Bacterial virulence factors as targets for new antibacterials: an overview Dr. Ariel Blocker Cellular and Molecular Medicine & Biochemistry Antibiotic availability timeline Nature Rev. Micro. 5, 175 (2007) Antibiotic resistance • Mechanisms of antibiotic resistance: -Target modification (direct mutation, upregulation, alternative pathway) -Antibiotic modification -Altered permeability/efflux See Poster presentation • Acquisition of resistance: -Spontaneous mutation (frequency of occurrence ~10-8 to 10-9) -And/or gene acquisition via plasmid/transposons (frequency of transfer 10-3 to 10-4) • The extent of the problem: within a few years resistance is observed to every single antibiotic ever introduced to clinical use Consequence: insidiously rising death rates Nature 406, 762 (2000) Why we are here I • The industrial pipeline of novel-mechanism antibacterials is presently empty especially for Gram-negative pathogens (Nat. Rev. Microbiol. 6, 29 (2007) and see Plenary Session II): 1) Since the mid 1990s, commercial genome mining has not worked (but see FP7 antiPathoGN in Poster session III) 2) Synthesis/discovery of novel chemical scaffolds that block established targets seems the only way forward now 3) At the rate of present resource investment it could take 5-10 years and 10-15 years to see a novel mechanism drug to treat Gram-positives and Gram negatives, respectively Why we are here II • It is increasingly apparent that present antibiotics have significant negative effects in patients because: 1) They non-specifically displace the resident microflora -e.g. Clostridium difficile/ Candida secondary infections are increasing problems -Possible serious long-term effects on immune system development (see Plenary Session I) 2) They are often initially microbial compounds, can damage cells and/or reduce viable counts and as such they can act as signalling molecules which can enhance antibiotic resistance, virulence, and mutation rates (see Plenary Session III) Besides new antibiotics, what are our options? • Phage or other “natural killer” therapy? • Vaccination! • Host defense antimicrobial peptide derivatisation or development? Nature Biotech. 24, 1551 (2006) Curr. Opin. Microbiol. 9, 489 (2006) • Drug development strategies based on our present understanding of bacterial virulence strategies (Plenary Session III) Advantages/disadvantages of antivirulence drugs Pluses: • Targets are intrinsically unique to pathogens and should hence spare the natural flora • There should be little “natural resistance” to any such drugs/reagents already developed by bacteria present in the natural/host environment • Antivirulence drugs should not directly kill the pathogen, only cripple it, allowing host defense systems to clear it. This might slow evolution of resistance • May act synergistically with conventional antibiotics, because they act through independent mechanisms to block in vivo bacterial replication or survival Minuses: • Most antivirulence drugs are unlikely to be as “broad-spectrum” as any of the present commonly used antibiotics • As virulence gene expression is a function of time and space in vivo, some antivirulence drugs may not work if administered at the wrong point in the infection. We need to identify “pathogenesis bottlenecks” and specifically target those Nat. Rev. Microbiol. 6, 17 (2008) What are the possible virulence targets? • Toxins • Adhesion (when virulence specific) • Virulence-dedicated secretion systems • Virulence-dedicated two-component regulatory systems • Quorum sensing and biofilm generation systems (see Plenary session III talk, Tuesday pm) Different types of toxins Individual secreted toxins are amongst the most pathogenic virulence factors that bacteria produce “Anti-toxins” • Antisera raised against inactivated toxin forms (“toxoids”) are one of the oldest known form of antibacterial therapy. • Neutralising IgG fractions were obtained from horses or humans and administered post-exposure to C. tetani, C. diphteriae or C. botulinum toxins while infection with these organisms is cleared. • Humanised monoclonal antibodies are great alternative to horse sera! • In 2003 the FDA approved BabyBIG, a human Botulism Immune Globulin for treatment of infant botulism. • Presently, there are six other such types of antibodies in clinical trials, it making it the most pursued/advanced antivirulence strategy. • But, how to target toxins directly delivered into cells is non-obvious and for diseases which are rare or where the toxin(s) only in part contributes to the disease symptoms or its rare complications (e.g. dysentery), the market may be too small to justify industrial interest. High diversity of adhesion structures • Found first in Gram- and now in Gram+ • Afimbrial adhesins: monomeric proteins or protein complexes at cell surface (e.g. Hap autotransporter of H. influenzae or Dr of E. coli) • Fimbrial adhesins: heteropolymeric extracellular fibers called pili or fimbriae (6 different types described in E. coli) • Their expression can vary depending on site/time point of Pili of of UPEC adhering to bladder cells infection • Bind diverse host cell factors (surface/extracellular matrix carbohydrates or protein) Pili function represents a “bottleneck” in UPEC infection • Both Type 1 and P pili key to the initial attachment of UPEC to bladder cells •Both are assembled by the “chaperone-usher pathway”, for which there are 15 other operons in E. coli and which are wide- spread in other Gram- •Thus, inhibiting this pathway has “broad spectrum potential” Molecular mechanism of chaperone- usher pathway Donor strand exchange determined through crystalisation of most pilin and chaperone subunits in this pathway (Scott Hultgren and Gabriel Waksman) Rational design of “Pilicides” Fred Almqvist, Waksman and Hultgren PNAS 103, 17897 (2006) The chaperone R8-K112 donor strand exchange cleft region was originally targeted, followed by compounds 1a and 1b with two substituents (R1 and R2) that bind P and type 1 pili chaperones, respectively. However, poor water solubility limited their utility. Substitution of the open position in the 2-pyridone scaffold (R3) resolved this issue, resulting in 2- pyridones, 2a–2d, where 2a–2c bound chaperones in the low millimolar range and 2d is a negative control. However, crystal structure of the PapD-2c complex revealed that 2c binds to the surface of the chaperone interacting Growth in pilicides inhibits hemagglutination, with the usher so it can only prevent host cell adhesion and biofilm formation in vitro. pili assembly, not diassemble them Two-component response systems (TCRSs) • Dominant system by which bacteria respond to their environment • 4000 different ones identified in 400 sequenced genomes, only a few are virulence specific • Consist of 1) receiver histidine kinase that is activated by extracellular signals and 2) response regulator that transmits the signal to an intracellular target that modulates gene expression • Broad-spectrum inhibitors could non-selectively target TCRSs common to many bacteria • More selective inhibitors are required to inhibit expression of specific virulence genes Regulation of V. cholerae virulence gene expression Environmental signals: temperature, bile salts, pH Low cell density sensing cascade John Mekalanos, Victor DiRita Infec. Immun. 75, 5542 (2007) Identification of Virstatin, a ToxT inhibitor • HTS of a 50,000-compound small molecule library • Screening used V. cholerae strain carrying a chromosomally integrated tetA resistance gene controlled by the ctx promoter • 101 hits, 15 minimally generally toxic to bacteria • 4-[N-(1,8- naphthalimide)]-n-butyric acid (virstatin) was selected for further study • Inhibition of ctx and tcp (but not toxRS, tcpPH or toxT) expression confirmed in WT strains at 5-50 µM drug • Global transcriptional profiling used to verify that only ToxT-regulated genes where affected • ToxT drug-resistant mutant isolated, in dimerisation domain • Tested in in vivo model: orogastric administration of virstatin protects infant mice from intestinal colonization by V. cholerae. Even delayed administration reduced bacterial recovery by 3 logs Mekalanos Science 310, 670 (2005) Secretion systems involved in virulence Gram- and Gram+ • General secretion pathway (GSP) or •Basic knowledge still fairly rudimentary sec system (into and across IM to (molecular genetics was harder). periplasm) • Type I secretion (ABC-transporters) •Several systems similar to Gram- predicted from • Type II secretion (main terminal branch genome sequences (sec, Tat, ABC, T4SS, flagella and type IV pili). Probably many undiscovered of GSP and type IV pili) new ones. • Type III secretion (flagella and virulence) •ESAT-6/ESX-1/Snm secretion system in • Type IV secretion (conjugation and Mycobacterium, Staphyloccus (and probably virulence) etc…). Called Type VII. • Type V secretion (autotransporters) • Type VI secretion (very new!) • Tat-pathway (twin arginine transport), Caveat: Those systems mostly equivalent to ΔpH pathway of involved in virulence may not be chloroplasts. virulence-dedicated. Genetic, structural and functional similarities by flagella and virulence T3SS The most highly conserved part is the export machinery F0-like? F1-ATPase like Where does our work fit in? Recent papers on small molecule T3SS inhibitors (Plenary and poster sessions III) Initial screening of 9,400 chemicals for Yersinia yopE gene expression (Kauppi et al., 2003) Inhibition of Yersinia motility (Kauppi et al., 2003) Inhibition of Yersinia Yop secretion (Nordfelth et al.,