu. Phe Glu Arg Trp Lieu. Ser Glin Ala Cys Glu Ala Lys Lieu Ala 35 4 O 45 Asp Pro Thr Ala Met Val Val Ala Thr Val Asp Glu. His Gly Glin Pro SO 55 6 O Tyr Glin Arg Ile Val Lieu Lleu Lys His Tyr Asp Glu Lys Gly Met Val 65 70 7s 8O Phe Tyr Thr Asn Lieu. Gly Ser Arg Lys Ala His Glin Ile Glu Asn. Asn 85 90 95 Pro Arg Val Ser Lieu. Leu Phe Pro Trp His Thr Lieu. Glu Arg Glin Val 1OO 105 11 O US 2010/0028334 A1 Feb. 4, 2010 57

- Continued Met Val Ile Gly Lys Ala Glu Arg Lieu. Ser Thr Lieu. Glu Val Met Lys 115 12 O 125 Tyr Phe His Ser Arg Pro Arg Asp Ser Glin Ile Gly Ala Trp Val Ser 13 O 135 14 O Lys Glin Ser Ser Arg Ile Ser Ala Arg Gly Ile Lieu. Glu Ser Llys Phe 145 150 155 160 Lieu. Glu Lieu Lys Glin Llys Phe Glin Glin Gly Glu Val Pro Lieu Pro Ser 1.65 17O 17s Phe Trp Gly Gly Phe Arg Val Ser Lieu. Glu Glin Ile Glu Phe Trp Glin 18O 185 19 O Gly Gly Glu. His Arg Lieu. His Asp Arg Phe Lieu. Tyr Glin Arg Glu Asn 195 2OO 2O5 Asp Ala Trp Llys Ile Asp Arg Lieu Ala Pro 21 O 215

<210s, SEQ ID NO 24 &211s LENGTH: 226 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 24 Met Ile Asin Val Glin Asn Val Ser Lys Thr Phe Ile Lieu. His Glin Glin 1. 5 1O 15 ASn Gly Val Arg Lieu. Pro Val Lieu. ASn Arg Ala Ser Lieu. Thr Val Asn 2O 25 3O Ala Gly Glu. Cys Val Val Lieu. His Gly His Ser Gly Ser Gly Lys Ser 35 4 O 45 Thir Lieu. Lieu. Arg Ser Lieu. Tyr Ala Asn Tyr Lieu Pro Asp Glu Gly Glin SO 55 6 O Ile Glin Ile Llys His Gly Asp Glu Trp Val Asp Lieu Val Thir Ala Pro 65 70 7s 8O Ala Arg Llys Val Val Glu Ile Arg Llys Thir Thr Val Gly Trp Val Ser 85 90 95 Glin Phe Lieu. Arg Val Ile Pro Arg Ile Ser Ala Lieu. Glu Val Val Met 1OO 105 11 O Glin Pro Lieu. Lieu. Asp Thr Gly Val Pro Arg Glu Ala Cys Ala Ala Lys 115 12 O 125 Ala Ala Arg Lieu. Lieu. Thir Arg Lieu. Asn Val Pro Glu Arg Lieu. Trp His 13 O 135 14 O Lieu Ala Pro Ser Thr Phe Ser Gly Gly Glu Gln Glin Arg Val Asn Ile 145 150 155 160 Ala Arg Gly Phe Ile Val Asp Tyr Pro Ile Lieu Lleu Lieu. Asp Glu Pro 1.65 17O 17s Thir Ala Ser Lieu. Asp Ala Lys Asn. Ser Ala Ala Val Val Glu Lieu. Ile 18O 185 19 O Arg Glu Ala Lys Thr Arg Gly Ala Ala Ile Val Gly Ile Phe His Asp 195 2OO 2O5 Glu Ala Val Arg Asn Asp Wall Ala Asp Arg Lieu. His Pro Met Gly Ala 21 O 215 22O

Ser Ser 225

<210s, SEQ ID NO 25 US 2010/0028334 A1 Feb. 4, 2010 58

- Continued

&211s LENGTH: 439 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 25 Met Ser Glin Ala Lys Ser Asn Llys Met Gly Val Val Glin Lieu. Thir Ile 1. 5 1O 15 Lieu. Thir Met Val Asn Met Met Gly Ser Gly Ile Ile Met Leu Pro Thr 2O 25 3O Lys Lieu Ala Glu Val Gly. Thir Ile Ser Ile Ile Ser Trp Leu Val Thr 35 4 O 45 Ala Val Gly Ser Met Ala Lieu Ala Trp Ala Phe Ala Lys Cys Gly Met SO 55 6 O Phe Ser Arg Llys Ser Gly Gly Met Gly Gly Tyr Ala Glu Tyr Ala Phe 65 70 7s 8O Gly Lys Ser Gly Asn Phe Met Ala Asn Tyr Thr Tyr Gly Val Ser Lieu. 85 90 95 Lieu. Ile Ala Asn. Wall Ala Ile Ala Ile Ser Ala Val Gly Tyr Gly Thr 1OO 105 11 O Glu Lieu. Lieu. Gly Ala Ser Lieu. Ser Pro Val Glin Ile Gly Lieu Ala Thr 115 12 O 125 Ile Gly Val Lieu. Trp Ile Cys Thr Val Ala Asn. Phe Gly Gly Ala Arg 13 O 135 14 O Ile Thr Gly Glin Ile Ser Ser Ile Thr Val Trp Gly Val Ile Ile Pro 145 150 155 160 Val Val Gly Lieu. Cys Ile Ile Gly Trp Phe Trp Phe Ser Pro Thr Lieu. 1.65 17O 17s Tyr Val Asp Ser Trp Asn Pro His His Ala Pro Phe Phe Ser Ala Val 18O 185 19 O Gly Ser Ser Ile Ala Met Thr Lieu. Trp Ala Phe Leu Gly Lieu. Glu Ser 195 2OO 2O5 Ala Cys Ala Asn. Thir Asp Val Val Glu Asn Pro Glu Arg Asn Val Pro 21 O 215 22O Ile Ala Val Lieu. Gly Gly Thr Lieu. Gly Ala Ala Val Ile Tyr Ile Val 225 23 O 235 24 O Ser Thr Asn Val Ile Ala Gly Ile Val Pro Asn Met Glu Lieu Ala Asn 245 250 255 Ser Thr Ala Pro Phe Gly Lieu Ala Phe Ala Gln Met Phe Thr Pro Glu 26 O 265 27 O Val Gly Llys Val Ile Met Ala Leu Met Val Met Ser Cys Cys Gly Ser 27s 28O 285 Lieu. Leu Gly Trp Glin Phe Thr Ile Ala Glin Val Phe Lys Ser Ser Ser 29 O 295 3 OO Asp Glu Gly Tyr Phe Pro Lys Ile Phe Ser Arg Val Thr Llys Val Asp 3. OS 310 315 32O Ala Pro Val Glin Gly Met Lieu. Thir Ile Val Ile Ile Glin Ser Gly Lieu. 3.25 330 335

Ala Lieu Met Thir Ile Ser Pro Ser Lieu. Asn. Ser Glin Phe Asn. Wall Lieu. 34 O 345 35. O Val Asn Lieu Ala Val Val Thr Asn Ile Ile Pro Tyr Ile Leu Ser Met 355 360 365 Ala Ala Lieu Val Ile Ile Glin Llys Val Ala Asn Val Pro Pro Ser Lys US 2010/0028334 A1 Feb. 4, 2010 59

- Continued

37 O 375 38O Ala Lys Val Ala Asn Phe Val Ala Phe Val Gly Ala Met Tyr Ser Phe 385 390 395 4 OO Tyr Ala Leu Tyr Ser Ser Gly Glu Glu Ala Met Leu Tyr Gly Ser Ile 4 OS 41O 415 Val Thr Phe Leu Gly Trp Thr Lieu. Tyr Gly Lieu Val Ser Pro Arg Phe 42O 425 43 O Glu Lieu Lys Asn Llys His Gly 435

<210s, SEQ ID NO 26 &211s LENGTH: 219 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 26 Met Thr Glin Asp Glu Lieu Lys Lys Ala Val Gly Trp Ala Ala Lieu. Glin 1. 5 1O 15 Tyr Val Glin Pro Gly. Thir Ile Val Gly Val Gly Thr Gly Ser Thr Ala 2O 25 3O Ala His Phe Ile Asp Ala Lieu. Gly. Thir Met Lys Gly Glin Ile Glu Gly 35 4 O 45 Ala Val Ser Ser Ser Asp Ala Ser Thr Glu Lys Lieu Lys Ser Lieu. Gly SO 55 6 O Ile His Val Phe Asp Lieu. Asn. Glu Val Asp Ser Lieu. Gly Ile Tyr Val 65 70 7s 8O Asp Gly Ala Asp Glu Ile Asn Gly. His Met Gln Met Ile Lys Gly Gly 85 90 95 Gly Ala Ala Lieu. Thir Arg Glu Lys Ile Ile Ala Ser Val Ala Glu Lys 1OO 105 11 O Phe Ile Cys Ile Ala Asp Ala Ser Lys Glin Val Asp Ile Lieu. Gly Lys 115 12 O 125 Phe Pro Leu Pro Val Glu Val Ile Pro Met Ala Arg Ser Ala Val Ala 13 O 135 14 O Arg Glin Lieu Val Llys Lieu. Gly Gly Arg Pro Glu Tyr Arg Glin Gly Val 145 150 155 160 Val Thr Asp Asn Gly Asn Val Ile Lieu. Asp Wal His Gly Met Glu Ile 1.65 17O 17s Lieu. Asp Pro Ile Ala Met Glu Asn Ala Ile Asn Ala Ile Pro Gly Val 18O 185 19 O Val Thr Val Gly Lieu. Phe Ala Asn Arg Gly Ala Asp Wall Ala Lieu. Ile 195 2OO 2O5 Gly Thr Pro Asp Gly Val Lys Thr Ile Val Lys 21 O 215

<210s, SEQ ID NO 27 &211s LENGTH: 405 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 27 Met Ser Ser Val Asp Ile Lieu Val Pro Asp Lieu Pro Glu Ser Val Ala 1. 5 1O 15 Asp Ala Thr Val Ala Thir Trp His Llys Llys Pro Gly Asp Ala Val Val US 2010/0028334 A1 Feb. 4, 2010 60

- Continued

2O 25 3O Arg Asp Glu Val Lieu Val Glu Ile Glu Thir Asp Llys Val Val Lieu. Glu 35 4 O 45 Val Pro Ala Ser Ala Asp Gly Ile Lieu. Asp Ala Val Lieu. Glu Asp Glu SO 55 6 O Gly. Thir Thr Val Thir Ser Arg Glin Ile Lieu. Gly Arg Lieu. Arg Glu Gly 65 70 7s 8O Asn Ser Ala Gly Lys Glu Thir Ser Ala Lys Ser Glu Glu Lys Ala Ser 85 90 95 Thr Pro Ala Glin Arg Glin Glin Ala Ser Lieu. Glu Glu Glin Asn. Asn Asp 1OO 105 11 O Ala Lieu. Ser Pro Ala Ile Arg Arg Lieu. Lieu Ala Glu. His Asn Lieu. Asp 115 12 O 125 Ala Ser Ala Ile Lys Gly Thr Gly Val Gly Gly Arg Lieu. Thir Arg Glu 13 O 135 14 O Asp Val Glu Lys His Lieu Ala Lys Ala Pro Ala Lys Glu Ser Ala Pro 145 150 155 160 Ala Ala Ala Ala Pro Ala Ala Glin Pro Ala Lieu Ala Ala Arg Ser Glu 1.65 17O 17s Lys Arg Val Pro Met Thr Arg Lieu. Arg Lys Arg Val Ala Glu Arg Lieu. 18O 185 19 O Lieu. Glu Ala Lys Asn Ser Thr Ala Met Lieu. Thir Thr Phe Asin Glu Val 195 2OO 2O5 Asn Met Llys Pro Ile Met Asp Lieu. Arg Lys Glin Tyr Gly Glu Ala Phe 21 O 215 22O Glu Lys Arg His Gly Ile Arg Lieu. Gly Phe Met Ser Phe Tyr Val Lys 225 23 O 235 24 O Ala Val Val Glu Ala Lieu Lys Arg Tyr Pro Glu Val Asn Ala Ser Ile 245 250 255 Asp Gly Asp Asp Val Val Tyr His Asn Tyr Phe Asp Wal Ser Met Ala 26 O 265 27 O Val Ser Thr Pro Arg Gly Lieu Val Thr Pro Val Lieu. Arg Asp Val Asp 27s 28O 285 Thir Lieu. Gly Met Ala Asp Ile Glu Lys Lys Ile Lys Glu Lieu Ala Val 29 O 295 3 OO Lys Gly Arg Asp Gly Llys Lieu. Thr Val Glu Asp Lieu. Thr Gly Gly Asn 3. OS 310 315 32O Phe Thir Ile Thr Asn Gly Gly Val Phe Gly Ser Leu Met Ser Thr Pro 3.25 330 335 Ile Ile Asn Pro Pro Glin Ser Ala Ile Lieu. Gly Met His Ala Ile Llys 34 O 345 35. O Asp Arg Pro Met Ala Val Asn Gly Glin Val Glu Ile Leu Pro Met Met 355 360 365 Tyr Lieu Ala Lieu. Ser Tyr Asp His Arg Lieu. Ile Asp Gly Arg Glu Ser 37 O 375 38O Val Gly Phe Lieu Val Thir Ile Lys Glu Lieu. Lieu. Glu Asp Pro Thr Arg 385 390 395 4 OO Lieu. Lieu. Lieu. Asp Wall 4 OS

<210s, SEQ ID NO 28 US 2010/0028334 A1 Feb. 4, 2010 61

- Continued

&211s LENGTH: 127 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 28 Met Lieu. His Glin Glin Arg Asn. Glin His Ala Arg Lieu. Ile Pro Val Glu 1. 5 1O 15 Lieu. Tyr Met Ser Asp Llys Ile Ile His Lieu. Thir Asp Asp Ser Phe Asp 2O 25 3O Thir Asp Val Lieu Lys Ala Asp Gly Ala Ile Lieu Val Asp Phe Trp Ala 35 4 O 45 Glu Trp Cys Gly Pro Cys Llys Met Ile Ala Pro Ile Lieu. Asp Glu Ile SO 55 6 O Ala Asp Glu Tyr Glin Gly Llys Lieu. Thr Val Ala Lys Lieu. Asn. Ile Asp 65 70 7s 8O Gln Asn Pro Gly Thr Ala Pro Llys Tyr Gly Ile Arg Gly Ile Pro Thr 85 90 95 Lieu. Lieu. Lieu. Phe Lys Asn Gly Glu Val Ala Ala Thr Llys Val Gly Ala 1OO 105 11 O Lieu. Ser Lys Gly Glin Lieu Lys Glu Phe Lieu. Asp Ala Asn Lieu Ala 115 12 O 125

<210s, SEQ ID NO 29 &211s LENGTH: 95 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 29 Met Lieu. His Thr Lieu. His Arg Ser Pro Trp Lieu. Thr Asp Phe Ala Ala 1. 5 1O 15 Lieu. Lieu. Arg Lieu Lleu Ser Glu Gly Asp Glu Lieu Lleu Lleu Lieu. Glin Asp 2O 25 3O Gly Val Thir Ala Ala Val Asp Gly Asn Arg Tyr Lieu. Glu Ser Lieu. Arg 35 4 O 45 Asn Ala Pro Ile Llys Val Tyr Ala Lieu. Asn. Glu Asp Lieu. Ile Ala Arg SO 55 6 O Gly Lieu. Thr Gly Glin Ile Ser Asn Asp Ile Ile Lieu. Ile Asp Tyr Thr 65 70 7s 8O Asp Phe Val Arg Lieu. Thr Val Llys His Pro Ser Gln Met Ala Trp 85 90 95

<210s, SEQ ID NO 3 O &211s LENGTH: 109 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 30 Met Lieu. Ile Phe Glu Gly Lys Glu Ile Glu Thir Asp Thr Glu Gly Tyr 1. 5 1O 15 Lieu Lys Glu Ser Ser Glin Trp Ser Glu Pro Lieu Ala Val Val Ile Ala 2O 25 3O Glu Asn. Glu Gly Ile Ser Lieu. Ser Pro Glu. His Trp Glu Val Val Arg 35 4 O 45 Phe Val Arg Asp Phe Tyr Lieu. Glu Phe Asn Thr Ser Pro Ala Ile Arg SO 55 6 O US 2010/0028334 A1 Feb. 4, 2010 62

- Continued Met Lieu Val Lys Ala Met Ala Asn Llys Phe Gly Glu Glu Lys Gly Asn 65 70 7s 8O Ser Arg Tyr Lieu. Tyr Arg Lieu. Phe Pro Lys Gly Pro Ala Lys Glin Ala 85 90 95 Thir Lys Ile Ala Gly Lieu Pro Llys Pro Wall Lys Cys Ile 1OO 105

<210s, SEQ ID NO 31 &211s LENGTH: 251 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 31 Met Val Asp Llys Ser Glin Glu Thir Thr His Phe Gly Phe Glin Thr Val 1. 5 1O 15 Ala Lys Glu Glin Lys Ala Asp Met Val Ala His Val Phe His Ser Val 2O 25 3O Ala Ser Lys Tyr Asp Wal Met Asn Asp Lieu Met Ser Phe Gly Ile His 35 4 O 45 Arg Lieu. Trp Lys Arg Phe Thir Ile Asp Cys Ser Gly Val Arg Arg Gly SO 55 6 O Glin Thr Val Lieu. Asp Lieu Ala Gly Gly. Thr Gly Asp Lieu. Thir Ala Lys 65 70 7s 8O Phe Ser Arg Lieu Val Gly Glu. Thr Gly Llys Val Val Lieu. Ala Asp Ile 85 90 95 Asn Glu Ser Met Pro Llys Met Gly Arg Glu Lys Lieu. Arg Asn. Ile Gly 1OO 105 11 O Val Ile Gly Asn Val Glu Tyr Val Glin Ala Asn Ala Glu Ala Lieu Pro 115 12 O 125 Phe Pro Asp Asn Thr Phe Asp Cys Ile Thr Ile Ser Phe Gly Lieu. Arg 13 O 135 14 O Asn Val Thir Asp Lys Asp Lys Ala Lieu. Arg Ser Met Tyr Arg Val Lieu 145 150 155 160 Llys Pro Gly Gly Arg Lieu. Lieu Val Lieu. Glu Phe Ser Llys Pro Ile Ile 1.65 17O 17s Glu Pro Leu Ser Lys Ala Tyr Asp Ala Tyr Ser Phe His Val Leu Pro 18O 185 19 O Arg Ile Gly Ser Lieu Val Ala Asn Asp Ala Asp Ser Tyr Arg Tyr Lieu 195 2OO 2O5 Ala Glu Ser Ile Arg Met His Pro Asp Glin Asp Thir Lieu Lys Ala Met 21 O 215 22O Met Glin Asp Ala Gly Phe Glu Ser Val Asp Tyr Tyr Asn Lieu. Thir Ala 225 23 O 235 24 O Gly Val Val Ala Lieu. His Arg Gly Tyr Llys Phe 245 250

<210s, SEQ ID NO 32 &211s LENGTH: 392 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 32 Met Ser Val Ile Ile Val Gly Gly Gly Met Ala Gly Ala Thr Lieu Ala 1. 5 1O 15 US 2010/0028334 A1 Feb. 4, 2010 63

- Continued Lieu Ala Ile Ser Arg Lieu. Ser His Gly Ala Lieu Pro Val His Lieu. Ile 2O 25 3O Glu Ala Thir Ala Pro Glu Ser His Ala His Pro Gly Phe Asp Gly Arg 35 4 O 45 Ala Ile Ala Lieu Ala Ala Gly Thr Cys Glin Glin Lieu Ala Arg Ile Gly SO 55 6 O Val Trp Glin Ser Leu Ala Asp Cys Ala Thr Ala Ile Thr Thr Val His 65 70 7s 8O Val Ser Asp Arg Gly. His Ala Gly Phe Val Thir Lieu Ala Ala Glu Asp 85 90 95 Tyr Glin Lieu Ala Ala Lieu. Gly Glin Val Val Glu Lieu. His Asn Val Gly 1OO 105 11 O Glin Arg Lieu. Phe Ala Lieu. Lieu. Arg Lys Ala Pro Gly Val Thir Lieu. His 115 12 O 125 Cys Pro Asp Arg Val Ala Asn. Wall Ala Arg Thr Glin Ser His Val Glu 13 O 135 14 O Val Thir Lieu. Glu Ser Gly Glu Thir Lieu. Thr Gly Arg Val Lieu Val Ala 145 150 155 160 Ala Asp Gly Thr His Ser Ala Lieu Ala Thr Ala Cys Gly Val Asp Trip 1.65 17O 17s Glin Glin Glu Pro Tyr Glu Gln Lieu Ala Val Ile Ala Asn. Wall Ala Thr 18O 185 19 O Ser Val Ala His Glu Gly Arg Ala Phe Glu Arg Phe Thr Glin His Gly 195 2OO 2O5 Pro Lieu Ala Met Lieu Pro Met Ser Asp Gly Arg Cys Ser Lieu Val Trp 21 O 215 22O Cys His Pro Lieu. Glu Arg Arg Glu Glu Val Lieu. Ser Trp Ser Asp Glu 225 23 O 235 24 O Llys Phe Cys Arg Glu Lieu. Glin Ser Ala Phe Gly Trp Arg Lieu. Gly Lys 245 250 255 Ile Thr His Ala Gly Lys Arg Ser Ala Tyr Pro Lieu Ala Lieu. Thir His 26 O 265 27 O Ala Ala Arg Ser Ile Thr His Arg Thr Val Lieu Val Gly Asn Ala Ala 27s 28O 285 Gln Thr Lieu. His Pro Ile Ala Gly Glin Gly Phe Asn Lieu. Gly Met Arg 29 O 295 3 OO Asp Wal Met Ser Lieu Ala Glu Thir Lieu. Thr Glin Ala Glin Glu Arg Gly 3. OS 310 315 32O Glu Asp Met Gly Asp Tyr Gly Val Lieu. Cys Arg Tyr Glin Glin Arg Arg 3.25 330 335 Glin Ser Asp Arg Glu Ala Thir Ile Gly Val Thr Asp Ser Lieu Val His 34 O 345 35. O Lieu. Phe Ala Asn Arg Trp Ala Pro Lieu Val Val Gly Arg Asn. Ile Gly 355 360 365 Lieu Met Thr Met Glu Lieu Phe Thr Pro Ala Arg Asp Val Leu Ala Glin 37 O 375 38O Arg Thr Lieu. Gly Trp Val Ala Arg 385 390

<210s, SEQ ID NO 33 &211s LENGTH: 513 212. TYPE: PRT US 2010/0028334 A1 Feb. 4, 2010 64

- Continued

<213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 33 Met Glin Lieu. Asn. Ser Thr Glu Ile Ser Glu Lieu. Ile Lys Glin Arg Ile 1. 5 1O 15 Ala Glin Phe Asin Val Val Ser Glu Ala His Asn Glu Gly Thr Ile Val 2O 25 3O Ser Val Ser Asp Gly Val Ile Arg Ile His Gly Lieu Ala Asp Cys Met 35 4 O 45 Glin Gly Glu Met Ile Ser Lieu Pro Gly Asn Arg Tyr Ala Ile Ala Lieu. SO 55 6 O Asn Lieu. Glu Arg Asp Ser Val Gly Ala Val Val Met Gly Pro Tyr Ala 65 70 7s 8O Asp Lieu Ala Glu Gly Met Llys Val Lys Cys Thr Gly Arg Ile Lieu. Glu 85 90 95 Val Pro Val Gly Arg Gly Lieu. Lieu. Gly Arg Val Val Asn. Thir Lieu. Gly 1OO 105 11 O Ala Pro Ile Asp Gly Lys Gly Pro Lieu. Asp His Asp Gly Phe Ser Ala 115 12 O 125 Val Glu Ala Ile Ala Pro Gly Val Ile Glu Arg Glin Ser Val Asp Glin 13 O 135 14 O Pro Val Glin Thr Gly Tyr Lys Ala Val Asp Ser Met Ile Pro Ile Gly 145 150 155 160 Arg Gly Glin Arg Glu Lieu. Ile Ile Gly Asp Arg Glin Thr Gly Lys Thr 1.65 17O 17s Ala Lieu Ala Ile Asp Ala Ile Ile Asin Glin Arg Asp Ser Gly Ile Llys 18O 185 19 O Cys Ile Tyr Val Ala Ile Gly Gln Lys Ala Ser Thr Ile Ser Asn Val 195 2OO 2O5 Val Arg Llys Lieu. Glu Glu. His Gly Ala Lieu Ala Asn. Thir Ile Val Val 21 O 215 22O Val Ala Thir Ala Ser Glu Ser Ala Ala Lieu. Glin Tyr Lieu Ala Pro Tyr 225 23 O 235 24 O Ala Gly Cys Ala Met Gly Glu Tyr Phe Arg Asp Arg Gly Glu Asp Ala 245 250 255 Lieu. Ile Ile Tyr Asp Asp Lieu. Ser Lys Glin Ala Val Ala Tyr Arg Glin 26 O 265 27 O Ile Ser Lieu. Lieu. Lieu. Arg Arg Pro Pro Gly Arg Glu Ala Phe Pro Gly 27s 28O 285 Asp Val Phe Tyr Lieu. His Ser Arg Lieu. Lieu. Glu Arg Ala Ala Arg Val 29 O 295 3 OO Asn Ala Glu Tyr Val Glu Ala Phe Thir Lys Gly Glu Wall Lys Gly Lys 3. OS 310 315 32O Thr Gly Ser Lieu. Thir Ala Leu Pro Ile Ile Glu Thr Glin Ala Gly Asp 3.25 330 335 Val Ser Ala Phe Val Pro Thr Asn Val Ile Ser Ile Thr Asp Gly Glin 34 O 345 35. O Ile Phe Lieu. Glu Thir Asn Lieu. Phe Asn Ala Gly Ile Arg Pro Ala Val 355 360 365 Asn Pro Gly Ile Ser Val Ser Arg Val Gly Gly Ala Ala Glin Thir Lys 37 O 375 38O US 2010/0028334 A1 Feb. 4, 2010 65

- Continued Ile Met Lys Llys Lieu. Ser Gly Gly Ile Arg Thr Ala Lieu Ala Glin Tyr 385 390 395 4 OO Arg Glu Lieu Ala Ala Phe Ser Glin Phe Ala Ser Asp Lieu. Asp Asp Ala 4 OS 41O 415 Thir Arg Lys Glin Lieu. Asp His Gly Glin Llys Val Thr Glu Lieu. Lieu Lys 42O 425 43 O Gln Lys Glin Tyr Ala Pro Met Ser Val Ala Glin Glin Ser Leu Val Lieu. 435 44 O 445 Phe Ala Ala Glu Arg Gly Tyr Lieu Ala Asp Val Glu Lieu. Ser Lys Ile 450 45.5 460 Gly Ser Phe Glu Ala Ala Lieu. Lieu Ala Tyr Val Asp Arg Asp His Ala 465 470 47s 48O Pro Leu Met Glin Glu Ile Asn Gln Thr Gly Gly Tyr Asn Asp Glu Ile 485 490 495 Glu Gly Lys Lieu Lys Gly Ile Lieu. Asp Ser Phe Lys Ala Thr Glin Ser SOO 505 51O Trp

<210s, SEQ ID NO 34 &211s LENGTH: 392 212. TYPE: PRT <213> ORGANISM: Escherichia coli

< 4 OO > SEQUENCE: 34 Met Ser Val Ile Ile Val Gly Gly Gly Met Ala Gly Ala Thr Lieu Ala 1. 5 1O 15 Lieu Ala Ile Ser Arg Lieu. Ser His Gly Ala Lieu Pro Val His Lieu. Ile 2O 25 3O Glu Ala Thir Ala Pro Glu Ser His Ala His Pro Gly Phe Asp Gly Arg 35 4 O 45 Ala Ile Ala Lieu Ala Ala Gly Thr Cys Glin Glin Lieu Ala Arg Ile Gly SO 55 6 O Val Trp Glin Ser Leu Ala Asp Cys Ala Thr Ala Ile Thr Thr Val His 65 70 7s 8O Val Ser Asp Arg Gly. His Ala Gly Phe Val Thir Lieu Ala Ala Glu Asp 85 90 95 Tyr Glin Lieu Ala Ala Lieu. Gly Glin Val Val Glu Lieu. His Asn Val Gly 1OO 105 11 O Glin Arg Lieu. Phe Ala Lieu. Lieu. Arg Lys Ala Pro Gly Val Thir Lieu. His 115 12 O 125 Cys Pro Asp Arg Val Ala Asn. Wall Ala Arg Thr Glin Ser His Val Glu 13 O 135 14 O Val Thir Lieu. Glu Ser Gly Glu Thir Lieu. Thr Gly Arg Val Lieu Val Ala 145 150 155 160 Ala Asp Gly Thr His Ser Ala Lieu Ala Thr Ala Cys Gly Val Asp Trip 1.65 17O 17s Glin Glin Glu Pro Tyr Glu Gln Lieu Ala Val Ile Ala Asn. Wall Ala Thr 18O 185 19 O Ser Val Ala His Glu Gly Arg Ala Phe Glu Arg Phe Thr Glin His Gly 195 2OO 2O5 Pro Lieu Ala Met Lieu Pro Met Ser Asp Gly Arg Cys Ser Lieu Val Trp 21 O 215 22O US 2010/0028334 A1 Feb. 4, 2010 66

- Continued Cys His Pro Lieu. Glu Arg Arg Glu Glu Val Lieu. Ser Trp Ser Asp Glu 225 23 O 235 24 O Llys Phe Cys Arg Glu Lieu. Glin Ser Ala Phe Gly Trp Arg Lieu. Gly Lys 245 250 255 Ile Thr His Ala Gly Lys Arg Ser Ala Tyr Pro Lieu Ala Lieu. Thir His 26 O 265 27 O Ala Ala Arg Ser Ile Thr His Arg Thr Val Lieu Val Gly Asn Ala Ala 27s 28O 285 Gln Thr Lieu. His Pro Ile Ala Gly Glin Gly Phe Asn Lieu. Gly Met Arg 29 O 295 3 OO Asp Wal Met Ser Lieu Ala Glu Thir Lieu. Thr Glin Ala Glin Glu Arg Gly 3. OS 310 315 32O Glu Asp Met Gly Asp Tyr Gly Val Lieu. Cys Arg Tyr Glin Glin Arg Arg 3.25 330 335 Glin Ser Asp Arg Glu Ala Thir Ile Gly Val Thr Asp Ser Lieu Val His 34 O 345 35. O Lieu. Phe Ala Asn Arg Trp Ala Pro Lieu Val Val Gly Arg Asn. Ile Gly 355 360 365 Lieu Met Thr Met Glu Lieu Phe Thr Pro Ala Arg Asp Val Leu Ala Glin 37 O 375 38O Arg Thr Lieu. Gly Trp Val Ala Arg 385 390

<210s, SEQ ID NO 35 &211s LENGTH: 316 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 35 Met His Cys Lys Gly Ala Cys Met Llys Pro Lieu. Lieu. Asp Wall Lieu Met 1. 5 1O 15 Ile Lieu. Asp Ala Lieu. Glu Lys Glu Gly Ser Phe Ala Ala Ala Ser Ala 2O 25 3O Lys Lieu. Tyr Lys Thr Pro Ser Ala Leu Ser Tyr Thr Val His Llys Lieu. 35 4 O 45 Glu Ser Asp Lieu. Asn. Ile Glin Lieu. Lieu. Asp Arg Ser Gly His Arg Ala SO 55 6 O Llys Phe Thr Arg Thr Gly Llys Met Lieu. Lieu. Glu Lys Gly Arg Glu Val 65 70 7s 8O Lieu. His Thr Val Arg Glu Lieu. Glu Lys Glin Ala Ile Llys Lieu. His Glu 85 90 95 Gly Trp Glu Asn Glu Lieu Val Ile Gly Val Asp Asp Thr Phe Pro Phe 1OO 105 11 O Ser Lieu. Leu Ala Pro Leu. Ile Glu Ala Phe Tyr Gln His His Ser Val 115 12 O 125 Thir Arg Lieu Lys Phe Ile Asn Gly Val Lieu. Gly Gly Ser Trp Asp Ala 13 O 135 14 O Lieu. Thr Glin Gly Arg Ala Asp Ile Ile Val Gly Ala Met His Glu Pro 145 150 155 160 Pro Ser Ser Ser Glu Phe Gly Phe Ser Arg Lieu. Gly Asp Leu Glu Gln 1.65 17O 17s

Wall Phe Ala Wall Ala Pro His His Pro Leu Ala Lieu. Glu Glu Glu Pro 18O 185 19 O US 2010/0028334 A1 Feb. 4, 2010 67

- Continued

Lieu. Asn Arg Arg Ile Ile Lys Arg Tyr Arg Ala Ile Val Val Gly Asp 195 2OO 2O5 Thir Ala Glin Ala Gly Ala Ser Thr Ala Ser Glin Lieu. Lieu. Asp Glu Glin 21 O 215 22O Glu Ala Ile Thr Val Phe Asp Phe Llys Thir Lys Lieu. Glu Lieu. Glin Ile 225 23 O 235 24 O Ser Gly Lieu. Gly Cys Gly Tyr Lieu Pro Arg Tyr Lieu Ala Glin Arg Phe 245 250 255 Lieu. Asp Ser Gly Ala Lieu. Ile Glu Lys Llys Val Val Ala Glin Thr Lieu. 26 O 265 27 O Phe Glu Pro Val Trp Ile Gly Trp Asn Glu Gln Thr Ala Gly Lieu Ala 27s 28O 285 Ser Gly Trp Trp Arg Asp Glu Ile Lieu Ala Asn. Ser Ala Ile Ala Gly 29 O 295 3 OO Val Tyr Ala Lys Ser Asp Asp Gly Lys Ser Ala Ile 3. OS 310 315

<210s, SEQ ID NO 36 &211s LENGTH: 382 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 36 Met Ala Ala Ser Thr Phe Phe Ile Pro Ser Val Asn Val Ile Gly Ala 1. 5 1O 15 Asp Ser Lieu. Thir Asp Ala Met Asn Met Met Ala Asp Tyr Gly Phe Thr 2O 25 3O Arg Thr Lieu. Ile Val Thr Asp Asn Met Lieu. Thir Lys Lieu. Gly Met Ala 35 4 O 45 Gly Asp Val Glin Lys Ala Lieu. Glu Glu Arg Asn. Ile Phe Ser Val Ile SO 55 6 O Tyr Asp Gly Thr Glin Pro Asn Pro Thr Thr Glu Asn Val Ala Ala Gly 65 70 7s 8O Lieu Lys Lieu. Lieu Lys Glu Asn. Asn. Cys Asp Ser Val Ile Ser Lieu. Gly 85 90 95 Gly Gly Ser Pro His Asp Cys Ala Lys Gly Ile Ala Lieu Val Ala Ala 1OO 105 11 O Asn Gly Gly Asp Ile Arg Asp Tyr Glu Gly Val Asp Arg Ser Ala Lys 115 12 O 125 Pro Gln Leu Pro Met Ile Ala Ile Asn. Thir Thr Ala Gly Thr Ala Ser 13 O 135 14 O Glu Met Thr Arg Phe Cys Ile Ile Thr Asp Glu Ala Arg His Ile Llys 145 150 155 160 Met Ala Ile Val Asp Llys His Val Thr Pro Lieu Lleu Ser Val Asn Asp 1.65 17O 17s Ser Ser Leu Met Ile Gly Met Pro Llys Ser Lieu. Thir Ala Ala Thr Gly 18O 185 19 O Met Asp Ala Lieu. Thir His Ala Ile Glu Ala Tyr Val Ser Ile Ala Ala 195 2OO 2O5 Thr Pro Ile Thir Asp Ala Cys Ala Lieu Lys Ala Val Thr Met Ile Ala 21 O 215 22O Glu Asn Lieu Pro Lieu Ala Val Glu Asp Gly Ser Asn Ala Lys Ala Arg US 2010/0028334 A1 Feb. 4, 2010 68

- Continued

225 23 O 235 24 O Glu Ala Met Ala Tyr Ala Glin Phe Lieu Ala Gly Met Ala Phe Asn. Asn 245 250 255 Ala Ser Lieu. Gly Tyr Val His Ala Met Ala His Glin Lieu. Gly Gly Phe 26 O 265 27 O Tyr Asn Lieu Pro His Gly Val Cys Asn Ala Val Lieu Lleu Pro His Val 27s 28O 285 Glin Val Phe Asin Ser Llys Val Ala Ala Ala Arg Lieu. Arg Asp Cys Ala 29 O 295 3 OO Ala Ala Met Gly Val Asn Val Thr Gly Lys Asn Asp Ala Glu Gly Ala 3. OS 310 315 32O Glu Ala Cys Ile Asn Ala Ile Arg Glu Lieu Ala Lys Llys Val Asp Ile 3.25 330 335 Pro Ala Gly Lieu. Arg Asp Lieu. Asn. Wall Lys Glu Glu Asp Phe Ala Val 34 O 345 35. O Lieu Ala Thr Asn Ala Lieu Lys Asp Ala Cys Gly Phe Thr Asn Pro Ile 355 360 365 Glin Ala Thr His Glu Glu Ile Val Ala Ile Tyr Ala Ala Arg 37 O 375 38O

<210s, SEQ ID NO 37 &211s LENGTH: 571 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OO > SEQUENCE: 37 Met Asin Ser Leu Glin Ile Leu Ser Phe Val Gly Phe Thr Lieu. Leu Val 1. 5 1O 15 Ala Val Ile Thr Trp Trp Llys Val Arg Llys Thr Asp Thr Gly Ser Glin 2O 25 3O Glin Gly Tyr Phe Lieu Ala Gly Arg Ser Lieu Lys Ala Pro Val Ile Ala 35 4 O 45 Ala Ser Lieu Met Lieu. Thir Asn Lieu. Ser Thr Glu Gln Lieu Val Gly Lieu SO 55 6 O Ser Gly Glin Ala Tyr Lys Ser Gly Met Ser Val Met Gly Trp Glu Val 65 70 7s 8O Thir Ser Ala Val Thr Lieu. Ile Phe Leu Ala Lieu. Ile Phe Leu Pro Arg 85 90 95 Tyr Lieu Lys Arg Gly Ile Ala Thir Ile Pro Asp Phe Lieu. Glu Glu Arg 1OO 105 11 O Tyr Asp Llys Thir Thr Arg Ile Ile Ile Asp Phe Cys Phe Lieu. Ile Ala 115 12 O 125 Thr Gly Val Cys Phe Leu Pro Ile Val Lieu. Tyr Ser Gly Ala Leu Ala 13 O 135 14 O Lieu. Asn Ser Leu Phe His Val Gly Glu Ser Leu Glin Ile Ser His Gly 145 150 155 160 Ala Ala Ile Trp Lieu. Lieu Val Ile Lieu. Lieu. Gly Lieu Ala Gly Ile Lieu 1.65 17O 17s Tyr Ala Val Ile Gly Gly Lieu. Arg Ala Met Ala Val Ala Asp Ser Ile 18O 185 19 O Asn Gly Ile Gly Lieu Val Ile Gly Gly Lieu Met Val Pro Val Phe Gly 195 2OO 2O5 US 2010/0028334 A1 Feb. 4, 2010 69

- Continued Lieu. Ile Ala Met Gly Lys Gly Ser Phe Met Glin Gly Ile Glu Gln Leu 21 O 215 22O Thir Thr Val His Ala Glu Lys Lieu. Asn Ser Ile Gly Gly Pro Thr Asp 225 23 O 235 24 O Pro Leu Pro Ile Gly Ala Ala Phe Thr Gly Lieu. Ile Leu Val Asn Thr 245 250 255 Phe Tyr Trp Cys Thr Asn Glin Gly Ile Val Glin Arg Thr Lieu. Ala Ser 26 O 265 27 O Llys Ser Lieu Ala Glu Gly Glin Lys Gly Ala Lieu. Lieu. Thir Ala Val Lieu. 27s 28O 285 Llys Met Lieu. Asp Pro Lieu Val Lieu Val Lieu Pro Gly Lieu. Ile Ala Phe 29 O 295 3 OO His Lieu. Tyr Glin Asp Lieu Pro Lys Ala Asp Met Ala Tyr Pro Thir Lieu. 3. OS 310 315 32O Val Asn Asn Val Lieu Pro Val Pro Met Val Gly Phe Phe Gly Ala Val 3.25 330 335 Lieu. Phe Gly Ala Val Ile Ser Thr Phe Asin Gly Phe Lieu. Asn Ser Ala 34 O 345 35. O Ser Thr Lieu Phe Ser Met Gly Ile Tyr Arg Arg Ile Ile Asin Glin Asn 355 360 365 Ala Glu Pro Glin Gln Leu Val Thr Val Gly Arg Llys Phe Gly Phe Phe 37 O 375 38O Ile Ala Ile Val Ser Val Lieu Val Ala Pro Trp Ile Ala Asn Ala Pro 385 390 395 4 OO Glin Gly Lieu. Tyr Ser Trp Met Lys Glin Lieu. Asn Gly Ile Tyr Asn. Wall 4 OS 41O 415 Pro Leu Val Thir Ile Ile Ile Met Gly Phe Phe Phe Pro Arg Ile Pro 42O 425 43 O Ala Lieu Ala Ala Lys Val Ala Met Gly Ile Gly Ile Ile Ser Tyr Ile 435 44 O 445 Thir Ile Asn Tyr Lieu Val Llys Phe Asp Phe His Phe Lieu. Tyr Val Lieu. 450 45.5 460 Ala Cys Thr Phe Cys Ile Asn Val Val Val Met Leu Val Ile Gly Phe 465 470 47s 48O Ile Llys Pro Arg Ala Thr Pro Phe Thr Phe Lys Asp Ala Phe Ala Val 485 490 495 Asp Met Llys Pro Trp Lys Asn. Wall Lys Ile Ala Ser Ile Gly Ile Lieu SOO 505 51O Phe Ala Met Ile Gly Val Tyr Ala Gly Leu Ala Glu Phe Gly Gly Tyr 515 52O 525 Gly Thr Arg Trp Leu Ala Met Ile Ser Tyr Phe Ile Ala Ala Val Val 53 O 535 54 O Ile Val Tyr Lieu. Ile Phe Asp Ser Trp Arg His Arg His Asp Pro Ala 5.45 550 555 560 Val Thr Phe Thr Pro Asp Gly Lys Asp Ser Leu 565 st O

<210s, SEQ ID NO 38 &211s LENGTH: 3 OO 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 38 US 2010/0028334 A1 Feb. 4, 2010 70

- Continued

Met Thr Met Ile Arg Val Ala Cys Val Gly Ile Thr Val Met Asp Arg 1. 5 1O 15 Ile Tyr Tyr Val Glu Gly Lieu Pro Thr Glu Ser Gly Lys Tyr Val Ala 2O 25 3O Arg Asn Tyr Thr Glu Val Gly Gly Gly Pro Ala Ala Thr Ala Ala Val 35 4 O 45 Ala Ala Ala Arg Lieu. Gly Ala Glin Val Asp Phe Ile Gly Arg Val Gly SO 55 6 O Asp Asp Asp Thr Gly Asn. Ser Lieu. Lieu Ala Glu Lieu. Glu Ser Trp Gly 65 70 7s 8O Val Asn. Thir Arg Tyr Thr Lys Arg Tyr Asn Glin Ala Lys Ser Ser Glin 85 90 95 Ser Ala Ile Met Val Asp Thir Lys Gly Glu Arg Ile Ile Ile Asn Tyr 1OO 105 11 O Pro Ser Pro Asp Lieu Lleu Pro Asp Ala Glu Trp Lieu. Glu Glu Ile Asp 115 12 O 125 Phe Ser Glin Trp Asp Val Val Lieu Ala Asp Val Arg Trp His Asp Gly 13 O 135 14 O Ala Lys Lys Ala Phe Thr Lieu Ala Arg Glin Ala Gly Wal Met Thr Val 145 150 155 160 Lieu. Asp Gly Asp Ile Thr Pro Glin Asp Ile Ser Glu Lieu Val Ala Lieu. 1.65 17O 17s Ser Asp His Ala Ala Phe Ser Glu Pro Gly Lieu Ala Arg Lieu. Thr Gly 18O 185 19 O Val Lys Glu Met Ala Ser Ala Lieu Lys Glin Ala Glin Thr Lieu. Thir Asn 195 2OO 2O5 Gly His Val Tyr Val Thr Glin Gly Ser Ala Gly Cys Asp Trp Leu Glu 21 O 215 22O Asn Gly Gly Arg Gln His Glin Pro Ala Phe Llys Val Asp Val Val Asp 225 23 O 235 24 O Thir Thr Gly Ala Gly Asp Val Phe His Gly Ala Lieu Ala Val Ala Lieu. 245 250 255 Ala Thir Ser Gly Asp Lieu Ala Glu Ser Val Arg Phe Ala Ser Gly Val 26 O 265 27 O Ala Ala Lieu Lys Cys Thr Arg Pro Gly Gly Arg Ala Gly Ile Pro Asp 27s 28O 285 Cys Asp Gln Thr Arg Ser Phe Leu Ser Leu Phe Val 29 O 295 3 OO

<210s, SEQ ID NO 39 &211s LENGTH: 412 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 39 Met Met Tyr Gly Val Tyr Arg Ala Met Lys Lieu Pro Ile Tyr Lieu. Asp 1. 5 1O 15 Tyr Ser Ala Thr Thr Pro Val Asp Pro Arg Val Ala Glu Lys Met Met 2O 25 3O Glin Phe Met Thr Met Asp Gly Thr Phe Gly ASn Pro Ala Ser Arg Ser 35 4 O 45 His Arg Phe Gly Trp Glin Ala Glu Glu Ala Val Asp Ile Ala Arg Asn US 2010/0028334 A1 Feb. 4, 2010 71

- Continued

SO 55 6 O Glin Ile Ala Asp Lieu Val Gly Ala Asp Pro Arg Glu Ile Val Phe Thr 65 70 7s 8O Ser Gly Ala Thr Glu Ser Asp Asn Lieu Ala Ile Lys Gly Ala Ala Asn 85 90 95 Phe Tyr Gln Lys Lys Gly Lys His Ile Ile Thr Ser Lys Thr Glu. His 1OO 105 11 O Lys Ala Val Lieu. Asp Thr Cys Arg Glin Lieu. Glu Arg Glu Gly Phe Glu 115 12 O 125 Val Thr Tyr Lieu Ala Pro Glin Arg Asn Gly Ile Ile Asp Lieu Lys Glu 13 O 135 14 O Lieu. Glu Ala Ala Met Arg Asp Asp Thir Ile Lieu Val Ser Ile Met His 145 150 155 160 Val Asn. Asn. Glu Ile Gly Val Val Glin Asp Ile Ala Ala Ile Gly Glu 1.65 17O 17s Met Cys Arg Ala Arg Gly Ile Ile Tyr His Val Asp Ala Thr Glin Ser 18O 185 19 O Val Gly Lys Lieu Pro Ile Asp Lieu. Ser Glin Lieu Lys Val Asp Lieu Met 195 2OO 2O5 Ser Phe Ser Gly His Lys Ile Tyr Gly Pro Lys Gly Ile Gly Ala Leu 21 O 215 22O Tyr Val Arg Arg Llys Pro Arg Val Arg Ile Glu Ala Gln Met His Gly 225 23 O 235 24 O Gly Gly His Glu Arg Gly Met Arg Ser Gly Thr Lieu Pro Val His Glin 245 250 255 Ile Val Gly Met Gly Glu Ala Tyr Arg Ile Ala Lys Glu Glu Met Ala 26 O 265 27 O Thr Glu Met Glu Arg Lieu. Arg Gly Lieu. Arg Asn Arg Lieu. Trp Asn Gly 27s 28O 285 Ile Lys Asp Ile Glu Glu Val Tyr Lieu. Asn Gly Asp Lieu. Glu. His Gly 29 O 295 3 OO Ala Pro Asn. Ile Lieu. Asn Val Ser Phe Asn Tyr Val Glu Gly Glu Ser 3. OS 310 315 32O Lieu. Ile Met Ala Lieu Lys Asp Lieu Ala Val Ser Ser Gly Ser Ala Cys 3.25 330 335 Thir Ser Ala Ser Lieu. Glu Pro Ser Tyr Val Lieu. Arg Ala Lieu. Gly Lieu. 34 O 345 35. O Asn Asp Glu Lieu Ala His Ser Ser Ile Arg Phe Ser Lieu. Gly Arg Phe 355 360 365 Thir Thr Glu Glu Glu Ile Asp Tyr Thr Ile Glu Lieu Val Arg Llys Ser 37 O 375 38O Ile Gly Arg Lieu. Arg Asp Lieu. Ser Pro Lieu. Trp Glu Met Tyr Lys Glin 385 390 395 4 OO Gly Val Asp Lieu. Asn. Ser Ile Glu Trp Ala His His 4 OS 41O

<210s, SEQ ID NO 4 O &211s LENGTH: 345 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 4 O US 2010/0028334 A1 Feb. 4, 2010 72

- Continued Met His Gly Asn Ser Glu Met Gln Lys Ile Asn Gln Thr Ser Ala Met 1. 5 1O 15 Pro Glu Lys Thr Asp Val His Trp Ser Gly Arg Phe Ser Val Ala Pro 2O 25 3O Met Lieu. Asp Trp Thr Asp Arg His Cys Arg Tyr Phe Lieu. Arg Lieu. Lieu. 35 4 O 45 Ser Arg Asn Thr Lieu. Leu Tyr Thr Glu Met Val Thr Thr Gly Ala Ile SO 55 6 O Ile His Gly Lys Gly Asp Tyr Lieu Ala Tyr Ser Glu Glu Glu. His Pro 65 70 7s 8O Val Ala Lieu. Glin Lieu. Gly Gly Ser Asp Pro Ala Ala Lieu Ala Glin Cys 85 90 95 Ala Lys Lieu Ala Glu Ala Arg Gly Tyr Asp Glu Ile Asn Lieu. Asn Val 1OO 105 11 O Gly Cys Pro Ser Asp Arg Val Glin Asn Gly Met Phe Gly Ala Cys Lieu. 115 12 O 125 Met Gly Asn Ala Glin Lieu Val Ala Asp Cys Val Lys Ala Met Arg Asp 13 O 135 14 O Val Val Ser Ile Pro Val Thr Val Lys Thr Arg Ile Gly Ile Asp Asp 145 150 155 160 Gln Asp Ser Tyr Glu Phe Lieu. Cys Asp Phe Ile Asn Thr Val Ser Gly 1.65 17O 17s Lys Gly Glu. Cys Glu Met Phe Ile Ile His Ala Arg Lys Ala Trp Lieu. 18O 185 19 O Ser Gly Lieu. Ser Pro Llys Glu Asn Arg Glu Ile Pro Pro Lieu. Asp Tyr 195 2OO 2O5 Pro Arg Val Tyr Gln Leu Lys Arg Asp Phe Pro His Lieu. Thr Met Ser 21 O 215 22O Ile Asin Gly Gly Ile Llys Ser Lieu. Glu Glu Ala Lys Ala His Lieu. Glin 225 23 O 235 24 O His Met Asp Gly Val Met Val Gly Arg Glu Ala Tyr Glin Asn Pro Gly 245 250 255 Ile Lieu Ala Ala Val Asp Arg Glu Ile Phe Gly Ser Ser Asp Thr Asp 26 O 265 27 O Ala Asp Pro Val Ala Val Val Arg Ala Met Tyr Pro Tyr Ile Glu Arg 27s 28O 285 Glu Lieu Ser Glin Gly Thr Tyr Lieu. Gly His Ile Thr Arg His Met Leu 29 O 295 3 OO Gly Lieu. Phe Glin Gly Ile Pro Gly Ala Arg Glin Trp Arg Arg Tyr Lieu. 3. OS 310 315 32O Ser Glu Asn Ala His Lys Ala Gly Ala Asp Ile Asn Val Lieu. Glu. His 3.25 330 335 Ala Lieu Lys Lieu Val Ala Asp Lys Arg 34 O 345

<210s, SEQ ID NO 41 &211s LENGTH: 217 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 41 Met Lieu. Cys Wall Lys Asn. Wal Ser Lieu. Arg Lieu Pro Glu Ser Arg Lieu. 1. 5 1O 15 US 2010/0028334 A1 Feb. 4, 2010 73

- Continued

Lell Thir Asn Wall Asn Phe Thr Val Asp Gly Asp Ile Wall Thir Lieu. 25

Met Gly Pro Ser Gly Gly Lys Ser Thir Luell Phe Ser Trp Met Ile 35 4 O 45

Gly Ala Luell Ala Glu Glin Phe Ser Thir Gly Glu Lell Trp Luell Asn SO 55 6 O

Glu Glin Arg Ile Asp Ile Leul Pro Thir Ala Glin Arg Glin Ile Gly Ile 65 70 8O

Lell Phe Glin Asp Ala Lieu. Luell Phe Asp Glin Phe Ser Wall Gly Glin Asn 85 90 95

Lieu. Luell Luell Ala Leul Pro Ala Thir Lieu. Gly Asn Ala Arg Arg Asn 1OO 105 11 O

Ala Wall Asn Asp Ala Lieu. Glu Arg Ser Gly Lieu. Glu Gly Ala Phe His 115 12 O 125

Glin Asp Pro Ala Thr Lell Ser Gly Gly Glin Arg Ala Arg Wall Ala Lieu 13 O 135 14 O

Lell Arg Ala Lieu. Luell Ala Glin Pro Ala Lieu. Lieu. Lieu Asp Glu Pro 145 150 155 160

Phe Ser Arg Lieu. Asp Val Ala Lieu Arg Asp Asn. Phe Arg Glin Trp Val 1.65 17O 17s

Phe Ser Glu Wall Arg Ala Lell Ala Ile Pro Wal Wall Glin Wall Thir His 18O 185 19 O

Asp Lieu. Glin Asp Val Pro Ala Asp Ser Ser Val Lieu. Asp Met Ala Glin 195 2O5

Trp Ser Glu Asn Tyr Asn Lys Luell Arg 21 O 215

SEQ ID NO 42 LENGTH: 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 42 Gly Gly Gly Gly Cys 1. 5

1. A composition comprising an antimicrobial agent and an 7. The composition of claim 5, wherein the polymyxin is enhancer to the antimicrobial agent, wherein the enhancer to selected from polymyxin B1, polymyxin B2, and a mixture of the antimicrobial agent is an inhibitor of a gene product that polymyxin B1 and polymyxin B2. by inactivating the gene product potentiates the effectiveness 8. The composition of claim 5, wherein the polymyxin is of the antimicrobial agent. selected from colistin A, colistin B, and a mixture of colistin 2. The composition of claim 1, wherein the antimicrobial A and colistin B. agent is an antimicrobial peptide. 9. The composition of claim 5, wherein the polymyxin is in 3. The composition of claim 2, wherein the antimicrobial the form of a colistin salt. peptide is a lipopeptide. 10. The composition of claim 9, wherein the colistin salt is a methane Sulphonate and/or Sulfate salt. 4. The composition of claim3, wherein the lipopeptide is a 11. The composition of claim 1, wherein the gene product cyclic lipopeptide. is selected from a group consisting of agaA, atp A, atpC, atpB. 5. The composition of claim 4, wherein the cyclic lipopep atpD, atpE, atpG, atpH, betB, csdA, csdB, fepC, guaA, guaB, tide is a polymyxin class of antibiotic or derivative thereof. iscS, kdgK, lip A, lySA, mnmA, nuvC, papa, pdxH. phn L. 6. The composition of claim 5, wherein the polymyxin is pot. rpiA, suc3, trxA, tusB (YheL), tusB. ubiE, ubiH, uncA, selected from the group of polymyxin A, B1, B2, D1, D2, E1 visB, yeeY, yiaY, yidK, yihV, yfhC), yibn and/or ynjD or and/or E2, F, G, M, P S and/or T homologues, variants or fragments thereof. US 2010/0028334 A1 Feb. 4, 2010 74

12. The composition of claim 1, wherein the inhibitor is 26. (canceled) selected from a group consisting of mefloquine, Venturicidin 27. The composition of claim 23, wherein the multi-drug A, diary guinoline, betaine aldehyde chloride, acivein, psico resistant microorganism is resistant to at least one member of furaine, buthionine Sulfoximine, diaminopemelic acid, the polymyxin class of antibiotics or derivatives or analogues 4-phospho-D-erythronhydroxamic acid, motexafin gado thereof. linium and/or Xycitrin or modified versions or analogues 28. (canceled) thereof. 29. (canceled) 13. The composition of claim 1, wherein the gene product 30. (canceled) is atp A, atpF or atpH or homologues, variants or fragments 31. The composition of claim 1 further comprising a phar thereof, and the inhibitor is mefloquine and/or venturicidin A maceutically acceptable carrier. and/or diarycuinoline or modified versions or analogues 32. (canceled) thereof. 33. The composition of claim 1, where the amount of the 14. The composition of claim 1, wherein the gene product antimicrobial agents is at least 25% less than the same anti is betB or homologues or variants thereof, and the inhibitor is microbial agent in an isogenic cell except for the addition of betaine aldehyde chloride or modified versions or analogues the enhancer of antimicrobial agent without reduction of anti thereof. microbial effect. 15. The composition of claim 1, wherein the gene product 34. (canceled) is guaA or guaB or homologues or variants thereof, and the 35. A method of treatment and/or prophylaxis of an infec inhibitor is acivin and/or psicofluranine or modified versions tion caused by an microorganism comprising steps of admin or analogues thereof. istering to a subject in need thereof an effective amount of the 16. The composition of claim 1, wherein the gene product composition according to claim 1. is LipA or homologues or variants thereof, and the inhibitoris 36. The method of claim 35, wherein the subject is mam buthionine Sulfoximine or modified versions or analogues malian, avian, amphibian or a plant. thereof. 37. The method of claim 36, wherein the mammalian is 17. The composition of claim 1, wherein the gene product human. is LySA or homologues or variants thereof, and the inhibitoris 38.-41. (canceled) diaminopimelic acid or modified versions or analogues 42. The method of claim 35, wherein the infection is thereof. selected from the group consisting of bacterial wound infec 18. The composition of claim 1, wherein the gene product tions, mucosal infections, enteric infections, septic condi is rpiA or homologues or variants thereof, and the inhibitor is tions, infectious in airways, cerebrospinal fluid, blood, eyes 4-phospho-D-erythronhydrixamic acid or modified versions and skin. or analogues thereof. 43.-51. (canceled) 19. The composition of claim 1, wherein the gene product 52. A method for identifying gene products, wherein inac is trx A or homologues or variants thereof, and the inhibitor is tivation of the gene products potentates antimicrobial agent motexafin gadolinium and/or Xycitrin acid or modified ver activity, the method comprising the steps of sions or analogues thereof. (a) mutating one or more genes in a cell, 20. The composition of claim 1, wherein the inhibitor (b) contacting the cell with the antimicrobial agent, comprises a small molecule, nucleic acid, nucleic acid ana (c) incubating the cell for a sufficient amount of time to logue, peptide, ribosome, antibody, and variants and frag allow for growth, and; ments thereof. (d) assessing the number of cells, wherein the number of 21. (canceled) cells is compared to steps (a)-(c) performed on a non 22. (canceled) mutated cell, wherein the decrease in numbers of cells 23. The composition of claim 1, wherein the microorgan identifies a gene product that when inactivated potenti ism is selected from the group consisting of a bacterium, a ates antimicrobial peptide activity. gram-positive bacterium, a gram-negative bacterium, a multi 53. The method of claim 52, wherein the cell is bacterium. drug resistant microorganism. 54-58. (canceled) 24. (canceled) 25. (canceled)