Stability of Resistance and Extended Potential of Targeting the Folate Synthesis

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 45

Microbial Responses to Antibiotics – Stability of Resistance and Extended Potential of Targeting the Folate Synthesis

MARIA JÖNSSON

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This thesis is based on the following papers, which will be referred to in the text by their roman numerals.

I Clarithromycin treatment selects for persistent macrolide- resistant bacteria in throat commensal flora. Maria Jönsson, Yvonne Qvarnström, Lars Engstrand and Göte Swedberg. Inter- national Journal of Antimicrobial Agents 25 (2005) 68 -74.

II Mutations and Horizontal Transmission Have Contributed to Sulfonamide Resistance in Streptococcus pyogenes. Maria Jönsson. Katrin Ström and Göte Swedberg.Microbial Drug Re- sistance 9 (2) 2003. 147 - 153.

III Hydroxymethyldihydropterin pyrophosphokinase from Plasmodium falciparum complements a folK-knockout mutant in E. coli when expressed as a separate polypeptide detached from dihydropteroate synthase. Maria Jönsson and Göte Swedberg. Molecular and Biochemical Parasitology. 2005 Mar;140(1):123-5

IV Further characterisation of the bifunctional HPPK-DHPS from Plasmodium falciparum. Maria Jönsson, Yvonne Qvarn- ström, Therese Rosenling and Göte Swedberg. (in manuscript).

Reprints were made with permission from the publishers.

Contents

Introduction...... 9 Resistance to antibiotics...... 9 What they are, and how they work ...... 9 So, how does resistance spread?...... 11 Why is resistance interesting? ...... 12 The commensal flora can act as a reservoir for resistance genes ...... 13 Drug use and problems ...... 14 Folate biosynthesis ...... 15 One carbon metabolism ...... 15 Folate biosynthesis ...... 16 The enzymes ...... 18 Drugs targeting this pathway...... 22 Polyfunctionality in proteins ...... 26 Polyfunctional proteins...... 26 Substrate channelling...... 26 Aims of this thesis...... 28 Results and discussion ...... 29 Emergence and spread of antibiotic resistance...... 29 Current sulfonamide targets ...... 32 Possible new drug targets...... 37 Construction of a folK-knockout in E. coli...... 41 Concluding remarks...... 42 Populärvetenskaplig sammanfattning ...... 43 Vad är antibiotikaresistens?...... 43 Vad händer vid en antibiotikabehandling? ...... 43 Folsyra ...... 44 Acknowledgements...... 45 References...... 47

Abbreviations

Enzymes

HPPK hydroxymethyl dihydropterin pyrophosphokinase DHPS dihydropteroate synthase DHNA dihydroneopterin aldolase GTPCH GTP cyclohydrolase DHFS dihydrofolate synthase FPGS folylpolyglutamate synthase DHFR dihydrofolate reductase TS thymidylate synthase

Compounds pABA p-amino benzoic acid HMDP hydroxymethyl dihydropterin HMDPP hydroxymethyl dihydropterin pyrophosphate THF tetrahydrofolate DHF dihydrofolate H4 tetrahydro -

Gene designations folA dihydrofolate reductase folK hydroxymethylpterin pyrophosphokinase folP dihydropteroate synthase thyA thymidylate synthase thyX thymidylate synthase mef(A) macrolide efflux

Introduction

Resistance to antibiotics

What they are, and how they work The word antibiotic derives from Greek, and means “the opposite of life”. The term normally refers to a substance used to get rid of microbes causing an infection. One important "fact" to keep in mind is that antibiotics work rather like a shotgun aimed at a person in a crowd. The target will probably be hit, but so will many other people surrounding him, like in this case the harmless bacteria in the body. An antibiotic substance can be of various types that interfere with the cell machinery. It can bind to the active site1 of a protein, and thereby stop a sub- strate2 from binding, or it can mimic the substrate but make the protein produce inactive product. Another way might be to bind elsewhere on the protein, hindering it in its work. Some antibiotics kill the target organism. Oth- ers do not, but instead slow down its growth so it loses in competition by others, or can be removed by the immune system (Figure 1). Microorganisms are quite good at counteracting the antibiotics, in other words, they become resistant. The basics of antibiotic resistance is not complicated: all it is really about is slight modification of the proteins affected by an antibiotic. There are several ways to do this. You can modify the targeted proteins so that they are unaffected or less affected by the antibiotic substance. This is the case in for example resistance to sulfonamides, as discussed below, and in the kind of resistance to macrolides encoded by rRNA methylase genes. An elaborate version of protein modification is to develop or acquire a new protein doing the same thing, but that does not bind the antibiotic. An example of this is again found in the folate pathway, where resistance to sulfonamides can be achieved by importing a drug resistant dihydropteroate synthase, that can work in parallel with the cell’s own enzyme. It is also possible to make another protein modify the antibiotic and

1 The active site is the place in a protein where the reaction catalysed takes place. 2 A substrate is a compound used by a protein to produce a product.

9 inactivate it, as is the case in penicillin resistance caused by E-lactamases, or maybe pump it out of the cell (80). The last case is again exemplified in this thesis, in the case of the macrolide resistance gene mef(A), that encodes an efflux pump.

RNA synthesis Protein synthesis Rifampicin Cell wall synthesis Macrolides Linezolid Penicillin Chloramphenicol

DNA synthesis Metabolic functions Metronidazole Sulfonamides Quinolones Trimethoprim

Figure 1. Antibiotics target many processes in the cell. Underneath each important process are listed some of the antibiotics targeted there

Modifying the proteins is again not as complicated as it may seem. It all boils down to slight modifications of the protein template: DNA. A few base pairs in the DNA sequence are replaced, deleted, or inserted, thus causing the amino acid sequence to change. These changes are normally kept to a minimum, as protein function is often quite dependent on its sequence, like a misplaced letter in a sentence does not affect comprehension very much, but many misspellings would. These modified, and thereby resistant, proteins do not only appear during a treatment with antibiotics. They arise all the time. The reason we do not see them that often is because they are not always the most economical, en- ergywise, and could therefore be a cost for the organism. It is often not until the organism is exposed to the antibiotic, and thereby put under selective pressure (is put into a situation where it needs the modified protein), that the modified, or with another word, mutated, proteins are efficient. The organism carrying them in that situation has a better survival value than one with a normal protein.

10 So, how does resistance spread? Spread of resistance is really as simple as its development, but instead of focusing on the protein, we focus on its blueprint: the gene. Genes are stretches of DNA, and DNA can be transferred between organisms. For sim- plicity I will focus on bacterial genes, as they are the main subject of this thesis. It is not practical for each and every bacterium to evolve resistant proteins when there is need. Instead, they transfer already mutated genes among each other.

a lysis

c phage

lysis

Figure 2. a) Transformation is the process in which free DNA is taken up by a bacterium. It can be incorporated in the chromosome (as shown), or remain as a circular plasmid. b) Conjugation is the transfer of DNA through cell-cell contact. c) Trans- duction is phage-mediated DNA transfer.

Horizontal transfer Bacterial genes can, and do, move between bacteria. Genes located on the normal chromosome can move to another host when the bacterium dies, and its DNA is released into the surroundings (Figure 2a). This process is called

11 transformation (80). Genes can also be located on transposons3, or on plasmids (circular “mini-chromosomes”). These elements can then move from one bacterium to another by a process called conjugation (Figure 2b). A third mechanism of gene transfer is transduction, where phage4 can incorporate host DNA by mistake, and then move it to other bacteria (Figure 2b). Of course, horizontal transfer of genes does not only occur with resistance genes. There is increasing evidence that an important part of the diversity of bacterial genomes was generated by lateral shuffling of genes, thereby reducing the need for creating new genes for new functions (78). This ultimately allowed bacteria to occupy virtually every ecological niche there is.

Clonal expansion A consequence of acquiring an advantage such as a resistance gene is a phenomenon called clonal expansion. This means that one single bacterium multiplies and becomes more prominent in a population, instead of the usual situation where there is a mix of clones. The bacterium carrying a resistance gene has gained a selective advantage when the antibiotic is present, and can then multiply faster than its competitors. The result is an increase in the frac- tion of the population belonging to that specific clone. This could cause problems, especially if the expanding clone carries resistance genes for more than one antibiotic.

Why is resistance interesting? Personally, what I find so interesting with resistance to antibiotics is the way it offers us a way to study evolution. Evolution is all about genes, and thereby proteins, changing a little at a time. The modifications give or remove advantages when different types of selective pressure (like a treatment with antibiotics) is applied. Resistance to antibiotics would, however, be much less interesting to many people, if it were not for the fact that it ap- pears faster than humans can combat it with antibiotics. The war began about seventy years ago with the discovery of penicillin (1928) and sulfonamides (1932). Resistance to these drugs appeared quickly (2), and although we now have many different kinds of antibiotics, the bacteria still become resistant to new ones like linezolid within a matter of months (for an example, see Bozdogan and Appelbaum 2004 (19)).

3 Transposons are pieces of DNA encoding proteins that can move the transposon to different places in the genome. They can do this in a variety of ways, including “cut and paste” and making a copy of itself in the new location. 4 Phage are viruses that target bacteria.

12 The commensal flora can act as a reservoir for resistance genes

The reservoir function The commensal flora is another name for the bacteria we have all the time in our bodies. As an indication of its relevance, it can be noted that an average human being carries around ten times as many bacteria as there are cells in the body5. Most of these bacteria are harmless, and some even beneficial to us. The commensal flora can be seen as a gene pool, where genes conferring resistance to different agents are present in small numbers all the time. As- suming this, two things can happen when a population is subjected to selective pressure, such as a treatment with antibiotics. The first is a clonal expansion of individuals resistant to the antibiotic used. Those few bacteria that harbour a gene giving resistance to the antibiotic in question will have a selective advantage, and thus the possibility to grow faster and better than those lacking the resistance gene. The second possibility is lateral transfer of resistance genes. There is an immense plasticity in microbial genomes, partly caused by the acquisition of genes from the environment, that is, horizontal transfer. The commensal flora may function as a reservoir of resistance genes, and under selective pressure spread these genes to other species, including pathogens. The genes that are transferred can stay on plasmids, or they can be inte- grated in the chromosome and become a more permanent part of the bacterium (80; 85). An example of the last scenario is paper II in this thesis, where we present evidence that parts of the folate synthesis operon in some S. pyogenes strains might well have been acquired through horizontal transfer. As the amount of knowledge about the transfer of resistance genes in- creases, so does the interest in the commensal flora. Many studies where the resistance properties of these bacteria are studied have been published. Alarmingly, the picture they present is that of an increasing amount of resistance genes among bacteria that have no real reason to carry them (40; 55; 99; 100). A similar study carried out on material collected in the beginning of the century (before antibiotics were invented) showed hardly any resistance genes (27; 47). A fascinating piece of evidence supporting the hypothesis that resistance spreads in response to antibiotic treatment is a small study made on Pneu- mocystis DHPS from primates, where none of the mutations usually associated with sulfonamide resistance were found. The primates in question had not been exposed to the antibiotic (28). In contrast, extensive use of sulfonamides in humans led to rapid resistance development and spread of resistance genes (48).

5 Prokaryotes are smaller than human cells, so space is not a problem.

13 Drug use and problems Numerous studies have reported a positive correlation between use of antibiotics and amount of resistance. A striking example is the study discussed in paper I in this thesis, where it is demonstrated that treatment with macrolides selects for stably resistant microorganisms in the entire body (58; 104). The trend is currently to recommend reduction of the amount of antibiotics used in order to reduce resistance, and in relevant cases to alternate the recom- mended antibiotic for a certain infection (10). Naturally, this problem with resistance development applies not only to bacterial infections, but also to those caused by protozoa and other microbes. For example, development of antibiotic-resistant malaria parasites is the cause of increasing problems in combating this disease. A matter that could potentially be of concern is the fact that the same antibiotics are used for different diseases. An emerging problem in the "third world" is the use of a sulfonamide-trimethoprim combination treatment against bacterial diseases and as prophylaxis against for example Pneumo- cystis infections in HIV-infected patients. Since a similar combination (sulfonamide-pyrimethamine) is still an important drug in malaria treatment, there is a potential problem when treating both kinds of diseases. Pyrimethamine and trimethoprim cause mutual cross resistance in Plasmo- dium falciparum, and the different sulfa derivatives probably do too (56). High carriage of sulfonamide resistant Streptococcus pneumoniae (34) and Escherichia coli (62) has been noted in individuals having received the drug against malaria. Furthermore, HIV-patients in San Fransisco having received sulfonamide-trimethoprim against Pneumocystis infection also carried sulfonamide resistant bacteria (76). This calls for caution in choosing antimicrobials, so as to not render the antibiotics useless.

14 Folate biosynthesis Development and spread of resistance is an important topic to cover, but of equal importance is the continuous evaluation of new and potential drug targets. The folate pathway is the target of several antibiotics and has the potential for further exploitation. It is also deeply involved in the shuffling of one-carbon units essential for cell survival.

One carbon metabolism The cell is an intricate machine, where very small changes might make a big difference. The tiniest modifications, like adding or removing small functional entities such as methyl groups can have a large impact on the specificity of an enzyme or a substrate. One example is the amino acid glycine, which becomes the completely different amino acid serine if one methyl group is added to it. One carbon-metabolism is the name adopted for the generation and recy- cling of methyl and formyl groups. As an example of the importance of one- carbon units, it can be mentioned that in E. coli grown on glucose, 15%6 of the carbon is transformed into the simple amino acids glycine and serine or into one-carbon units. The most common one-carbon unit donors include S- adenosylmethionine and tetrahydrofolate. After donating a carbon unit (often a methyl, a formyl or a methylene group), the carrier has to be recycled to get it back. A major part of this process is therefore the regeneration of carbon-unit donors. In figure 3 I attempt to give an idea of the complex coordi- nation of the reactions involved. One-carbon units are essential cofactors in many processes in the cell. The most important pathways where transfer of one-carbon units is involved are found in the synthesis of nucleotides and amino acids. The DNA building block dTMP needs the one-carbon carrier methylenetetrahydrofolate, and DNA repair and transcription is dependent on hemimethylation7 of the DNA, involving the carbon-unit donor s-adenosylmethionine. The synthesis of the amino acids methionine, histidine and serine is also dependent on transfer of one-carbon units. Initiation of translation8 in many bacteria is dependent on formyl-methionine, where not only the methionine synthesis, but also the addition of the formyl group need one-carbon units. Lastly, many cofactors and vitamins such as thiamine (vitamin B) need one-carbon units in some stage of their synthesis (77).

6 Yes, that is a lot. Almost every process in the cell involves carbon in some way. 7 One strand of the DNA double helix is methylated after replication so that the cell can tell which is the new one. 8 The process of going from RNA to protein (the RNA sequence is translated into amino acids).

15 A deficiency in one-carbon units probably reduces biosynthesis of certain amino acids, and thereby slows the rate of protein translation (77). This has impact on the speed of cell growth, and ultimately on the fitness of the organism in that reduced growth also reduces the possibility to compete with other microorganisms.

DHNA Purine GTPCH metabolism HPPK Sulfonamides

DHPS Phenylalanine biosynthesis DHFS Pterin biosynthesis

DHFR

dihydrofolate Pyrimethamine tetrahydrofolate Trimethoprim

Methanogenesis One carbon pool Thymidine metabolism

Methionine Serine, threonine and glycine Pyrimidine metabolism metabolism metabolism

Figure 3. One-carbon metabolism. Tetrahydrofolate produced by the folate synthesis pathway(circled) is used by many processes in the cell. Inhibition by drugs in shown by |----.

Folate biosynthesis In mammals, folates are vitamins, meaning that they have to be provided in trace amounts from the outside (17). Bacteria, protozoa and plants on the other hand have a de novo synthesis. This makes the folate biosynthesis pathway an excellent target for antimicrobial drugs, since an adequate supply of folates is necessary for cell growth. Inhibiting this pathway slows microbial growth markedly, and can even kill in some cases. Some organisms, like Plasmodium falciparum, combine the de novo synthesis with folate salvage from the outside. An ability to salvage folates can be of importance when the de novo pathway is blocked by sulfonamides, as it allows an increased resistance to the drug. Wang and co-workers report

16 that the salvage pathway in P. falciparum seems to be dependent on the dhfr- ts gene, and on the type of folate salvaged. p-aminobenzoic acid (pABA) is the precursor most readily acquired, followed by folinic acid and folic acid (118).

Figure 4. The folate synthesis converts the nucleotide GTP to tetrahydrofolate. The first enzyme in the pathway, GTPCH and the second, a pyrophosphatase, synthesise 7,8-dihydroneopterin (2). DHNA converts (2) into 6-hydroxymethyl-7,8- dihydropterin (3). HPPK phosphorylates (3) with ATP as the phosphate donor, resulting in 6-hydroxymethyl-7,8-dihydropterin diphosphate (4). DHPS then condenses (4) with p-aminobenzoic acid (pABA) producing dihydropteroate. (5). DHFS adds a glutamate residues to (5), resulting in dihydrofolate (6). In the last step, (6) is reduced to tetrahydrofolate (7) by DHFR.

The biosynthesis pathway The aim of the pathway is the generation of dihydrofolate (DHF) and tetrahydrofolate (THF), which can then be converted to polyglutamated forms, useful in many other processes, and more prone to intracellular retention (17). The folate biosynthesis pathway converts GTP to dihydrofolate through a series of steps. It consists of two branches that converge to form folate: one where pABA is formed from chorismate, and another where hydroxymethyl dihydropterin pyrophosphate (HMDPP) is formed from GTP. pABA and HMDPP are then fused to become dihydropteroate. Dihydropteroate is in turn converted by addition of a glutamate moiety to dihydrofolate. Since dihydrofolate is not an active form of folate, it has in turn to be reduced to tetrahydrofolate by yet another enzyme: dihydrofolate reductase (39).

17 Miscellanea A rapidly developing field is that of metabolic engineering to increase folate content in cells. There are socio-economic factors behind this, since folate deficiency leads to severe disorders in humans. Producing foodstuffs already enriched for folates with a higher nutritional content could be a way of making sure fewer people are affected by such a deficiency. Most of the work has been done in the dairy starter bacterium Lactococcus lactis, (110-112) but a recent report also present folate fortification in tomatoes (29).

The enzymes

GTP cyclohydrolase GTP cyclohydrolase (GTPCH) performs the first committed step in the biosynthesis of folate. It catalyses the conversion of two GTP molecules to dihydroneopterin triphosphate and formate. The enzyme is found in most species, but the fate of the reaction product dihydroneopterin triphosphate dif- fers. In plants and microbes, that is in organisms with a de novo synthesis of folates, it goes on towards the synthesis of tetrahydrofolate. In animals, where folates have to be imported as vitamins, the product is instead used to make tetrahydrobiopterin. The pterin binding residues and the residues participating in catalysis are highly conserved across nearly all species. (114).

The step betweeen GTPCH and DHNA GTPCH generates dihydroneopterin triphosphate and the substrate of the next well-known enzyme, DHNA, is dihydroneopterin. This implies some mechanism for removing the triphosphate. The most probable mechanism seems to involve two enzymes, one removing a pyrophosphate, and the other the last phosphate. The pyrophosphatase has been described in E. coli, and was also recently discovered in L. lactis as belonging to a well known type of pyrophosphatase: the NUDIX enzymes (61; 108). The phosphatase is yet to be identified, but as many of the phosphatases are quite indiscriminate, it is less of a problem (39).

Dihydroneopterin aldolase Dihydroneopterin aldolase (DHNA) catalyses the transformation of dihydroneopterin to hydroxymethyl dihydropterin, and is the first of the enzymes committed purely to folate biosynthesis. It has been described in many organisms, both bacteria, protozoa and plants. As many of the folate enzymes, DHNA can appear both as a monofunctional unit (59; 109), and as part of polyfunctional polypeptides, as in Streptococcus pneumoniae and Pneumo- cystis spp. In both these cases the DHNA activity is coupled to HPPK, and in the case of Pneumocystis spp, also to DHPS (54; 72; 117).

18 The DHNA structure has been determined in E. coli and in Arabidopsis thaliana, and reveals that the enzyme is made up of two tetrameric rings joined head to head. The active sites are evenly spaced out, positioned in the “corners” of the molecule (12; 45). Pterin recognition involves several conserved amino acids, and occurs in a deep, narrow pocket, like it does in DHPS and several of the other folate enzymes (12; 45).

Hydroxymethyl dihydropterin pyrophosphokinase HPPK catalyses the addition of a pyrophosphate moiety to hydroxymethyl dihydropterin.

Enzyme structure The structure of HPPK has been solved for the E. coli, Haemophilus influenzae and Saccharomyces cerevisiae enzymes. The enzyme is an approximately 18 kDa monomer, where the fold is a four-stranded, antiparallel E- sheet. The enzyme has a rigid core containing the binding sites for both substrates. This core is surrounded by regions with less rigidity, including the highly flexible loops 2 and 3 that are crucial for catalysis (45; 64; 107; 113; 123).

Substrate binding and enzymatic mechanism The details of HPPK mechanism and binding of substrate has been studied most closely in the E. coli enzyme. The level of identity between the active sites in HPPK from different organisms is only about 65%, although up to 75% in prokaryotes (107). However, the nine residues involved in pterin binding are conserved in all species sequenced so far, and a recent computer model predicts them to be almost universally so (101; 107; 123). The enzymatic mechanism of HPPK has been studied in detail by several groups. The substrates bind sequentially. Until recently, the proposed mechanism of the first step was that binding of ATP induces a dramatic conformational change allowing hydroxymethyl dihydropterin (HMDP) to bind. Recently, however, this was questioned by Yang and co-workers, who instead propose that the conformation needed to bind HMDP is already there, since loops 2 and 3 are highly flexible, and that binding of ATP only functions as a stabilizer (124). HMDP binding in turn causes another conformational change, assembling the active site. HPPK then catalyses a phosphate transfer from ATP to HMDP, and releases hydroxymethyl dihydropterin-pyrophosphate (HMDPP) followed by AMP. HPPK has a remarkably low turnover number, and the rate limiting step seems to be product release rather than catalysis. This is corroborated by the fact that HPPK has a surprisingly strong affinity for its own product HMDPP, something which might have implications in the possible interactions with DHPS (36; 70; 121; 123).

19 Biosynthesis of pABA pABA derives initially from the shikimate pathway, the same pathway as the aromatic amino acids come from. More specifically, it is generated from chorismate, via a series of steps. In E. coli, an enzyme called ADC synthase, encoded by the genes pabA and pabB, converts chorismate and glutamine to amino-deoxychorismate (ADC) and glutamate. Another enzyme, glutamate lyase (encoded by pabC), then catalyses the transformation of ADC to pyruvate and pABA (39).

Dihydropteroate synthase DHPS catalyses the conversion of p-aminobenzoic acid and hydroxymethyl pterin pyrophosphate to dihydropteroate. It is thus the enzyme combining the two branches of the synthesis of folates. Since DHPS is the target for the sulfonamide class of drugs, it is one of the two most well studied enzymes in this pathway.

Enzyme structure The three dimensional structure of DHPS has been determined in five organisms: Escherichia coli (3), Staphylococcus aureus (43), Mycobacterium tuberculosis (9), Bacillus anthracis (8), and from the eukaryote Saccharomy- ces cerevisiae (64). The average DHPS monomer is approximately 30 kDa in size, with a relatively conserved overall fold. The 3D structure shows that the DHPS molecule generally adopts a TIM-barrel like fold, with the active site in the C-terminal end of the polypeptide. Its normal active form is that of a dimer, with the two active sites relatively far from each other (9). Interest- ingly, the structure, although not the amino acid sequence, is preserved to a great extent across species, enabling the identification of a DHPS homologue in the archaeon Methanococcus janaschii (122).

Substrate binding9 As in many enzymes, substrate binding in DHPS involves several conserved amino acids. The residues involved in binding the pterin moiety of HMDPP are almost absolutely conserved across species, while those binding the pyrophosphate part are allowed some variation. HMDPP binding occurs in a deep pocket inside the barrel structure, and involves amino acids located in nearly all the loops of the enzyme. Loop 2 is of particular interest, as it har- bours the essential arginine in position 60, and was crystallised in different conformations from E. coli and S. aureus. It is suggested to play an important part in enzyme catalysis (8; 9). Due to problems in crystallising an enzyme-substrate complex the exact binding site of pABA is not yet determined, and residues involved in binding

9 All numbers referring to the amino acid sequence derive from the S. pyogenes sequence and its numbering.

20 pABA are less well defined. Babaoglu et al do argue, based on a crystal structure of the enzyme in complex with a dihydropteroate analogue, that binding of pABA should involve the conserved Lys212 and Phe180 (3; 8). This reasoning seems to be confirmed by the B. subtilis structure, where DHPS in complex with sulfonamide shows sulfonamide binding to occur in almost the same place. Baca and co-workers suggest a channel lined by eleven amino acids, two of which also bind HMDPP, where pABA and sulfonamides bind. The residues in this channel vary slightly between species, leading to subtle variations in pABA binding, and differential inhibition by different sulfonamides (9; 53).

Enzymatic mechanism DHPS condenses pABA and HMDPP to dihydropteroate. As it does so, the pyrophosphate moiety is dispensed with. The steady state kinetics of the DHPS reaction has been studied in some detail, showing comparable substrate affinity levels in enzymes from different organisms (41; 84; 90). Very little is known about the exact mechanism of DHPS. Vinnicombe and Derrick have studied the binding order of the two substrates, and report an ordered mechanism where the HMDPP substrate binds first. Their data together with the structure of the S. aureus enzyme also suggest that only one of the two subunits is active at a time (43; 116). Babaoglu and co-workers again have a suggestion. They propose that binding of HMDPP causes conformational change of loop 2 assembling the pABA channel. After the catalytic step where dihydropteroate is synthesised, this loop reassumed its’ original position, thereby releasing the product and the residual pyrophosphate molecule (8).

Dihydrofolate synthase DHFS is the last of the folate enzymes unique to folate synthesizing organisms. In E. coli, and many other bacteria with a de novo folate synthesis, dihydrofolate synthase is part of a bifunctional unit also encoding folylpolyglutamate synthase (39). This is also the case in the protozoan P falciparum (94). In other folate synthesizing eukaryotes such as plants (26; 79) and yeast (94) these two activities are present, but as separate entities (66; 94). DHFS attaches one glutamate moiety to dihydropteroate, thereby making dihydrofolate. Folylpolyglutamate synthase converts tetrahydrofolate, or rather, methyl-tetrahydrofolate, to its polyglutamated forms (39). This process is important for cellular retention of folate, as folates with three or more glutamates do not pass through the cell membrane (17). Worth noting is that in non-folate synthesising organisms, FPGS occurs on its own, as polyglutamation of THF is an important process in all cells.

21 Dihydrofolate reductase Dihydrofolate reductase is the second of of the two most well studied enzymes in this pathway. This enzyme catalyses the reduction of dihydrofolate to the active form tetrahydrofolate. Like GTPCH, DHFR is present also in non-folate synthesizing organisms, since dihydrofolate can be generated from imported folic acid, and is also a product of thymidylate synthase. Theoretically, DHFR is the only source of tetrahydrofolate available to a bacterial cell (96), and is therefore a potential bottleneck in cell growth. Eu- karyotic cells can to some extent import methylTHF, and convert it to THF via methionine synthase (98). Since THF is such a central compound, some bacteria and archaea have derived alternate ways to produce tetrahydrofolate10. One such mechanism uses a variant of thymidylate synthase that generates THF instead of the usual DHF (82). Another possibility is coupling the DHFR activity to another enzyme like in H. pylori, where a weak DHFR activity is located near the N-terminal end of the DHPS enzyme (68). Like the other folate enzymes. DHFR tends to be monofunctional in bacteria, but coupled to thymidylate synthase in protozoa such as P. falciparum. Since this enzyme is an important drug target, not least in cancer therapy, its structure has been determined in detail in many species. The structural determinations revealed that DHFR resembles DHPS in that the structure, although not sequence, is conserved (69). As in many of the other folate enzymes, substrate recognition occurs in a deep pocket, and binding of one substrate causes a conformational change enabling the other to bind (96).

Drugs targeting this pathway

Dihydropteroate synthesis inhibitors

Current sulfonamide use Sulfonamides were among the first antibiotics developed (30). They inhibit the enzyme dihydropteroate synthase by mimicking the substrate p- aminobenzoic acid and thereby competing with this substrate. The first sulfonamide, sulfanilamide, was a metabolite of the dye Prontosil and was in common use during, and even before, the second World War. With time, other sulfa derivatives were developed, but due to side effects and increasing resistance development together with the development of other antimicrobials, their use decreased (1; 105). In a worldwide perspective, sulfa drugs are still among the most fre- quently used antibiotics. This status is probably due to their use as antimalar-

10 This is not in response to antibiotics, but instead a perfectly normal natural variation. Many of these organisms do not have dihydrofolate reductase.

22 ials in combination with pyrimethamine. They are also of importance as prophylactic treatment of Pneumocystis pneumonia in HIV patients, and for treating severe respiratory disease in children in Africa, Asia and in South America (48) Sulfonamides are commonly used in combination with dihydrofolate reductase inhibitors, as there has proven to be some synergistic effect when using them together. It is not quite clear how this synergistic effect is achieved, since dihydrofolate is not produced only by the folate synthesis pathway, but also as a product of thymidylate synthase. One possibility is that blocking the de novo folate synthesis leads to a depletion of DHF. DHFR will then be more prone to binding the drug pyrimethamine, since there is less substrate to compete with the drug.

Mechanism of sulfonamide resistance Sulfonamides are analogues of the DHPS substrate pABA, and resistance is achieved by mutating DHPS to reduce enzyme efficiency in binding the drug. The inhibition is competitive, so the target organisms are generally not killed but only hindered in growth. Since sulfonamides have been popular drugs, their target DHPS has been thoroughly investigated with respect to resistance mutations. In bacteria, there are several well-defined areas of the enzyme where mutations conferring resistance to sulfonamides occur (9; 33; 41; 59; 90) These sites seem in most cases to be positioned in or near conserved regions of the enzyme, and near residues involved in substrate binding. Resistance to sulfonamides is even more well studied in Plasmodium spp., the organisms causing different forms of malaria, since sulfonamide drugs have been important in treating this disease, and still remain so in some countries. A number of the amino acids known to be involved in sulfonamide resistance are the same as those in other microorganisms, underlin- ing the degree of conservation in the substrate binding sites (50; 51). The different resistance mutations can to some extent have an additive effect (16; 52). It has been proposed based on the M. tuberculosis structure that all the sulfonamide resistance mutations affect the channel where the substrates pass to the active site (9). This area is allowed some sequence (and thereby structural) variation, which leads to the fact that the different kinds of sulfonamide do not all inhibit all DHPS enzymes equally well (53). Despite the fact that sulfonamides inhibit DHPS, the drug can to some extent be metabolised by the enzyme. The reaction product sulfa- dihydropteroate is however a competitive inhibitor of DHFS in E. coli, and possibly of DHFR in yeast (87; 93). This toxicity can be countered by the yeast cell by increasing the production of dihydrofolate reductase (83; 87), but adding higher concentrations of the antibiotic will kill the cell.

23 Another way to achieve resistance to sulfonamides is to bypass the pathway entirely. Such an example is found in P. falciparum, where Wang and co-workers report salvage of folic acid from the host blood stream. Since folic acid has to be reduced before it can be metabolized by the cell, they also looked for such a folate reductase activity, and suggest that it is located in DHFR-TS (118). Finally, the suggestion that resistance to sulfonamides can be achieved by overexpression of p-aminobenzoic acid is often put forward. This is an at- tractive hypothesis, but so far, no microbial system where this is the case has been identified (25).

Inhibitors other than sulfonamides The pABA analogue p-aminosalicylic acis (pAS), a drug used as second-line treatment of tuberculosis, was recently shown to inhibit the folate pathway in mycobacteria (92). To date, there is no other class of drugs targeting DHPS. Some studies have been made of potential pterin analogues, but they are only at a very experimental stage (8; 67). There are also several other reports covering non- sulfonamide pABA analogues. They demonstrate that different substituents to the benzene ring can cause inhibition of DHPS, and that this inhibition is not universal (inhibits either the E. coli enzyme or the P. carinii enzyme) (57; 63). However, none of these have, to my knowledge, reached further than the experimental stage11.

Dihydrofolate reductase inhibitors There are many drugs targeting dihydrofolate reductase, and new ones are being continuously developed (23; 86). Due to small variations in the substrate binding sites of DHFR, it has been possible to develop DHFR inhibitors that target only one type of DHFR (mammalian, protozoan, bacterial), thereby achieving specificity and decreasing the risk with use. Metotrexate, a close analogue of folic acid, targets the mammalian enzyme, and is used mainly in cancer therapy. Trimethoprim and pyrimethamine target the bacterial and protozoal enzymes, respectively. All these drugs are competitive inhibitors of DHFR. They bind in almost the same conformation as the DHFR substrate dihydrofolate, but with the heterocyclic ring inverted to increase enzyme affinity for the drug (18). Inhibiting DHFR blocks the reduction of dihydrofolate to tetrahydrofolate. The single most important effect of this is the depletion of the DNA building block dTMP. Other effects include depletion of tetrahydrofolate in the cell leading to a greatly reduced one-carbon turnover, and a decrease in

11 Possibly because there is no money in developing drugs that will be used mainly in developing countries.

24 the amount of formylated methionines (f-Met) in the cell. As f-Met is especially important for translation initiation in bacteria, protein translation and thereby cell growth is markedly slowed (17). The effects on other cells are of course similar, as they all affect protein turnover in some way. Resistance to DHFR inhibitors is ultimately achieved by point mutations in the enzyme reducing the efficiency of drug binding. However, in this case it is not as easy as in that of DHPS to say that point mutations in the enzyme were selected for in response to the antibiotic. There are many variations in the DHFR enzyme, and different variants bind the inhibitory drugs unequally well, a fact aptly illustrated by the different inhibitors for the mammalian, protozoan and bacterial enzyme, respectively. Resistance can be, and is, thus often achieved by acquiring a drug resistant DHFR gene by horizontal transfer. It is also possible to indirectly cause resistance by relieving the depend- ence on thymine produced by thymidylate synthase, and thereby reducing the need for THF produced by DHFR. This can be done by becoming a thymine auxotroph (49; 105), and possibly by using an alternate thymidylate synthase, thyX, and thereby reducing the need for reducing dihydrofolate (65).

Other For the treatment of parasite and bacterial infections, the only antifolates are the ones described above. Some work has been done towards synthesizing inhibitors to hydroxymethyl dihydropterin pyrophosphokinase (HPPK) and DHNA, and has yielded some success (95; 102). In anticancer therapy, there is also the possibility of using thymidylate synthase inhibitors like 4-fluoro-uracil, but as they are not strictly antifolates, and otherwise beyond the scope of this thesis, I will not mention them further.

25 Polyfunctionality in proteins

Polyfunctional proteins Many of the enzymes participating in the folate biosynthesis are part of polyfunctional proteins. Examples include HPPK-DHPS and DHFR-TS in most protozoa, and HPPK-DHNA in S. pneumoniae. There are also some enzymes combining three activities on the same polypeptide, like the FAS proteins in for example yeast and Pneumocystis spp (54; 117). This kind of organisation might reflect an evolution from single proteins. A step on the way here could be the organisation of the genes in operons, such as the folate synthesis genes in Streptococcus pyogenes and other Gram-positive organisms (for an example see paper II in this thesis). There are reports showing that both some aminoacyl tRNA-synthetases and one of the steps in histidine biosynthesis are the products of gene fusions having generated bifunctional proteins (15; 20). The line of reasoning is that coordi- nation of the flow of metabolites is facilitated if the activities are clumped together. Brilli and Fani suggest that polyfunctionality in eukaryotes is due to the fact that there are no operons (producing polycistronic transcripts) in eukaryotic cells, and that polyfunctionality might therefore be a way to co- regulate gene products. The different activities present on each polypeptide can be more or less dependent on each other. In the P. falciparum DHFR-TS, the DHFR activity can function without TS, while the opposite is not the case (120). This is probably due to the fact that the very beginning of a polypeptide often is important for correct folding properties, and indeed, the TS moiety does function when provided with the beginning of DHFR.

Substrate channelling One convenient side-effect of expressing several activities in the same protein is that the active sites are relatively close to each other. In some proteins, this is used to push the product of one activity directly to the other site. Substrate channelling is a phenomenon involving the direct transfer of the product of one enzyme to the active site of another, without diffusion through the surroundings. Channelling behaviour is believed to be quite common, although there are surprisingly few fully described cases (5). The basic idea is that if intermediates are kept out of the bulk solution, unfavour- able conditions will be less of an impediment to the reaction. Catalytic efficiency may be retained while keeping substrate and product concentrations at low levels. Furthermore, reaction intermediates are isolated from competing reactions and unstable intermediates may be protected (106).

26 There are several ways to measure the occurrence of channelling (5), but they generally have in common that they determine the amount of intermediate in the bulk solution. Ideally, if there is channelling, one should not be able to find any such intermediate. Another possible way is to measure the transient time, that is, the time needed for the intermediate to move from one active site to another. Again, if there is channelling, there should be no time (or a very short time). Channelling of intermediates between two active sites has potential for exploitation as a drug target (6). If the interaction is necessary for full enzymatic behaviour, interfering with this reaction might reduce viability of the organism.

Channelling of folates Many of the enzymes involved in folate synthesis and utilization are part of polyfunctional entities. In eukaryotic cells, they are often also found together in different compartments in the cell. This suggests that there might be some kind of communication between the different activities, and the idea of substrate channelling has been put forward. The first evidence of channelling of folates was presented already in the 1980s, when it was demonstrated in the human bifunctional methyle- neH4folate dehydrogenase and methenylH4folate cyclohydrolase (88) and in the enzyme with the formidable name formiminoglutamate H4folate for- miminotransferase-formiminoH4folate cyclodeaminase (74). The latter has the peculiarity of channelling more efficiently the more glutamated the product is - possibly part of a mechanism ensuring that the optimal substrates get to the right place. There are also examples of folate systems where channelling of intermediates has been disproven. In Pisum sativum (that is, pea) the bifunctional HPPK-DHPS localised in the mitochondrion does not show any indication of this type of interaction (81). Channelling of intermediates has also been found in protozoans. The subunits of DHFR-TS in Leishmania major communicate in this way, by passing DHF through an electrostatic channel on the outside of the protein. The same enzyme from Cryptosporidium hominis however does not work in this way. Atreya and Andersen suggest that this be due to the existence of two distinct families of enzymes with structural and mechanistic differences (7; 71). Channelling of folates is also a possibility in the malarial DHFR-TS and HPPK-DHPS, although this still remains to be proven ((126), and this thesis). In conclusion, substrate channelling of folates is an area well worth inves- tigating further, as more and more of the folate enzymes are characterised structurally and mechanistically.

27 Aims of this thesis

The aims of this thesis were, apart from studying resistance to antibiotics in general (Paper I): 1. To investigate the role of the commensal flora as a resistance reservoir. 2. To study sulfonamide resistance of the S. pyogenes dihydropteroate synthase (Paper II) 3. Make a more detailed study of HPPK-DHPS from P. falciparum, with particular focus on HPPK (Papers III and IV). 4. Investigate the drug target potential of the HPPK part of the enzyme.

28 Results and discussion

Emergence and spread of antibiotic resistance

Response to an antibiotic treatment (Paper I) Increasing levels of microbial resistance to antibiotics has led to a rising interest in the commensal microbiota of both man and animals (4; 89; 104). There is an increasing understanding of the commensal flora as a factor in maintaining and transferring resistance to antimicrobials. The aim of the study discussed here was to look at the long term effects of a single antibiotic treatment on the human commensal flora. More than a hundred dyspeptic patients having been given a standard triple treatment with clarithromycin, omeprazole and metronidazole against stomach ulcer were followed for three years. All patients were screened for resistance to the macrolide clarithromycin. In addition to this, some patients were studied in more detail, and screened for the presence of genes conferring resistance to clarithromycin, and for effects on the clonal variation. In paper I, we present results from the throat samples (more specifically commensal D-streptococci and Neisseria spp). Nose and faeces samples were studied separately (103; 104). The results showed, not unsurprisingly, that resistance to clarithromycin increased markedly in response to the antibiotic treatment, but also that the resistance levels needed between one and three years to drop when the treatment had been completed (Figure 5).

29 Figure 5. Comparison of zone diameters for the streptococcal isolates. The number of isolates is depicted against their clarithromycin inhibition zones. Zone diameters are expressed in millimetres. Each graph shows the distribution of various inhibition zones from one time point during the study (columns), with the distribution of the A samples added for reference (dashed line). Stars indicate the statistical significance of the differences between the A samples and the B, C or D samples, respectively, as determined by one-way ANOVA and Tukey’s multiple comparison test. A - D cor- responds to the collection time: before, one week after, one year after or three years after treatment.

30 Several studies have noted the presence of macrolide resistant viridans streptococci in healthy people (55; 73; 99). Since the whole idea of antimicrobial therapy relies on there not being resistant microorganisms in the body, this is an alarming prospect. Our results showing a very slow drop in resistance levels, correlated with the relatively high use of antibiotics in many countries may explain this phenomenon (24). If it is indeed the case that resistance levels need several years to return to baseline level, then any treatment during this time would select for resistant bacteria, and push resistance levels up again. Levels of resistance would thus never be allowed to drop to low levels before a new antibiotic treatment was administered. In fact, pre-treatment levels of resistance were not reached until one to three years after the finished treatment. An interesting observation was that after three years without any antibiotic treatment whatsoever, the patients actually showed less resistance than they had done before the treatment began. This gives an indication of the timescale needed when discussing antimicrobial resistance.

Transfer of resistance genes One of the most prominent results of the above study was a dramatic increase in the frequency of the macrolide resistance determinant mef(A) in the streptococcal isolates, and the persistence of this gene for more than a year after treatment. Clonal expansion, as seen in the Neisseria spp. could not be the only answer, as there was a large mix of streptococcal clones. The most logical was to suspect some way of horizontal transmission. The mef(A) gene is normally located on a defective transferable element called MEGA (37). In work pursued as continuation of that mentioned above, we demonstrate that this MEGA element can be transferred by conjugation to the pathogen Streptococcus pyogenes. These results are doubly interesting, as S. pyogenes does not normally have the ability to receive genes by transformation (44), and because MEGA does not carry genes encoding any transfer mechanism. To explain these results, we studied the sequence up-and downstream of MEGA, and found that immediately following the downstream sequence was sequence homologous to a large, conjugative element, ICESt1, described in Streptococcus termophilus (21; 22). Work is currently going on to determine the exact extent of this ICESt element.

An example of horizontal transfer (Paper II) We show above that treatment with macrolides selects for stably resistant viridans streptococci. Such stability implies that the clones carrying the resistance genes have in some way compensated for the fitness cost, and can compete on equal terms with the rest of the microbial population (75). Stable resistance can stay in a population for a very long time, as is the case with sulfonamide resistance. Sulfonamides were widely used into the seventies,

31 but not since. Despite this, highly resistant clones were found during routine surveillance in 1985 (109). Resistance to sulfonamides is due to mutations in the DHPS gene. In several streptococcal species including S. pyogenes, this gene is part of an operon encoding genes necessary for de novo folate biosynthesis. When comparing the nucleotide sequence of this operon from different sulfonamide sensitive and sulfonamide resistant Streptococcus pyogenes strains, it was obvious that the operon consisted of sequence parts derived from more than one origin. Parts with perfect homology were interspersed with regions with 15-30 % sequence dissimilarity. The highest degree of variation was seen in the most resistant strains, implying that the sulfonamide resistance is due to the sequence variation. This kind of mosaicism has also been noted in penicillin binding proteins in S. pneumoniae, and in the 16S rRNA genes in the same organism (31; 97). It is generally believed that mosaic genes are formed by horizontal transfer. Spontaneous mutation is not probable, as too many amino acids differ (in comparison, nine S. aureus isolates from different geographical locations showed only small differences (43). In some cases (for an example see (91)) it is possible to tell from which organism the sequence derived. If however the sequence has deviated too much from the original, as might be the current case, this is not always possible. Although horizontal transfer is the most probable explanation for the sequence variation mentioned above, it remains an interesting question where it came from, since S. pyogenes is generally not believed competent for transformation. Conjugation and transduction still remain possible mechanisms, as there are several prophages in the S. pyogenes genome, and because we show above that macrolide resistance can be transferred from commensal streptococci to S. pyogenes by conjugation.

Current sulfonamide targets

The importance of specific amino acids for sulfonamide resistance (Paper II) The substrate binding sites of DHPS and other folate biosynthesis enzymes are conserved in many species (9; 51). This gives a hint that small changes in sequence might cause significant changes in substrate and/or inhibitor binding properties. In the DHPS enzyme, it is well established that specific amino acids are involved in sulfonamide resistance (8; 51). The mosaic operons discussed above were closely studied to determine any obvious amino acid changes that might be the cause of the high resistance.

32 Ec ------MKLFAQGTSLDLSHPHVMGILNVTPDSFSDGGTHNSLIDAVKHANLSSFSF 46 Mt ------MSPAPVQVMGVLNVTDDSFSDGGCYLDLDDAVKHGLASSFSF 37 Sp ------MKIGKFVIEGNAAIMGILNVTPDSFSDGGSYTTVQKALDHVEQSSFSF 43 Sa ------MTKTKIMGILNVTPDSFSDGGKFNNVESAVTRVKASSFSF 35 Pf KEQYNINIKENNKRIYVLKDRISYLKEKTNIVGILNVNYDSFSDGGIFVEPKRAVQRMFESSFSF 420 ::*:***. ******* . *: :

Ec MINAGATIIDVGGESTRPGAAEVSVEEELQRVIPVVEAIAQRF------EVWIS 94 Mt MAAAGAGIVDVGGESSRPGATRVDPAVETSRVIPVVKELAAQ------GITVS 84 Sp MIADGAKIIDVGGESTRPGCQFVSATDEIDRVVPVIKAIKENY------DILIS 91 Sa MMDEGADIIDVGGVSTRPGHEMITVEEELNRVLPVVEAIVGF------DVKIS 82 Pf MINEGASVIDIGGESSGPFVIPNPKISERDLVVPVLQLFQKEWNDIKNKIVKCDAKPIIS 480 * ** ::*:** *: * * . *:**:: : :*

Ec VDTSKPEVIRESAKVG-AHIINDIR-SLSEPGALEAAAETG--LPVCLMHMQGNPKTMQE 150 Mt IDTMRADVARAALQNG-AQMVNDVSGGRADPAMGPLLAEAD--VPWVLMHWRAVSADTPH 141 Sp IDTYKTETARAALEAG-ADILNDVWAGLYDGQMFALAAEYD--APIILMHNQDEE----- 143 Sa VDTFRSEVAEACLKLG-VDIINDQWAGLYDHRMFQVVAKYD--AEIVLMHNGNGN----- 134 Pf IDTINYNVFKECVDNDLVDILNDISACTNNPEIIKLLKKKNKFYSVVLMHKRGNPHTMDK 540 :** . :...... ::** . : : . ***

Ec AP-KYDDVFAEVNRYFIEQIARCEQAGIAKEKLLLDPGFGFGKNLSHNYSLLARLAEFHH 209 Mt VPVRYGNVVAEVRADLLASVADAVAAGVDPARLVLDPGLGFAKTAQHNWAILHALPELVA 201 Sp ---VYQEVTQDVCDFLGNRAQAALDAGVPKNNIWVDPGFGFAKSVQQNTELLKGLDRVCQ 200 Sa ---RDEPVVEEMLTSLLAQAHQAKIAGIPSNKIWLDPGIGFAKTRNEEAEVMARLDELVA 191 Pf LT-NYDNLVYDIKNYLEQRLNFLVLNGIPRYRILFDIGLGFAKKHDQSIKLLQNIH--VY 597 : :: : *: .: .* *:**.*. ... :: :

Ec FNLPLLVGMSRKSMIGQLLN-----VGPSE-RLSGS------239 Mt TGIPVLVGASRKRFLGALLAGPDGVMRPTDGRDTAT------237 Sp LGYPVLFGISRKRVVDALLGG----NTKAKERDGAT------232 Sa TEYPVLLATSRKRFTKEMMGYD---TTPVE-RDEVT------223 Pf DEYPLFIGYSRKRFIAHCMNDQNVVINTQQKLHDEQQNENKNIVDKSHNWMFQMNYMRKD 657 *::.. *** . : .

Ec ------LACAVIAAMQGAHIIRVHDVKETVEAMRVVEATLSAKENKRYE- 282 Mt ------AVISALAALHGAWGVRVHDVRASVDAIKVVEAWMGAERIERDG- 280 Sp ------AALSAYALGKGCQIVRVHDVKANQDIVAVLSQLM------266 Sa ------AATTAYGIMKGVRAVRVHNVELNAKLAKGIDFLKENENARHNFS 267 Pf KDQLLYQKNICGGLAIASYSYYKKVDLIRVHDVLETKSVLDVLTKIDQV------706 . : . : :***:* . . :

Figure 6. Multiple alignment of dhps from several species. Boxes show residues involved in sulfonamide resistance. Worth noting is that they tend to be located near conserved regions of the enzyme. Arrows indicate positions 67 and 213, respectively. Stars and dots underneath the alignment indicate the degree of homology. Species names are abbreviated as follows: Ec: Escherichia coli; Mt: Mycobacterium tuberculosis; Sp: Streptococcus pyogenes; Sa: Staphylococcus aureus; Pf: Plasmo- dium falciparum.

Pos 213 Position 213 is located near a stretch of conserved amino acids, in the beginning of loop 7. This region of the enzyme is involved in pterin binding and has several residues known to be of importance for sulfonamide resistance. One of these is the structural arginine in position 213 (Figure 6) (8). When comparing the sequence from several sulfonamide sensitive and resistant strains, it was notable that some resistant strains did not have an ar-

33 ginine in position 213, but a glycine. To investigate the importance of this amino acid difference, we used site directed mutagenesis to change the amino acid from an arginine to a glycine, or vice versa. This substitution resulted in a change in the MIC for sulfathiazole (Table 1). Although of importance, it was clear that this amino acid could not be solely responsible for the resistance to sulfonamides, as mutating it could neither revert nor restore the resistance completely.

Insertions after pos 67 Two of the highly resistant strains described above did have an arginine in position 213. This meant that the reason for the resistance had to be found elsewhere in the protein. These two strains proved to have a two amino acid insertion in the conserved part of helix 2. Insertions in this region have been shown to confer high sulfonamide resistance in H. influenzae and in S. pneumoniae (33; 41), and mutation of pos 61 to confer high level resistance to N. meningitidis (90). Deletion of these two amino acids in strain G72 resulted in a tenfold lowering of the MIC value (Table 1).

These two cases clearly show that one amino acid change can be of importance for the enzyme as a whole, and influence, or even cause, resistance to an antibiotic. The next step was to determine whether these changes also affected substrate binding.

Table 1. Comparison of the kinetic properties of sulfonamide susceptible and resistant S. pyogenes strains.

Strain pos 213Insertion MIC for Stz Km for p-AB, Km for pteridine Ki for Stz after mM µM µM µM pos67 G56 Gly No 2 8.5 55.46 7.6 G56 GĺR Arg No 1 1.49 4.16 0.26 G1 Arg No <0,02 1.44 25 0.05 G1 RĺG Gly No <1 2.3 16.4 2.5 G72 Arg Yes 2 18.33 41.8 3.15 G52 Arg Yes 2 8.21 16.2 3.19 G72'VA Arg No 0,25 n.d. n.d. n.d.

Effects on substrate binding (Paper II) If the regions around pos 213 and pos 67 are important for sulfonamide resistance, it is logical that they should also have some effect on substrate 12 binding. Determination of the kinetic parameters Km and Ki for the sub-

12 Km and Ki are kinetic constants signifying the enzyme’s affinity for its substrates and inhibitors, respectively. High values correspond to low affinity, and vice versa.

34 strates HMDPP and pABA and for the inhibitor sulfathiazole in the strains described above demonstrates that this is indeed the case (Table 1). Position 213 is located in a region of the enzyme involved in both pterin and pABA binding, and seems to be one of the key residues in binding pABA (8; 9). Changing the glycine in the sulfonamide resistant strain to the wild-type arginine reverts the pABA Km to the value of the wild-type, which is to be expected of an amino acid closely implied in substrate binding. The back mutation, from arginine to glycine done in the strain G1, does not quite have the same profound effect, suggesting that the G56 strain has in some way compensated for its bad pABA binding. It can be expected that the insertion after position 67 have a similar effect, since this loop is crucial for catalysis (8). These experiments still remain to be carried out, as we encountered difficulties in overexpressing the protein from the deletion mutant.

Studying the possibilities of channelling

The Km determinations in the S. pyogenes DHPS showed that this enzyme has elevated Km for its substrate dihydropteridine pyrophosphate. A possible explanation for this might be a dependency on the enzyme producing this substrate: HPPK. Since we could not detect any strong or long lasting such interaction, we suspected that a more transient type of interaction such as channelling of product from one enzyme to the other might be the case. However, due to problems in cloning the streptococcal enzymes, these experiments could not be carried out. We therefore wanted to use the bifunctional HPPK-DHPS from P. falciparum as a positive control for the system. The rationale behind this was that channelling had been shown to occur in the bifunctional enzyme DHFR-TS in Leishmania major (71), and it seemed logical that this also be the case in another protozoan bifunctional enzyme like PfHPPK-DHPS. Isotope dilution experiments (106) performed with increasing concentrations of unlabelled dihydropteroate pyrophosphate did however indicate that there is no measurable amount of channelling between the two active sites. As described below, the two activities do however still seem to depend on each other for full functionality. The absence of channelling in PfHPPK-DHPS need not necessarily ex- clude the possibility of channelling occurring between the enzymes in a different organism, such as S. pyogenes. Such interactions could be due to species differences rather than to different enzymes. This idea is supported by the fact that for example DHFR-TS shows channelling in L. major (71), but not in Cryptosporidium (7). Another indication of some kind of interaction is the fact that HPPK and DHPS are placed in close proximity in the genome of many organisms. This kind of organisation has been thought to support the idea of an interaction between the enzymes involved (32).

35 The DHPS of PfHPPK-DHPS part does not function on its own (Paper IV) The DHPS part of the P. falciparum bifunctional HPPK-DHPS is quite well studied, and much is known about how inhibitors such as sulfonamides act on it. However, to date only few attempts have been made of other potential ways of inhibiting the enzyme (for an example, see (86)). One aim of the work presented in paper 4 was therefore to cast more light on how the different parts of this enzyme "cooperate", with the idea in mind that further characterisation of the enzyme could facilitate development of new inhibitors.

1 HPPK 1465 DHPS 2647

a b c d e

Figure 7. The different PfHPPK-DHPS clones. (a) and (b) correspond to clones where the the boxed regions in figure 8. are deleted (c) - (d) show the extent of the DHPS clones.

The first step was to determine whether the DHPS part of the enzyme could function at all if it lacked the HPPK sequence preceding it, so we subcloned the DHPS part of the sequence and transformed it into a folP13 knockout (Figure 7) (35). The result was complementation failure, indicating that the DHPS activity of the enzyme is crucially dependent on the HPPK part for functionality. The DHPS half of the enzyme was then cloned with different amounts of the C-terminal end of the HPPK-part (Figure 7), in order to determine whether the presence of HPPK sequence would promote correct folding and restoration of some DHPS activity. Complementation tests performed in E. coli C600'folP were again negative, indicating that either more of HPPK, or different parts of HPPK, are needed for even a residue of DHPS activity.

13 folP is the gene encoding DHPS.

36 Interestingly, in the recently described Pneumocystis jirovecii trifunctional FAS polypeptide, the 11 N-terminal amino acids seem to be necessary for DHPS activity (54), and the 22 N-terminal residues are equally important in the Pneumocystis carinii DHPS (11). The is also the case in the P. falciparum DHFR-TS, where the 14 N-terminal amino acids of the DHFR moiety are crucial for TS activity (120). Following this line of reasoning, DHPS activity could be restored to the P. falciparum enzyme by including the N- terminal end of HPPK instead of the C-terminal end.

Possible new drug targets

The HPPK part functions on its own (Paper III) This bifunctional folate enzyme HPPK-DHPS is already the target for sulfonamides. However, the increasing amount of resistance to this drug has made it necessary to look for new drug targets. One possible such target is the previously unexploited HPPK-part of the enzyme, and we have therefore begun studying PfHPPK-DHPS in further detail. Our main interest was to see whether or not it was possible to separate the two functions from each other, and we accordingly subcloned the hppk part of the gene for expression studies. Comparisons with other enzymes showed that for example the DHNA and HPPK activities from the Pneumocystis carinii trifunctional FAS polypeptide could be expressed separately without losing activity (11). To see that this truncated version of the protein actually worked, we used an E. coli strain where the folK14 gene had been knocked out (Paper III). The hppk subclone complemented the knockout, but did not restore all the “vitality” of the original clone. Determination of the protein activity levels using radioactively labelled pABA confirmed that the truncated protein did indeed seem to function less well. We therefore studied the expression levels of the protein, and found them to be lower than for the full length peptide, but not so low as to explain all the loss of activity. The conclusions we drew were that the HPPK moiety of the bifunctional enzyme can function on its own, but is dependent on DHPS for full activity.

14 folK encodes HPPK.

37 * * Ec ------MTVAYIAIGSNLASPLEQVNAALKALGDIPESHILTVSSFYRTPPL* 46 Mt ------MTRVVLSVGSNLGDRLARLRSVADGLGDA----LIAASPIYEADPW 42 Sp ------MTIVYLSLGTNMGDRAAYLQKALEALADLPQTRLLAQSSIYETTAW 46 Sa ------MIQAYLGLGSNIGDRESQLNDAIKILNEYDGINVSNISPIYETAPV 46 Pf METIQELILSEENKTNIAVLNLGTNDRRNAVLILETALHLVEKYLGKIINTSYLYETVPE 60 Pv ---MEDSNTGGTTPRNIAVLNFGTNDKKNCVTILETALYLTEKYLGKIINSSYIYETVPE 57 Pb ------ENNKGNIVVLNFGTTDKTNAVTILEAALYLTEKYIGKIINTSYMYETVPE 50 Pc ------ENNKGNIVVLNFGTTDKTNAVTILETALYLTEKYIGKIINTSYMYETVPE 50 . : .*:. : . * : : * :*.: .

Ec GPQDQPD------53 Mt GGVEQGQ------49 Sp GKTGQAD------53 Sa GYTEQPN------53 Pf YIVLDKKESCEKINKDCRIYDVNYINELMQNLEESKYE----ENKELIDKCEEYETFLKNCEKINKDCRIYDVNYINELMQNLEES 116 Pv YIVLDEENKIGEVTEGEPPRDISWIGDLIPTVENSRYE----ESEDLIYECKELEVFLKN 113 Pb YVVLDKSDIPKNIIGEDDPYDGSSLNDLVKGLEKSKYENVFQGEENLVSQCEEYERFLNN 110 Pc YIVLDKSNIPNNIIGEDDPYDVSSLNELVNGLENSKYENVFQEDESLVSQCEEYEIFLNN 110 : .

Ec ------YLNAAVALET-TLA* * 66 Mt ------FLNAVLIADDPTCE 63 Sp ------FLNMACQLDT-QLT 660 Sa ------FLNLCVEIQT-TLT 66 Pf GKVDNSILKEVNVENYLLECNNIIVKNDEIMKNNLSKYKDKYYTSYFYNLTVVVKTFVND 176 Pv EKINESIIREVSVEDYENEARRIIKRNDEIMKKNLEQSKDKYYTSYFFNLTVVVRTFVED 173 Pb KDLFENKIKQISTEKYESETSNIIKENDEIMKINLEKHKNKYYTSYFYNLVVVFKCFIDD 170 Pc KDLFENKIKQISVEEYKTEASNIIKENDEIMKINLEKHKNKYYTNYFYNLAVVFKSFIDD 170 : * # # # #* # # Ec PEELLNHTQQIELQQG--RVRK--AERWGPRTLDLDIMLFGN------104 Mt PREWLRRAQEFERAAG--RVR---GQRWGPRNLDVDLIACYQTSATEALV------108 Sp AADFLKETQAIEQSLG--RVR---HEKWGSRTIDIDILLFGE------103 Sa VLQLLECCLKTEECLH--RIR---KERWGPRTLDVDILLYGE------103 Pf PLSMLVVIKYIEELMKRENVK--EKEKFENRIIDIDILFFNDFTIFMKNIKLEKNMIYKI 234 Pv PLAMLVILKYIEQIMKRRESKKGQGEIFQNRMIAIDILFFNNYPIFEKSISLKGEDIYKI 233 Pb PLNLLVILKYIEHLMKRKNSK--EVEKFENRLIDIDILFFNNYTIFEKNINLTKNDLYTI 228 Pc PLHLLVILKYIEHLMKRKNSK--EVEKFENRLIDIDILFFNNYTIFEKNINLTKDSLYAI 228 * * . : : : * : :*:: :

Ec ------Mt ------Sp ------Sa ------Pf LSKYIHLERDIKNGNDNMSKVNMDKDINLNNNNNIKKKNNNDIDCDCVDQKMNNHVNNKN 294 Pv ITKYIHIN------HTSDQN 247 Pb MCKYINIEYDNSS------SDNCNKL 248 Pc MCKYINIECD------DNCNKR 244 # ## # Ec ------ETINTERLTVPHYDMKNRGFMLWPLFEIAPELVF------* 138 Mt ------EVTARENHLTLPHPLAHLRAFVLIPWIAVDPTAQL------143 Sp ------EVYDTKELKVPHPYMTERAFVLIPLLELQPDLKL------137 Sa ------EMIDLPKLSVPHPRMNERAFVLIPLNDIAANVVE------137 Pf YINSFRDPQEIINNMVDNIEFLSIPHVYTTHRYSILLCLNDMIPEYKHNVLNNTIRCLYN 354 Pv RLD------IIQNLGDKIEFLCIPHVYTKYRYSILLCLNDIIPEYKHSTFEEAIRSTYN 300 Pb SRN------IEEIKDNIKFLSIPHVYTKHRYSILLCLNDIMPQYKHNALKETINKSHE 300 Pc LKD------IEQIKDNIKFLSIPHVYTKHRYSILLCLNDIIPNYKHNTLKETINNLYE 296 * :** * :* : .

Ec ------PDGETLREILTSLAVLQPAIWY------160 Mt ------TVAGCPRPVTRLLAELEPADRDSVRLFRPSFDLNSRHPVSRAPES- 188 Sp ------PPN--HKFLRDYLAALDQSDITLFSAQQTEF------166 Sa ------PRS--KLKVKDLVFVDDSVKRYK------158 Pf KYVSRMKEQYNINIKENNKRIYVLKDRISYLKEKTNIVGILNVNYDSFSDGGIFVEPKRA 414 Pv SYVESFEEKYHINIRKNNKRLYVLKDKVSYLKERTHIVGILNVNYDSFSDGGLFVDPVKA 360 Pb EFITSFSRLYNTCIKKYNKRLYVLKNEVVCLKEKTDIGGILNTNYNSFSDGGLFVKPNIA 360 Pc EFITNFSKLYNTCIKKYNKRLYVLKNEVLYLKEKTNIVGILNTNYNSFSDGGLFVKPDIA 356 :

38 Figure 8. Multiple sequence alignment of hppk. Stars and hashes above the alignment indicate pterin and ATP binding residues, respectively. Boxes show the deleted parts of PfHPPK-DHPS. Stars and dots underneath the alignment indicate the degree of homology. Species names are abbreviated as follows: Ec: Escherichia coli; Mt: Mycobacterium tuberculosis; Sp: Streptococcus pyogenes; Sa: Staphylococcus aureus; Pf: Plasmodium falciparum; Pv: Plasmodium vivax; Pb: Plasmodium berg- hei; Pc: Plasmodium chabaudi,

This again parallels the DHFR-TS case, as the DHFR from P. falciparum can be expressed as an active polypeptide separated from TS, but its activity is enhanced when the TS activity is added (42).

How much of HPPK is actually necessary? (Paper IV) Depending on the time since a gene fusion event occurred, a genome can become more or less reorganised, and retain or remove DNA from genes. This could or could not affect the protein structure. When comparing the HPPK sequence from several bacterial species with that of P. falciparum, it became clear that there were two regions in the enzyme that had no homology with bacteria (115). One of these proved to be present in several other plasmodial species, but a part of the other was apparently unique to P. falciparum. When comparing this sequence with that of E. coli, where the 3D structure has been determined in great detail (123) the predicted three- dimensional structure of these regions is two long, flexible loops, protruding from the core of the enzyme. Fitting them into the E. coli sequence puts both in semi-conserved regions of the enzyme, and the second near several residues involved in substrate binding (123). In order to investigate the importance of these "insertions", we constructed a set of deletion mutants encom- passing different amounts of sequence. Deleting the entire first region rendered a non-functional polypeptide. Preliminary data from deleting the last region suggests that this also is crucial for enzyme function. As concluded from the E. coli and H. influenzae structures, the first insertion is located in the core of the enzyme, between two sets of pterin binding residues, and removing it does obviously disrupt the enzyme. It can be speculated that a long loop in an otherwise rigid core ought to be of some structural importance. The second insertion seems to be in a region of the enzyme involved in ATP binding, and it is not surprising that deleting it removes all functionality. We first thought the sequence divergence of little importance, as the puta- tive loop regions lacked homology in other species. However, since deleting them had such a devastating effect on enzyme function, it seems more probable that they have some hitherto unknown plasmodium-specific effect. In the recently described structure of the bifunctional DHFR-TS from P. falciparum, Yuvaniyama and coworkers suggest that similar insertions in this enzyme might be of importance for interdomain communication (127). This

39 is a possibility in the current case, but effects on stability, catalysis etc can not be excluded without structural characterization of the enzyme.

Development of a spectrophotometrical assay for HPPK activity (Paper IV) All previous assays for HPPK activity are coupled to DHPS (14; 123). In order to facilitate kinetic studies of HPPK alone, I developed a new assay, where I coupled the HPPK activity to oxidation of NADPH to NADP+. (Fig- ure 9) This degeneration of NADPH is possible to monitor in a spectropho- tometer at 340 nm. The ratio of produced AMP to oxidized NADP+ is 1:1, enabling a direct measurement of the HPPK activity. Decoupling HPPK and DHPS activity in this way provides a possibility to study interactions between the two enzymes, and, since the assay is coclorimetric, to get an online view of the kinetics of the HPPK reaction.

HPPK hydroxymethylpterin hydroxymethylpterin- pyrophosphate

ATP AMP

AK AMP + ATP 2 ADP PK ADP + PEP ATP + pyruvate LDH pyruvate + NADPH NADP+ + lactate

Figure 9. The coupled reaction for determination of HPPK activity. The AMP produced by HPPK is used by adenylate synthase (AK) to make ADP, which in turn serves as a substrate for pyruvate kinase (PK). Finally, the pyruvate produced in this reaction is used by lactate dehydrogenase (LDH) to oxidize NADPH. The system does not require the addition of extra ATP, as all ATP used by AK is regenerated by PK.

40 Construction of a folK-knockout in E. coli When working with proteins, it is convenient not to have a background con- tamination of endogenous protein activity. This is often achieved by knock- ing out the gene corresponding to the protein of interest. Another advantage of having a knockout mutant is that it can be used for complementation tests, and thereby give an quick and easy way to determine the functionality of a polypeptide. In order to facilitate studies of HPPK expression, the gene encoding this protein was therefore knocked out in E. coli C600 and in E. coli BL21 (DE3). The mutant was made by inserting a tetracyclin resistance determinant into folK using recombineering (125). Briefly, this method replaces one gene by another using an E. coli strain overexpressing the lambda recombi- nase. To be sure that the folK gene would not be functional following acci- dental excision of the resistance gene, we also deleted part of the gene. One problem with knockouts of essential genes, such as those involved in the folate synthesis, is the viability of the knockout. A knockout of a truly essential function needs to be supported in some way to retain the capacity for life, either by the gene on a plasmid, or by supplemented growth medium. The folate synthesis genes have been thought to be essential, as they make up the organism’s only way of synthesising THF. This will however have to be rethought, as viable knockouts of more and more of these genes are produced. Successful knockouts have been established of both DHFR (46) and DHPS (35) in E. coli, and there are two recent reports of HPPK (60; 68). The genes encoding DHPS and DHFS have also been knocked out in yeast, but the knockouts require folates supplied in the medium (13), and it was not possible to knock out DHPS in P. falciparum (119). This might mirror the increased complexity of protozoans compared to bacteria. A possible way for these organisms to survive might be due to a salvage pathway for external folates like in P. falciparum, or, more probable, by some way of bypassing the folate pathway and generating tetrahydrofolate by different mechanisms. Such a mechanism has been suggested in the group of organisms carrying the alternative thymidylate synthase gene thyX, as many of them lack DHFR, but still somehow generate reduced folates as a substrate for thymidylate synthase (82). Yet another way is to have several proteins performing the same activity, like the E. coli folA and folM, encoding different dihydrofolate reductases (38).

41 Concluding remarks

Resistance to antibiotics can and does spread between bacteria. This is a well-known fact, that curiously enough has not always been taken into ac- count when discussing resistance development. It is being remedied as the importance of the commensal flora as a reservoir of resistance genes becomes more thoroughly investigated.

An antibiotic treatment does not only affect the organisms it is supposed to target. A treatment with macrolides against stomach ulcer led to a sub- stantial increase of macrolide resistant viridans streptococci in the throat. This kind of situation can lead to problems if an antibiotic is used against more than one disease, since a treatment can select for resistance in the commensal flora.

It takes a long time to rid the body of resistant bacteria. In the above study, resistant throat streptococci were present for at least a year after the finished treatment, and resistance levels after three years were lower than before the treatment. This suggests that many people carry residues of previous antibiotic treatments in the form of resistant commensals.

Both mutations and horizontal transfer contribute to sulfonamide resistance in S. pyogenes. Horizontal transfer can lead to stable resistance, where the organism has adapted to carrying a resistance gene and competes at equal levels with others The mosaic folate operon in S. pyogenes is believed to have appeared through horizontal transfer. Resistance to sulfonamides was conferred in part by mutations in position 67 and 213. These residues have later proved to be of catalytic importance.

In a bifunctional enzyme, some parts of the enzyme are dependent on other parts for full function. Two long regions of the plasmodial HPPK- DHPS proved necessary for enzyme function. They are located in the HPPK part, and have little homology in other species. Furthermore, the DHPS part of the enzyme could not be separated from HPPK without loss of activity, and the activity could not be restored by adding C-terminal HPPK sequence. Since there are no drugs aimed at HPPK, further understanding of its structure and mechanism make way for the development of such antimicrobials.

42 Populärvetenskaplig sammanfattning

Min avhandling handlar om antibiotikaresistens. Den kan delas in i tre delar: x Vad händer vid en antibiotikabehandling? x DHPS - ett gammalt mål för antibiotika. x HPPK - ett potentiellt nytt mål för antibiotika.

Vad är antibiotikaresistens? Antibiotika är ofta inriktade på att stoppa en livsnödvändig process i mikroorganismen. Sådana processer utförs av proteiner. Resistens mot antibiotika handlar sålunda om att förändra dessa proteiner så att de inte påverkas av antibiotikasubstansen. För att förstå hur detta går till behöver man gå ett steg längre ned, och studera ritningen till proteiner: DNA. Om man förändrar DNA, dvs ritningen, förändrar man även slutprodukten: proteinet. Det är förhållandevis enklet för en mikroororganism att förändra sitt DNA. Ytterligare en fördel för mikroorganismen är att både bakterier och parasiter kan byta DNA med varandra, och därmed sprida egenskaper. Det går väldigt fort att sprida egenskaper (gener) på det här viset, och mikroor- ganismerna är inte särskilt benägna att göra sig av med dem efteråt. Detta kan leda till problem vid en senare antibiotikabehandling, eftersom de orga- nismer man vill ta kål på inte dör.

Vad händer vid en antibiotikabehandling? Människokroppen innehåller till vardags en mängd bakterier, som brukar gå under namnet normalflora. Dessa bakterier är i regel ofarliga, och en del kan till och med göra nytta. En antibiotikabehandling påverkar inte bara de farliga bakterier den skall påverka, utan även normalfloran. Normalfloran kan ”dela med sig” av resistensegenskaperna till farliga bakterier. Vi studerade närmare hur normalfloran i halsen påverkas av en antibiotikabehandling mot magsår. Halsfloran är viktig, eftersom den ger skydd mot bakterier som ger sjukdomar. Vi kunde visa att direkt efter be- handlingen var en stor del av de ofarliga halsbakterierna resistenta mot det antibiotikum som användes, och att de fortfarande var resistenta ett år senare. Efter tre års tid var de dock känsliga igen. Detta betyder att en behandling med samma antibiotikum mindre än tre år efter den första skulle riskera att inte fungera.

43 Folsyra Folsyra är ett vitamin, som är helt nödvändigt för många processer i en cell. Bakterier och parasiter kan inte ta upp folsyra ur maten, som människan kan, utan måste tillverka den själva. Denna tillverkningsprocess är därför en bra måltavla för antibiotika, eftersom människocellerna inte påverkas (särskilt mycket). Sulfa är ett antibiotikum som är riktat mot tillverkningen av folsyra. Sulfa används idag framför allt mot malaria och andra parasitsjukdomar. Det hämmar ett protein som heter dihydropteroatsyntetas (DHPS). Resistens mot sulfa uppnås genom att förändra utseendet på DHPS, så att det inte påverkas lika mycket av sulfa. Vi visar i ett av arbetena i den här avhandlingen att det krävs mycket små förändringar i proteinet för att det skall binda sulfa mycket sämre. Vi visar också att delar av den gen som är ritning för proteinet DHPS i bakterien Streptococcus pyogenes troligen har ”lånats in” från en annan bakterie, och att det är just i dessa inlånade delar som den lilla föränd- ringen finns. Eftersom mikroorganismer är så bra på att utveckla resistens krävs det hela tiden nya antibiotika. Vi har studerat en potentiell måltavla för ett så- dant nytt antibiotikum, återigen i folsyrasyntesen. I malariaparasiten Plas- modium sitter DHPS ihop med HPPK, som tillverkar en av de byggstenar som DHPS använder. Det finns inte några antibiotika riktade mot HPPK. Vi har analyserat proteinet, och försökt förstå vilka delar som är nödvändiga för att det skall fungera. När vi vet det, går det sedan att utveckla hämmare mot just dessa bitar.

44 Acknowledgements

There are a number of people without whom this thesis would have been much more difficult to write, and to whom I would therefore like to give my most sincere thanks:

My supervisor, Göte Swedberg, for always being there to answer even the silliest questions. I tend to have a lot of those...

Yvonne, for being such a great example, and knowing so much. Labwork would have been much more difficult if you hadn’t always already optimized everything.

Carolina. You are the best office-and labmate possible.

Ola and Lars. You kept asking me those questions that forced me to think ;)

The rest of Farmaceutisk Mikrobiologi: Kristina, Claës, Ewa for support and help in every way.

Anh-Tri and Maarten. B9:3 (and IMBIM, for that matter) would not have been the same without you.

The IPhAB gang: Åsa, Ulrika, Ellenor, Tijs, Anders&Niklas, Carolyn, Per, Stina, Katarina.

The people I taught with: Yvonne and Carolina (you get mentioned twice - there was so much teaching...), Maarten, Anne, Nadja, Helena W, Cecilia, and not least Göte for giving me the opportunity to teach!

All the students I had during these five years: Åsa, Björn, Laila, Elisabeth, Adriano (and Peter), Jordi, Rocio, Riika, Liz, Eva, Annika, Therese. An awful lot of labwork would have remained undone had it not been for you...

All the other people in B9:3 (and not least C10), for making IMBIM such a great place to be.

45 Eva, Helena, Linda, for being the best of friends from 1995 onwards, and for making sure I knew there was a world outside the BMC.

Upsalafandom, for making sure that I got decent beer at least once a month, and for guaranteeing that there never was any “copious free time”. SweCon 2003 did wonders for my licentiate thesis! (A special thanks to Sten, for informing me that Word puts all formatting into the last character in a para- graph. That solved so many problems!)

All my friends at Teologen. You did make the last three years truly enjoy- able (we will get through the greek!).

Kalmarkören (Sofia, Stefan, Janne, Pär, Klara, Sara, Anna, Karin), for filling up the spare time not covered by Upsalafandom.

My family. You were always there. (I especially want to thank my parents for those peptalks in grade four, convincing me that studying was worth the loans..)

Samuel, for being my link to reality during the past five years. And of course, for all those “Men, hur funkar det här? Förklara!

Filemon. So, you got to see the last stages of thesis-writing, after all. (Och jag som hade tänkt ha dig i erratalistan;))

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