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A Strategy to Identify Novel Antimicrobial Compounds – A Bioinformatics and HTS Approach Sara Garbom Department of Molecular Biology Umeå University Umeå, Sweden, 2006 Copyright © 2006 by Sara Garbom ISBN 91-7264-190-8 PRINTED BY ARKITEKTKOPIA AB, UMEÅ, SWEDEN, 2006 2 Ju mer man tänker, ju mer inser man att det inte finns något enkelt svar /Nalle Puh 3 Table of Contents List of Papers ................................................................ 6 Abstract ....................................................................... 7 Abbreviations ................................................................ 8 Introduction.................................................................. 9 THE THREAT OF BACTERIAL INFECTIONS................................................. 9 ANTIBIOTICS AND RESISTANCE..........................................................9 MECHANISMS OF ANTIBIOTICS AND THE DEVELOPMENT OF RESISTANCE .................. 9 THE RIBOSOME AND TRANSLATION..................................................... 12 IDENTIFICATION OF TARGETS AND ANTIMICROBIAL COMPOUNDS .................... 15 DISCOVERING ANTIMICROBIAL COMPOUNDS ............................................ 15 WHAT IS A GOOD TARGET?............................................................ 16 TARGETING VIRULENCE AND IN VIVO SURVIVAL GENES ................................. 16 EXPERIMENTAL APPROACHES TO IDENTIFY TARGETS ................................... 17 GENOME COMPARISONS TO IDENTIFY TARGETS ........................................ 18 EVOLUTION AND CONDENSATION OF MICROBIAL GENOMES .......................... 20 MINIMAL GENOME CONCEPT ........................................................... 20 GENOME CONDENSATION .............................................................. 20 YERSINIA—OUR MODEL SYSTEM ...................................................... 23 YERSINIAE ............................................................................. 23 INVASION AND THE BATTLE AGAINST THE IMMUNE SYSTEM ............................. 23 REGULATION OF THE T3SS ............................................................ 27 YERSINIA—AN INTRACELLULAR PATHOGEN?............................................ 28 General and Specific Aims .............................................. 30 Results and Discussion ................................................... 31 IDENTIFICATION OF NOVEL VIRULENCE-ASSOCIATED GENES (PAPER I).............. 31 EVALUATION OF THE VAGS IN A MOUSE MODEL OF INFECTION YIELDED 5 ATTENUATED MUTANTS ............................................................................. 32 VAGA ................................................................................. 33 VAGH ................................................................................. 34 PHENOTYPIC CHARACTERIZATION OF THE VAGH MUTANT (PAPER II) ............... 34 VAGH IS A SAM-DEPENDENT METHYLTRANSFERASE ................................... 35 MICROARRAY AND 2D-GEL ANALYSIS AND A LINK TO THE T3SS ....................... 35 4 THE VAGH MUTANT BEHAVES LIKE A T3SS STRAIN IN THE MOUSE MODEL OF INFECTION AND IN THE INTERACTION WITH MACROPHAGES ........................................ 36 VAGH AND THE CONNECTION TO THE T3SS IN MORE DETAIL (PAPER III) ........... 37 SCREENING FOR VAGH INHIBITORS (PAPER IV) ...................................... 39 Conclusions................................................................. 41 Tack!!!....................................................................... 42 References ................................................................. 44 5 List of Papers This thesis is based on the following publications referred to in the text by their roman numerals (I-IV). I Sara Garbom, Åke Forsberg, Hans Wolf-Watz, Britt-Marie Kihlberg Identification of novel virulence-associated genes via genome analysis of hypothetical genes Infect Immun. 2004 Mar;72(3):1333-40. Reprinted with permission from the publisher. II Sara Garbom, Martina Olofsson, Ann-Catrin Björnfot, Manoj Kumar Srivastava Victoria L. Robinson, Petra C.F. Oyston, Richard W. Titbull, Hans Wolf-Watz Phenotypic characterization of a virulence associated protein, VagH, of Yersinia pseudotuberculosis reveals a tight link between VagH and the T3SS Submitted manuscript III Sara Garbom, Hans Wolf-Watz The negative regulator YopD regulates LcrF expression on the translational level as studied by a vagH mutant of Yersinia Manuscript IV Sara Garbom, Martina Olofsson, Hans Wolf-Watz, Mikael Elofsson Small molecule inhibitors of the methyltransferase VagH rescue cells from T3SSdependent toxicity Manuscript 6 Abstract Bacterial infections are again becoming difficult to treat because the microbes are growing increasingly resistant to the antibiotics in use today. The need for novel antimicrobial compounds is urgent and to achieve this new targets are crucial. In this thesis we present a strategy for identification of such targets via a bioinformatics approach. In our first study we compared proteins with unknown and hypothetical function of the spirochete Treponema pallidum to five other pathogens also causing chronic or persistent infections in humans (Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi and Streptococcus pneumoniae). T. pallidum was used as a starting point for the comparisons since this organism has a condensed genome (1.1 Mb). As we aimed at identifying conserved proteins important for in vivo survival or virulence of the pathogens we reasoned that T. pallidum would have deleted genes not important in the human host. This comparison yielded 17 ORFs conserved in all six pathogens, these were deleted in our model organism, Yersinia pseudotuberculosis, and the virulence of these mutant strains was evaluated in a mouse model of infection. Five genes were found to be essential for virulence and thus constitute possible antimicrobial drug targets. We have studied one of these virulence associated genes (vags), vagH, in more detail. Functional and phenotypic analysis revealed that VagH is an S-adenosyl-methionine dependent methyltransferase targeting Release factor 1 and 2 (RF1 and RF2). The analysis also showed that very few genes and proteins were differentially expressed in the vagH mutant compared to wild-type Yersinia. One major finding was that expression of the Type III secretion system effectors, the Yops, were down regulated in a vagH mutant. We dissected this phenotype further and found that the down regulation was due to lowered amounts of the positive regulator LcrF. This can be suppressed either by a deletion of yopD or by over expression of the Ribosomal Recycling Factor (RRF). These results indicate that YopD in addition to its role in translational regulation of the Yops also plays a part in the regulation of LcrF translation. We suggest also that the translation of LcrF is particularly sensitive to the amount of translation competent ribosomes and that one effect of a vagH mutation in Y. pseudotuberculosis is that the number of free ribosomes is reduced; this in turn reduces the amount of LcrF produced thereby causing a down regulation of the T3SS. This down regulation is likely the cause of the attenuated virulence of the vagH mutant. Finally, we set up a high throughput screening assay to screen a library of small molecules for compounds with inhibiting the VagH methyltransferase activity. Five such compounds were identified and two were found to inhibit VagH also in bacterial culture. Furthermore, analogues to one of the compounds showed improved inhibitory properties and inhibited the T3SS-dependent cytotoxic response induced by Y. pseudotuberculosis on HeLa cells. We have successfully identified five novel targets for antimicrobial compounds and in addition we have discovered a new class of molecules with antimicrobial properties. Keywords: Antibiotic resistance, Yersinia, T3SS, virulence associated genes, VagH, HemK/PrmC, Release factors, small molecular inhibitors, HTS. 7 Abbreviations • A, P, E-site Acceptor, Peptidyltransfer, Exit sites on the ribosome • A, U, T, G, C Adenine, Uracil, Thymine, Guanine, Cytosine • Ca Calcium • Da Dalton • EF-G, EF-TU Elongation factors • G, Q Glycin, Glutamine • GI Gastrointestinal • GTP, GDP Guanosine-5'-triphosphate, Guanosine-5'-diphosphate • 3 Tritium • HTS High Throughput Screen • ID50 50% Infectious dose • IF1, 2, 3 Initiation factors • IVET In Vivo Expression Technology • Kb, Mb Kilo-basepairs, Mega-basepairs • Lcr Low Calcium Response protein • M-cells Microfold cells • MDR Multi Drug Resistance • MTase Methyltransferase • PP Peyer´s Patches • RF1, RF2 Release Factor 1, Release Factor 2 • RNA Ribonucleic acid, mRNA – messenger, tRNA – transport H rRNA – ribosomal • RRF Ribosomal recycling factor • SAM S-adenosyl-methionine • SD Shine-Dalgarno • STM Signature Tagged Mutagenesis • T3SS Type III Secretion System • vag Virulence Associated Gene • Yop Yersinia Outer Protein 8 Introduction The Threat of Bacterial Infections Bacterial infections were a big problem before the introduction of antibiotics (penicillin) in the 1940s, and millions of people died annually from various bacterial infections. In the 1960s it was proclaimed that the threat of bacterial infections was a problem of the past and that the war against microbes was won. This has, however, unfortunately been proven wrong over the last decades with the appearance of microbes resistant to the antibiotics in use. Resistance to antibiotics is an emerging problem worldwide, and novel antibiotics are necessary to combat infections of resistant pathogens. This thesis deals with the possibilities of identifying novel antibacterial drug targets and compounds to meet the development of resistant pathogens. Antibiotics and Resistance Mechanisms of Antibiotics and the Development of Resistance Clinically important pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis are becoming multiresistant to antibiotics and difficult to treat [1]. In the past 30 years, only two new classes of antibiotics have reached the market—oxazolidinone and lipopeptide daptomycin—and most new antibiotics are simply modifications of or combinations of already present compounds [2]. Resistance toward penicillin was found already in the mid-1940s, within two years of the introduction of the compound on the market [3]. The reasons for the rapid development of resistance to antibiotics by microorganisms are manifold and a net outcome of the fitness increase and the cost of the alterations introduced to confer resistance. The alterations can be either mutations in genes or acquisition of new gene elements; the first is exemplified in the resistance toward rifampicin, which is conferred by a mutation in the gene encoding for the RNA polymerase subunit ß, rpoB [4], and the latter by the resistance to ß-lactams (that is, penicillin) by the introduction of a gene-expressing ß-lactamase [5]. Gene acquisition can occur by three different processes: transformation, transduction, or conjugation [6]. Resistance to a particular antibiotic can develop in alternative ways in different situations: a gene encoding ß-lactamase can be introduced conferring resistance; secondly, acquisition of efflux pumps prevent accumulation of the antibiotic inside the bacteria; thirdly, the 9 synthesis of the cell wall is altered so that the antibiotic no longer can bind; and finally, down regulation of porins that allow the influx of the antibiotics may occur—all of these alterations lead to resistance [7]. The antibiotics available today target many different aspects of cell metabolism and growth; the mechanisms and resistance mutations are summarized in Figure 3 and Table 1. A clinical case demonstrating the rapid spread of resistance involves a four-year-old girl who was severely ill with an Escherichia coli infection initially resistant to ampicillin and narrow-spectrum cephalosporins but not thirdgeneration cephalosporins. During the course of treatment (two months), the bacteria became resistant to the third-generation compounds through the acquisition of an additional ß-lactamase gene. A fraction of the bacteria had also lost the surface expression of the OmpF porin involved in the influx of the antibiotics, giving rise to additional resistance. The bacteria thus evolved different mechanisms of resistance in the blood of the infected child in a short period of time. [7] Some bacteria are naturally resistant to antibiotics, and the soil-living bacteria belonging to the Streptomyces genus are natural antibiotic producers as well as resistant to many of the antibiotics, both synthetic and natural compounds, in use today [8]. How, and if, the resistance elements can be transferred directly from soil bacteria to human pathogens is not known, but excessive use of vancomycin on farms is believed to have contributed to S. aureus resistance toward this compound, and the resistance elements could be transferred via soil bacteria [9]. Fig. 1. Antibiotics and targets. See Table 1 for references and more details. 10 Table 1. Antibiotics, mechanisms, and resistance Class/Antibiotic ß-lactams & cephalosporins Function Acetylates (and inhibits) active sites of transpeptidases involved in peptidoglycan synthesis [10]. Resistance mechanism Acquisition of a gene coding for ß-lactamase that disrupts the ring structure of the antibiotic [11] or mutations in the transpeptidases [12]. Glycopeptides Binds to D-Ala-D-ala dipeptide in the peptidoglycan layer of the cell wall of G+ bacteria, inhibiting cell growth. Peptide antibiotics that disrupt the outer membrane [14]. Modification of the peptidoglycan recognition site for the antibiotic [13]. Flouroquinolones Inhibits DNA replication by the targeting of DNA gyrase [16]. Mutations in DNA gyrase and drug efflux [17]. Rifampicin Inhibits mRNA synthesis by binding to RNA polymerase [18]. Binds to the ribosomal exit tunnel, inhibiting translation [19]. Inhibits the formation of the 50S subunit by binding to precursors [20]. Binds to the 30S subunit and blocks tRNA binding to the A site [24, 25]. Cationic inhibitor of translation. Binds to 16S RNA in the A site of the ribosome, affecting ribosome accuracy [27]. Mutations in the gene encoding RNA polymerase, rpoB [18]. Oxazolidinones Interacts with 23S RNA and inhibits translation [31]. Mutations in 23S RNA [31]. Trimethoprim & sulfonamides Inhibits the production of tetrahydrofolate, and thereby the production of purines, methionine, and thymidine [32]. Mutations in the target enzymes or introduction of an enzyme resistant to the compounds [32]. Polymyxins Macrolides Tetracyclins Aminoglycosides 11 Changes in the lipopolysacharide inhibit the binding of the antibiotic [15]. Methylation of the 23S RNA inhibits binding [21] or production of peptides binding the macrolides [22, 23]. Mainly drug efflux [26]. Modification of the antibiotic by acetylation, phosphorylation, or adenylation [8], or by mutations [28] and methylation [29, 30] of the 16S RNA. High-level resistance often requires that several consecutive mutations occur. The initial mutation is enough for a slight resistance toward the antimicrobial compound, allowing for sequential mutations in this population, conferring high-level resistance [7, 33]. The alterations introduced that provide resistance often decrease the overall fitness of the bacteria, and this can be compensated for by additional mutations, so-called compensatory mutations [33, 34]. The bacteria can also revert back to the original genotype, but this is less likely as there are many more possibilities for compensatory mutations compared to reversion [34]. The compensatory mutations can be intragenic and intergenic as well as result in increased amounts of a protein produced or duplication of the target to allow for multiple functions [34]. The introduction of compensatory mutations often stabilizes the original mutation as a strain with only the compensatory mutation having decreased fitness, but in combination with the original mutation, it leads to an increase of fitness [34]. It has also been shown that spread of resistance is delayed if the mutations necessary for resistance inflict a high fitness cost for the microbe; such mutations are unlikely to spread as the decrease in fitness would be too disadvantageous [35]. There is also data that combinations of different antibiotics and a multitude of targets delay development of resistance and make the drugs more efficient, as for trimethoprim and sulfamethoxazole [36, 37]. Both antibiotics target tetrahydrofolate synthesis but at different stages. The combination of the two drugs is more efficient, and the bacteria have to develop two resistance mechanisms to survive, thereby delaying resistance. The Ribosome and Translation The ribosome is a very large, complex structure, and its importance for bacterial survival has made it an excellent target for antibiotics, as seen in Table 1. With the increased knowledge about the structure of the ribosome, the process of translation has been elucidated. In many instances translational inhibitors, such as antibiotics, have been used to freeze the ribosome in a certain position before crystallization to clarify the different steps of translation. These studies have also revealed that although the ribosome is a much conserved structure, the mechanisms for inhibition of an antibiotic might differ between organisms [38]. Translation can be divided into four steps: initiation, elongation, termination and, finally, ribosome recycling. Each step requires the correct positioning of the ribosome and the recruitment of the right factors. Initiation of translation occurs before transcription has ended, making transcription and translation a coupled event in bacteria. 12 This requires three initiation factors (IF1, IF2, IF3), initiator tRNA and, of course, the ribosome and an mRNA with a Shine-Dalgarno (SD) sequence. The ribosome consists of two subunits (30S and 50S) (see Figure 2 for a schematic overview of ribosome structure) that dissociate prior to initiation. This dissociation is triggered by IF3 binding to the 30S subunit, and the action of IF3 is enhanced by IF1 [39, 40]. IF2, initiator tRNA, and mRNA thereafter associate with the small subunit. The SD sequence interacts with the anti-SD on the ribosome, and the start codon is adjusted so that it enters the P site of the ribosome [41]. This complex is unstable and undergoes changes forming the initiator 30S complex; thereafter IF1 and IF3 dissociate from the complex, and IF2 aids in the binding of the 30S to the 50S subunit, forming the 70S ribosome [42, 43]. The initiator tRNA is now positioned in the P site, IF2 is released, GTP is hydrolyzed to GDP in the process, and the initiator 70S complex is now ready for elongation. The A site of the ribosome now has the second codon of the mRNA in place; Ef-Tu:GTP bound to an elongator tRNA is recruited and bound to the second codon on the mRNA. The decoding process is very complex, as can be expected for such an important feature. Recent crystal structures have somewhat elucidated this event; conformational changes of the ribosome are induced when a tRNAEf-Tu:GTP is bound to the mRNA, and this allows elongation to proceed if there is a match between the tRNA and the mRNA in the A site [44-47]. Ef-Tu itself is also involved in this process, and after correct positioning, Ef-Tu:GTP is hydrolyzed to Ef-Tu:GDP and released. Thereafter the tRNA “swings” into the peptidyltransferase center, and a peptide bond is spontaneously and rapidly formed [48]. Crystal structures of the ribosome bound to a peptidyltransferase inhibitor (“Yarus inhibitor”) proved that it is the 23S rRNA that is responsible for this bond formation [49, 50]. At this time, the P-site tRNA is deacetylated, and the peptide chain has grown with one amino acid in the A site; for elongation to proceed, a translocation of the tRNAs in the A and P sites must occur so that the A site again is free to accept a new tRNA on the next codon. The translocation process is not fully understood, but it requires EF-G and GTP hydrolysis for efficient movement, and it likely includes a hybrid state with the tRNAs bound in both A/P and P/E sites simultaneously [51]. The elongation now proceeds until the ribosome reaches a stop codon on the mRNA (UAA; UGA or UAG). For termination of translation to occur, release factors are essential as no tRNAs exist for these codons. There are two class I release factors in bacteria: RF1 recognizing UAA and UAG, and RF2 recognizing UAA and UGA [52]. Eukaryotes, on the other hand, have only one class I release factor, eRF1, which recognizes all stop codons. RF1/2 binds to the stop codon in the A site and thereby promotes the release of the newly 13 synthesized peptide. A universally conserved essential motif, GGQ, the glutamine (Q) in this motif if post-translationally modified to a N5-glutamine by the SAM-dependent methyltransferase HemK [53-55]. This domain is believed to interact with the peptidyltransferase center, the methylation increases the efficiency of translational termination of the release factors although not essential for function [53]. Once the peptide is released, a class II release factor, RF3, induces the release of RF1/2 [56]. The complex ribosome machinery needs to be recycled for another round of translation; this is catalyzed by the binding of ribosome recycling factor (RRF), IF-3, and EF-G [57-59]. This process is not fully understood and it is not clear in what order the mRNA, peptidyl-tRNA, and ribosome subunits dissociate. During translation ribosomes can stall at for instance rare codons, if the amount of a certain tRNA is low, or if the mRNA contains secondary structures difficult to translate for some reason. Trans-translation is a process where stalled ribosomes are rescued by recruitment of SsrA and SmbP. SsrA can act as both a tRNA and an mRNA and when a ribosome has stalled the complex is recruited and SsrA, charged with an alanine, adds an alanine to the growing peptide and thereafter acts as an mRNA for the ribosome adding a specific tag to the peptide directing the faulty peptide to degradation. The ribosome is thereafter released and recycled as after normal termination. [60] Fig. 2. Structure of the ribosome subunits and the interaction with tRNA and mRNA.. (A) The individual subunits, 30S and 50S, with the active sites labeled A=entry, P=peptidyltransfer, E=exit. (B) The associated subunits, 70S, in complex with mRNA, and a tRNA seen from the side. 14 Identification of Targets and Antimicrobial Compounds Discovering Antimicrobial Compounds Early in drug discovery, most compounds were discovered through whole-cell screening, searching for compounds inhibiting growth; initially this method was very successful but later proved limiting in that most screens resulted in the same molecules identified. This lead to a shift in the strategies used, a shift toward target-based screening methods where high throughput screening (HTS) is applied to screen large collections of random molecules for inhibitory effects on, for instance, enzymatic activity. [61] To be able to perform HTS for compounds inhibiting a certain enzymatic activity, a potential target first has to be found; different ways to do this are discussed in the sections below. The process of identifying novel antimicrobial compounds is outlined in Figure 3. Fig. 3. Outline for finding potential antimicrobial drugs. First, a target is identified, which is followed by a functional analysis of the target and development of an assay to screen for compounds inhibiting the target protein function. When a lead compound has been identified, this can be further improved with chemical genetics and modification of the identified compounds. This is then followed by characterization of the compounds, mechanisms, toxicity, and so on. The advantages of a HTS strategy are that the targets of the compound found are known and that the mechanism of action is thereby easier to deduce. One disadvantage is that high throughput screens for inhibition of enzymatic activity often lead to compounds that are unable to enter the bacterial cell or are efficiently pumped out of the cell by already present efflux systems [62], whereas whole-cell screening systems avoid these molecules already at the screening stage. There are, however, ways to modify the molecules so that they are efficiently taken up by the bacteria, arguing that the whole-cell screening method has the disadvantage of missing inhibitory molecules not entering the cell [63]. It is also possible to modify an initial lead compound with weak inhibitory effects through structure-activity relationship (SAR) analysis. FabI is an enzyme involved in the metabolism of fatty acids, 15 and an initially identified lead compound inhibiting this enzyme exhibited low or no antibacterial activity. However, through SAR analysis specific inhibitors with a 350-fold higher activity were identified [64]. What Is a Good Target? The first step in performing a HTS is to find a target to pursue; different strategies to find targets are discussed in the following sections. First, one has to decide what a good target is. The general criteria for a good target are that it should exhibit a function essential for either growth or pathogenesis of the bacteria, be expressed during infection, and also have an enzymatic activity that can be modified [2]. It is also preferred if the target does not have a close human homologue to avoid toxicity problems further down the line. This does, however, seem to be rather gene specific—as exemplified by trimethoprim, a specific inhibitor of bacterial dihydrofolate reductase—even though the identity to the human homologue is 28% [65, 66]. In some cases the human homologue is closer to a particular bacterial drug target than the bacterial targets are to each other, and still the drug is effective and nontoxic [63]. Metabolic enzymes are considered good targets; in a study of the metabolic pathways essential for in vivo survival and pathogenesis of Salmonella, the authors concluded that very few novel broad-spectrum antimicrobial drug targets could be found in this group [67]. The study combined already published results with new in vivo expression data, virulence data, and metabolic networking. Enzymes with homologues in humans or no homologues in distantly related pathogens were discarded. The low amount of in vivo essential genes was attributed to metabolic robustness due to functional redundancy and to the host’s providing the microbe with nutrients, which renders the bacteria nondependent on its own metabolic pathways in vivo [67]. The apparent limitation in essential enzymes might be why so few novel antibiotics have been developed during the past 30 years [67]. However, there is much we do not know about microbes and their pathogenesis and in the group of genes with an unknown or hypothetical function, additional good targets may exist. This group (and other genes) probably contains suitable targets that are not metabolic enzymes but perhaps regulators, adhesins, virulence factors, and so on. Targeting Virulence and in vivo Survival Genes Most antibiotics today target proteins or functions essential for bacterial survival per se; another strategy is to target proteins directly involved in virulence, for example, type 3 secretion (T3S) components or proteins involved in in vivo survival of the pathogens but 16 not essential for growth outside the host. The strategy not to directly target growth of the microorganism is thought to delay the development of resistance [68]. All clinically used antibiotics today inhibit growth or kill bacteria in vitro [2], so the idea to target virulence genes or in vivo essential genes has not yet been evaluated. Nevertheless, compounds inhibiting the type 3 secretion system (T3SS) have been identified in a number of studies [69-72]. The targets for these small molecules are not known, but they have the potential to be effective against all microbes that use the T3SS in infection, that is, Salmonella spp., Yersinia spp., Shigella spp., Pseudomonas aerigunosa, E. coli, and Chlamydia spp. [73]. Molecules that inhibit the enzymatic activity of the phosphatase YopH secreted by Yersinia spp. have also been developed as a potential treatment for plague [74]. In another study, mice could be protected from infection of Vibrio cholerae by virstatin, as it inhibits the transcriptional regulator ToxT, which regulates virulence genes [75]. Quorum sensing (QS) is important for many pathogens for proper regulation of virulence genes and also survival inside the host; several studies have examined the potential of QS components as targets for antimicrobial compounds [76-79]. Another important feature for many pathogens in vivo is iron acquisition from the host during infection [80-82], and a study identified small molecules inhibiting the iron siderophore biosynthesis in Yersinia pestis and M. tuberculosis [83, 84]. These studies emphasize the potential of targeting either virulence genes or genes important for survival in the host. The selection pressure for development of resistance could be lower when the targets are not essential for growth per se and not important or nonexisting in the bacteria of the normal gut flora, thereby perhaps delaying the spread of resistance through horizontal gene transfer between microbes. Experimental Approaches to Identify Targets Experimental techniques to identify targets involved in virulence and not in vitro growth include signature-tagged mutagenesis (STM) and in vivo expression technology (IVET). STM is a technique introduced about 10 years ago making it possible to screen several mutants simultaneously for their importance in virulence [85]. A pool of random transposon mutants is used to infect an animal; the mutants not capable of colonizing the animal are considered to carry mutations in genes important for virulence. Each mutant carries a unique tag, the input and output pools of bacteria are compared, and the gene with an insertion resulting in a strain killed by the host is identified with sequencing. This technique has over the years been employed on 31 pathogens (until 2005) identifying 1,700 genes important for in vivo survival of the pathogens (Reviewed in [86] and [87]). The majority of 17 the genes identified are surface molecules (25%–30%), stressing their importance for pathogens in vivo. Also interesting is that up to 50% of the genes identified have an unknown function [87]. STM has also identified genes that are true virulence factors such as the SPI-2, encoding a second T3SS of Salmonella in a screen by Shea et al. [88]. The advantage of this technique is that it reduces the number of animals used and the identified genes are all potentially good targets for antimicrobials. IVET and other techniques evaluating the expression of genes in vivo have the limitation that after a gene is identified as being differentially expressed, the gene has to be disrupted and the virulence of the mutant evaluated. Also, genes that are equally expressed in vitro and in vivo can still be interesting for evaluation, and genes expressed only in vivo are perhaps not essential for the pathogen. Random transposon mutagenesis (or targeted mutagenesis) can also be used to identify genes essential for growth if that category of targets is of interest. Genome Comparisons to Identify Targets Another way to limit the amount of genes screened, and also the number of animals used, is to employ in silico methods. Today >350 microbial genomes are completely sequenced, and almost 600 additional projects are underway (NCBI web site). With the techniques available today, it is possible to sequence the genome of a microbe in a day or two. A lot of the genomes sequenced are genomes of pathogens, and this has revolutionized the possibilities to, via bioinformatics approaches, identify genes that constitute good antimicrobial drug targets. Comparisons of genomes of closely related pathogens and the subtraction of genes present in nonpathogenic bacteria or bacteria habituating a different environmental niche yield targets that are specific for the related pathogens. This can be used to, for example, identify targets important only for colonization of the lungs or the intestine. Antibacterial compounds directed toward these targets are narrow in spectrum. The identification of conserved genes in a wide variety of pathogens will instead yield targets that are suitable for broad–spectrum antibiotics. In the comparisons, it is beneficial to include microbes with condensed genomes to avoid identification of genes nonessential for in vivo survival as these genomes have deleted genes that are not necessary for survival in contact with a eukaryotic cell. The in silico analysis has to be followed by experiments determining whether the genes identified are indeed essential for survival or virulence of a pathogen. To do so, it is important to use an animal model where the expression of the target genes can be investigated and where the virulence of a mutant can be assessed. 18 With the increased amount of whole genome sequences of bacteria available, there was a great anticipation that the information within the sequences would instantly yield many new antimicrobial drug targets. This has not been the case. However, a few drug targets have been found or validated with the aid of genomics, such as peptide deformylase inhibitors, compounds targeting fatty-acid biosynthesis, and aminoacyl-tRNA synthetase inhibitors [1, 89]. Genome comparisons have also been used to screen genomes for already known targets to assess against which pathogens a certain antibiotic will be effective [1]. The publishing of the human genome presented the possibility to investigate whether the targets are present in the host and potential toxicity problems can thereby be avoided [67, 90]. But, as stated before, this might exclude potential targets to which the antimicrobial drug would not induce toxicity, even though a close homologue is present in humans [63]. Zheng et al. have created a database of essential genes (DEG), in which experimental data from a number of different studies have been incorporated into a searchable database [91]. This database is continuously updated and freely available, and it was used in a study where essential genes of P. aerigunosa were identified [92]. The clusters of orthologous groups (COG) database is a database in which proteins from different organisms are categorized into families based on function and homology, and it is here easy to analyze the potential spectrum of an antimicrobial drug directed toward a known target; eukaryotic genomes are also included, so the presence of eukaryotic homologues can be evaluated [93, 94]. 19 Evolution and Condensation of Microbial Genomes Minimal Genome Concept The concept of defining the minimal genome to sustain life has intrigued researchers for a long time, as it was believed that a core set of genes essential for life in all microorganisms could be identified. These genes would then constitute good antimicrobial drug targets, as they would be essential for growth of all bacteria. When the first two sequenced genomes of microbes, Haemophilus influenzae and Mycoplasma pneumoniae, were compared, the minimal genome was believed to consist of the 256 genes conserved between these two distantly related bacteria [95]. As more complete genome sequences are available, it is becoming evident that even fewer genes are conserved throughout evolution; only 74 genes are conserved in all microbes sequenced so far [96, 97]. There are insufficient universally conserved genes to maintain basic cellular processes such as transcription and translation, which means that microbes have evolved in different ways to minimize the size of the genome and that nonhomologous genes/proteins can have the same function [96, 98]. This complicates any attempt to define a minimal genome by computational methods. Using a more experimental approach, genes have been knocked out one by one in various organisms to determine which genes are essential and which should thus be included in the minimal genome [99-104]. This approach is limited in that a particular gene may not be essential if other genes are present, but a combination of deletions of two nonessential genes might lead to death. Because of this, some genes essential for sustaining life are missed, and therefore these methods do not really define the minimal set of genes. In fact, the minimal set of genes required for life will vary depending on the nutrients available and other growth conditions, such as temperature, oxygen, and osmolarity. It is perhaps not interesting to define the set of genes required to sustain life under the most favorable conditions but instead to study the naturally occurring genome condensation of obligate parasites in order to define the set of genes necessary to sustain life under different conditions [96, 97]. Genome Condensation The eukaryotic cell provides a very stable, nutrient-rich environment, which is taken advantage of by parasites living inside the cells. The microbe can simply benefit from the nutrient-rich environment, or the relationship can be beneficial for both partners as for the endosymbiont Buchnera aphidocola and its host, the aphids. The diet of the insect is very 20 low in amino acids, but this is instead provided for by the bacteria, and in return the bacteria receive other nutrients from the host [105-107]. Another example is Wigglesworthia glossinidia that colonizes tsetse flies and provides the host with vitamin B that the fly cannot acquire from its diet [108]. The genomes of the endosymbionts are very small and have evolved from much larger genomes. B. aphidicola is believed to have diverged from an ancestor of E. coli, from the time when the ancestor colonized the aphids approximately 75% of the coding capacity of the microbe has been lost [109]. Now most of the genomes of these species are around 600 kb, but recently the genome sequence of a Buchnera (Buchnera aphidicola BCc) with a genome size of 422kb was reported [110]. Interestingly, this strain has lost the capacity to synthesize tryptophan, an amino acid that the host cannot obtain from its diet and usually is provided for by a strain of B. aphidicola. This amino acid is instead likely provided for by a second endosymbiont, providing both the host and the B. aphidicola with this essential nutrient [110]. Another striking finding is the sequencing of Carsonella ruddii, this organism has a genome size of 160 kb encoding for only 182 genes [111]. This is by far the smallest genome sequenced to date and it is hypothesized that genes previously encoded for by the microorganism now instead has been integrated into the chromosome of the host as it has been for organelles such as the mitochondria [111]. The frequency of deletions and rearrangements was higher in the beginning of the hostmicrobe relationship, probably with large deletions of genes of unrelated function [106, 109]. The rate has decelerated over time, and now the genomes of the endosymbionts are more stable than their free-living relatives [96, 106, 109]. Genome reduction is driven by the host-microbe interaction; depending on the host, the microbe will lose genes in metabolic pathways no longer necessary, because the nutrients can be acquired from the host. In some cases, as described above for B. aphidicola and W. glossinidia, the microbes will retain genes important for the host to maintain the host-microbe interaction. These endosymbiont genomes have also reduced their mutational capacity by the deletion of recA and also the removal of repetitive sequences; this factor and also probably deletions of other recombination pathways make these genomes very stable [96, 106, 109]. The genomes of parasites living in contact only with eukaryotic cells have also undergone reductions. The spirochete Treponema pallidum is believed to have lost approximately 75% of its previous coding capacity; the size of the genome is now 1.1 Mb [112]. T. pallidum, the causative agent of syphilis, is an obligate human parasite that cannot be cultured continuously in vitro [113-115]. Through the interaction with the eukaryotic host cell, it has 21 deleted genes necessary for de novo nucleotide synthesis, cofactor synthesis, and fatty-acid synthesis [112]. A clear indication that a genome is in the process of adaptation is a large quantity of pseudogenes; the pseudogene is thereafter deleted resulting in a reduced genome size [116]. Gene elimination usually starts with a single base change, the insertion/deletion of a single base, or the introduction of insertion elements (IS) into a gene inactivating its expression, creating a psuedogene [116]. Gene rearrangements and deletions can also be large, deleting several genes with different functions in one event. The selective advantage of a small genome is not clear, but the reduction of genome size is probably the result of the pathogen living in a stable environment where many genes are useless and an increased genetic drift due to population bottlenecks [98]. The deletion of genes is not always beneficial for the microbe; even genes that are beneficial are inactivated by chance, and due to the small population size, these mutations can stabilize, even though there is no increase in fitness for this population but rather a decrease [117, 118]. Deletions are also balanced by acquisition of novel genes through horizontal gene transfer; this is also less frequent for microbes living in an environment where few other organisms exist, also contributing to the stability of these genomes. The microbes with small, condensed genomes are often difficult to study in the lab, and therefore model organisms are often used instead. In our studies, we have used Yersinia pseudotuberculosis as a model organism to study the virulence of a set of human pathogens, of which some are difficult to cultivate and modify. Y. pseudotuberculosis is a well-studied bacterium easy to cultivate in vitro with a number of genetic tools available for mutagenesis and modification. For a pathogen to be a useful model organism, it is also important that an animal model is available for the evaluation of virulence. Mice infected with Y. pseudotuberculosis develop a plague-like disease [119]. Animals can be infected via the oral, subcutaneous, intravenous, or intraperitoneal route, and the outcome is the same— systemic infection and death. The mouse model is, of course, not equivalent to infections in humans, but it is still an acceptable model for evaluating virulence attributes of pathogens. 22 Yersinia—Our Model System Yersiniae Y. pseudotuberculosis is a gram-negative, rod-shaped protobacteria belonging to the Enterobacteriacea family. The genus Yersinia consists of 11 species; of these, Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis are known to cause disease in humans [120, 121]. Y. pestis is, in contrast to the two other strains, a facultative pathogen with limited ability to survive outside its hosts (humans and rats) and perhaps the most famous of the three responsible for causing plague outbreaks in history. Y. enterocolitica and Y. pseudotuberculosis, on the other hand, cause enteritis and mesenteric adenitis that usually are self-limiting within two weeks in healthy individuals. In rare cases reactive arthritis is a complication of Yersinia infection [122, 123]. Although very similar to Y. pseudotuberculosis on the genetic level, Y. pestis causes a dramatically different disease. Interestingly, the two other strains—Y. enterocolitica and Y. pseudotuberculosis—that induce similar symptoms are evolutionary more distantly related [124]. Of the genes in Y. pseudotuberculosis (IP32953), 75% are >97% identical to their counterparts in Y. pestis (CO-92), and it is believed that Y. pestis evolved from Y. pseudotuberculosis very recently—between 1,500 and 20,000 years ago [125]. Y. pestis has acquired two additional plasmids by horizontal gene transfer since the divergence from Y. pseudotuberculosis: pPCP1 and pMT1. The plasmids encode genes important for tissue invasion [126], capsule formation [127], and infection of the flea vector [128, 129]. Y. pestis CO-92 also carries 147 unique pseudogenes compared to Y. pseudotuberculosis [130], indicating that the genome is in the process of eliminating genes not necessary for survival in the new niche. Genes in the process of being deleted encode for proteins involved in processes important for an enteropathogen but not for the new transmission route of Y. pestis via the flea vector such as genes important for adhesion and motility [131]. The increased virulence of Y. pestis is not easy to understand from genomic analysis; the reason for the difference in pathogenicity is likely a combination of gene inactivation and acquisition [130]. One common plasmid, and virulence feature, of the three pathogenic strains is a 70 kb virulence plasmid encoding a T3SS described in more detail below. Invasion and the Battle against the Immune System Y. pseudotuberculosis and Y. enterocolitica are transmitted via the oral-fecal route, and infected humans have usually ingested contaminated food or water. If infected via the oral 23 route, Y. pseudotuberculosis first encounters the epithelial-cell layer in the gastrointestinal (GI) tract. Microfold cells (M cells) are specialized cells in the GI tract that sample antigens in the lumen and allow macrophages to pass through from the underlying lymphoid tissue [132]. The M cells are positioned over the Peyer’s patches (PP) of the lymphoid tissue, and the sampling ability of the M cells is used by many bacteria to reach the lymphoid circulation [133]. Invasin is a 103 kDa adhesion molecule that via ß1-integrins binds to the M cells and promotes uptake of the bacteria; it is present in Y. enterocolitica and Y. pseudotuberculosis but not in Y. pestis [134]. Invasin is enough to promote uptake into cultured epithelial cells and turn a noninvasive Escherichia coli into an invasive organism [135]. If invasin is not present on the surface of Yersinia, the bacteria cannot efficiently bind to M cells, but are still capable of causing systemic disease [136, 137]. The route of entry in this case is not known, but it has recently been shown that Yersinia can multiply in the intestine and thereafter spread to spleen and liver in mice lacking PP [138]. In the same study, the authors also could show that the pool of bacteria first entering the PP of wildtype C57BL/6J mice was rapidly cleared from this site of infection, not reaching the liver and spleen in contrast to the pool of bacteria first replicating in the intestine. The authors could not conclude how the bacteria entered the deeper tissues, such as the liver and spleen if not via the PP. YadA is another adhesion molecule present on the surface of Yersinia. It is not required for virulence of Y. pseudotuberculosis. A yadAinv double mutant is at least as virulent as the wild-type strain [139, 140], and by the deletion of yadA in an inv background, the strain again can invade M cells by an unknown mechanism [141]. YadA binds to collagen, laminin, and fibronectin; it confers serum resistance; and YadA also causes auto-agglutination and fibril formations [142-144]. pH6 antigen is an additional adhesion molecule involved in hemagglutination and attachment to eukaryotic cells; it is essential for the virulence of Y. pestis but not the other Yersiniae likely due to the fact that Y. pestis lacks functional invasin and YadA [130, 145-147]. Apart from the adhesion molecules, other chromosomally encoded genes important for virulence have also been identified. STM of Y. pseudotuberculosis identified many genes involved in polysaccharide synthesis (LPS core and O antigen) and also genes encoding phospolipase A, RseA, a negative regulator of sigma 24 transcription factor, and proteins with unknown functions [148]. In addition, the ribosomal rescue system trans-translation has been shown to be important for virulence [149]. When Yersiniae reach the PP, liver, spleen, or other deep tissues, it has to combat the immune system of the host. The bacteria first encounter parts of the innate immune 24 system—for example, macrophages and neutrophils. Yersinia is viewed mainly as an extra cellular pathogen, although it can replicate inside macrophages (discussed below). To stay extracellular, it has a sophisticated system to avoid phagocytosis, T3SS. The T3SS is a secretion system that is conserved among a wide variety of gram-negative pathogens, ranging from the symbiotic microbe Rhizobium, which infects plants, to Y. pestis. It can play diverse roles in the pathogenesis; for Salmonella, it is important for the invasion of eukaryotic cells; for enteropathogenic E. coli (EPEC), it is involved in attachment to cells; and for Yersinia, it is used to avoid uptake [134, 150, 151]. Common characteristics of these secretion systems are: induction of secretion upon cell contact, delivery of toxins into the cytoplasm of the eukaryotic target cell, and also a needle-like structure protruding from the bacterial cell surface. The needle is attached to the basal body of the T3S machinery, spanning the inner and outer membranes of the bacteria creating a hollow conduit through which the toxins are believed to be secreted (see Figure 5). The T3S machinery has extensive homology to the components of the flagella, and it is believed to have developed from a flagella ancestor [73]. It is important to note that secretion through the needle-like structure and translocation via a pore in the eukaryotic cell membrane induced by Yersinia has not been shown conclusively. Therefore, other possibilities for the process of translocation from the bacterial cytoplasm into the eukaryotic target cell are possible. The process is, however, dependent on a functional T3S apparatus, and the toxins are transported into the target cell in a T3SS-dependent manner. The functions of the various toxins, the Yops and YpkA, have been studied extensively the past decades, and the cellular targets for most are now elucidated; this is summarized in Figure 4. The results of the effects of the toxins translocated are inhibition of phagocytosis, apoptosis, and inflammation. This enables the bacteria to stay extracellular, and to avoid detection and clearance by the immune system. No single Yop is essential for virulence in the mouse model of infection, although it is important at different stages. But the absence of both YopE and YopH in the same strain renders the bacteria unable to colonize mice [152]. The effects of YopE on epithelial cells can be easily evaluated in a cellular model system. The actin cytoskeletal rearrangements caused by the enzymatic activities of YopE can be scored morphologically by studying the effects on epithelial cells such as HeLa cells; see Figure 7. In vitro expression and secretion of the T3SS can be induced by the depletion of calcium from the growth media (Figure 5). The relevance of this calcium induction is not clear, but it provides a nice tool to study regulation and function of the T3SS in a test tube. 25 Fig. 4. The toxins and targets. YopH is a potent phosphatase (PTPase) [153] dephosphorylating a number of eukaryotic target molecules [154-159] leading to inhibition of phagocytosis [134], inactivation of T and B cells [160-162], and inhibition of Ca2+ signaling in response to Yersinia’s binding to ß1-integrins [163]. YopE is a GTPaseactivating protein (GAP) [164, 165] targeting RhoA and RacI in vivo [166]. The downregulation of these small GTPases lead to actin cytoskeleton rearrangements [164, 165, 167, 168], inhibition of phagocytosis [134, 169], prevention of Yersinia-induced pore formation in the eukaryotic cell membrane [170], and disruption of tight junctions [171]. YpkA is a Ser/Thr kinase [172] that binds to actin [173] and small GTPases [174, 175], which is probably the cause of the actin disruption properties of YpkA [174-176], and it also phosphorylates otubain 1 [177]. YopM is a scaffolding protein interacting with antitrypsin 1 [178] and two kinases modulating their activity [179]. It has also been shown to localize to the nucleus [180]. YopJ is an acetyltransferase (ACTase), and its activity inhibits NFκB-dependent apoptosis [181]. LcrV is not translocated but situated at the tip of the needle-like structure of Yersinia [182]. It interacts with toll-like receptor 2 (TLR2) and inhibits the secretion of IL-10, inhibiting the inflammatory response [183]. It is also essential for the translocation of the other toxins [184]. 26 Fig. 5. HeLa cells infected with Yersinia, (A) yopE mutant (B) wild-type. The actin cytoskeleton is disrupted by the action of YopE [185] and in infection with a yopE mutant strain the HeLa cell remains flat. (C) Calcium response of Yersinia. In media lacking calcium the expression of the Yops is induced. (D) Schematic picture of the T3Smachinery. (D) T3S-apparatuses purified from Salmonella and visualized by electron microscopy. Regulation of the T3SS LcrF is the main positive regulator of the T3SS in Yersinia. LcrF belongs to the AraCfamily of transcriptional regulators [186-188]. The transcription of this activator is regulated by changes in DNA topology and also on the level of translation by formation of a secondary structure hiding the SD-sequence [189, 190]. When bacteria are grown at 37°C, LcrF expression is high, which in turn activates the expression of effectors and machinery genes. At lower temperatures Ymo, a small histone like protein encoded for on the chromosome, inhibits transcription from the yop promoters [191, 192]. YmoA is degraded by ClpXP and Lon proteases at elevated temperature thereby relieving the transcriptional inhibition [192]. There is also an negative regulatory loop at work at 37°C before cell 27 contact and also when bacteria are grown in media containing calcium, this loop consists of the virulence plasmid encoded proteins LcrQ, YopD, and LcrH [193-195]. YopD is a multifunctional protein; in addition to its role in regulation, it is directly involved in the translocation process, and it is also translocated itself [185, 195, 196]. LcrH is a chaperone for YopD and also YopB in addition to its function in regulation [193, 197]. LcrQ does not have any other known roles besides regulation, and it is not under the regulatory control of LcrF [194]. It has been hypothesized that LcrQ, LcrH, and YopD together form a complex capable of inhibiting translation of the Yops until cell contact is achived. LcrQ is thereafter sequestered by SycH and secreted [198-201], the complex is dissolved and translation can occur [202, 203]. An additional regulatory level is imposed by the secretion machinery itself; if the machinery is not correctly assembled, the negative regulators are not secreted and the system remains repressed. Also, LcrG and YopN are believed to form lids on the inside and outside of the machinery, preventing premature secretion [204, 205]. Yersinia—an Intracellular Pathogen? It has been debated over the years whether Y. pseudotuberculosis has an intracellular stage in the infection process [206-209]. The discrepancies in the results obtained in cell culture between these studies are likely due to the different serotypes examined. YPIII (serogroup O3) is not able to replicate inside of J774.1 cells, while strains of serogroup O4 are. The difference between serogroups is also evident for Y. enterocolitica with some strains capable of intracellular replication and some not [210-213]. Y. pestis has for a long time been known to be able to replicate inside macrophages and neutrophils, and this ability is believed to be important early in infection before the T3SS has been turned on and the bacteria are able to avoid uptake [214-216]. The situation is different for the enteropathogens, because the orally ingested bacteria grow at the T3SS-inducing temperature (37°C) for some time before encountering neutrophils and macrophages. But even when the T3SS is induced, about 50% of the bacteria are internalized by macrophages [134], and the ability to survive and replicate inside the macrophages might be important for this fraction of bacteria [216]. There is also evidence of intracellular Yersinia when tissue sections from animals infected orally were analyzed early in infection (4 hours); the bacteria could then be found inside monocytic cells [217]. The relevance for this feature is not clear, but hypotheses include protecting from neutrophils, avoiding antigen presentation (thereby delaying the immune response), and using macrophages as a vehicle to reach deeper tissues [216]. The intracellular replication is not dependent on the plasmid-encoded 28 T3SS [208], and in addition to this, Y. pestis and Y. pseudotuberculosis harbor one more T3SS on the chromosome that is similar to the SPI-2 of Salmonella. In Salmonella this system is essential for survival inside macrophages [218], but when deleted in Yersinia, no effect on intracellular replication was observed [216]. The two component system PhoP/PhoQ is required for the ability to replicate inside macrophages, and a strain without phoP is attenuated in a mouse model of virulence, which suggests that intracellular replication is important in vivo [219]. 29 General and Specific Aims The aim of this thesis has been to develop a strategy for the identification of antimicrobial drug targets present in a wide spectrum of human pathogens. It also includes studying these target proteins in the model pathogen Y. pseudotuberculosis and evaluating their potential as drug targets. This process can be divided into the following steps: 1. Identification of targets (Paper I). 2. Target evaluation in a model system of virulence (Paper I). 3. Functional studies of targets (Papers II & III). 4. Development of an assay to perform a HTS for inhibitors (Papers II & IV). 30 Results and Discussion In this section, the main findings of the papers included in the thesis are presented and discussed. Identification of Novel Virulence-Associated Genes (Paper I) With the increasing threat of microbes’ becoming resistant to available antibiotics, it is essential to identify new targets for antimicrobial compounds and also to develop new strategies for finding such targets. Two different classes of targets exist: genes that are essential for growth of the microbe and those that are only essential for growth in vivo or for virulence of the pathogen. Bioinformatics can be of great help when selecting targets. In our first paper, we took a bioinformatics approach to identify novel virulence-associated genes (vags) that constitute possible antimicrobial drug targets. We compared the genomes of distantly related human pathogens, and the target genes identified are thus suitable for the development of broad-spectrum antibiotics. The pathogens we chose to compare were: Treponema pallidum, Borrelia burgdorferi, Neisseria gonorrhoeae, Helicobacter pylori, Streptococcus pneumoniae, and Yersinia pestis. T. pallidum, the causative agent of syphilis, was the starting point for our comparisons; it has a small and condensed genome (1.1 Mb) [112] that reflects its parasitic lifestyle. The condensed genome served as a good start for identifying genes essential for survival within the human host, and as we wanted to identify novel genes, we limited the starting gene pool to genes annotated as unknown or hypothetical (215 genes). To make the search nonbiased for codon usage, we used protein sequences and the BLASTP alignment tool in all of our comparisons. The sequences were compared to five additional microbes with parasitic lifestyles; the features of these are summarized in Table 2. We considered two sequences as homologous if the comparison yielded an E-value of <10-7, which resulted in 17 open reading frames that were conserved in all species (see Table 1, Paper I for a complete list). These proteins (Vags) are all potential targets for antimicrobial compounds, but to be interesting, the protein has to contribute to the in vivo survival of the pathogens. To evaluate this, we deleted the genes encoding these proteins in our model organism Yersinia pseudotuberculosis. This bacterium is a very close relative to Y. pestis; in fact, Y. pestis is believed to have evolved from Y. pseudotuberculosis less than 20,000 years ago [130, 220]. 31 Pathogen N. gonorrhoeae B. burgdorferi Table 2. Pathogens included in the comparison Disease Characteristics Gonorrhea Gram-negative bacteria, human specific, and host dependent. Lyme disease Spirochete, humanspecific pathogen, and host dependent. Small genome, 1.6 Mb. S. pneumoniae Pneumoniae, meningitis Gram positive. Part of the normal nasopharyngeal flora. H. pylori Stomach ulcers, gastric cancer T. pallidum Syphilis Y. pestis Plague Gram negative. Colonizes 80% of the population in the Western world. Spirochete, obligate parasite. Condensed genome, 1.1 Mb. Facultative pathogen. Recent clone from Y. pseudotuberculosis. Potential bio-weapon. Resistant to Penicillin, cephalosporins, quinolones [6]. Not a big problem. Resistance to erythromycin has been reported [221]. Penicillin, macrolides, cephalosporins, tetracyclines, and MDR strains [6]. Tetracycline, macrolides, quinolones [222]. Macrolides [223]. Rare cases of multi-drug and tetracycline resistance [224]. Evaluation of the vags in a Mouse Model of Infection Yielded 5 Attenuated Mutants Unlike its relative Y. pestis, Y. pseudotuberculosis can grow freely in the environment, and there is also a well-established animal model for evaluation of the virulence of this pathogen. Strains with mutations in 14 out of the 17 genes identified were constructed, and the virulence of these insertion mutants was evaluated in C57/BL6 mice infected via the oral route. Out of the 14 mutants investigated, 9 displayed an attenuated phenotype when compared to the wild-type strain. We could not create mutations in 3 of the genes, and these are likely essential for growth of Y. pseudotuberculosis in vitro. Because we were interested only in genes not essential in vitro but in vivo, these genes were not further analyzed. In this first screen, we created insertion mutants, and as bacteria often have their genes organized in operons, insertions in the first gene of an operon will probably silence all the downstream genes. We now created in-frame deletions of the 9 genes identified in this first 32 screen. We again infected mice orally with the mutant strains, and out of the 9, 5 were still attenuated (Paper I, Table 3). The mutants were also analyzed with respect to growth, Yop secretion, and ability to induce a cytotoxic response on HeLa cells. Yop secretion and cytotoxicity are important virulence traits for Y. pseudotuberculosis and dependent on a functional T3SS. The deletion mutants grew as wild-type in rich media, but the vagH mutant had a growth defect when grown in minimal defined media (MDM). In an attempt to validate our approach, we searched the literature for genes implicated in virulence by, for example, STM [85, 225-227] and selected capture of transcribed sequences (SCOTS) [228]. These genes were then analyzed in the same way as the hypothetical and unknown genes of T. pallidum. 99 proteins were compared; 5 were conserved in all species (Paper I, Table 2). Out of the 5 mutants constructed, 3 were attenuated in the mouse model. These results confirm that pathogens do rely on similar genes and functions for in vivo survival and virulence, and they also confirm that our in silico analysis resulted in genes important for the virulence of several microbes. VagA The vagA gene encodes for a universally conserved GTPase termed YchF in E. coli. The function for this GTPase is unknown, but a study regarding the bacteria Brucella melitensis 16M implicated that the homologue is involved in iron metabolism [229]. It was, however, not essential for virulence for this human pathogen causing chronic fever [229, 230]. In an STM analysis of Neisseria meningitides, an important cause of meningitis especially in children, the vagA homologue was found to be important for virulence [231]. Avian pathogenic E. coli (APEC) is also dependent on a functional vagA gene for virulence [232], and in Chlamydophila pneumoniae, the gene was upregulated in response to IFN-γ treatment [233]. In a study where essential genes in E. coli were identified with transposon mutagenesis, ychF was noted as essential [234] for growth in rich media. In our study the vagA mutant has a growth phenotype indistinguishable from wild-type, both in rich media and MDM (minimal defined media); this makes VagA a good antimicrobial drug target by our definition, together with the findings of vagA as being important for virulence in other organisms. In collaboration with the labs of M. Rehn and B. Henriques-Normark, the homologues of vagA have been deleted in Salmonella typhi and S. pneumoniae, and the resulting mutants are attenuated in animal models of virulence (unpublished results), further validating the potential of vagA as a target for broad-spectrum antibiotics and also the approach taken to identify genes important for virulence in a broad spectrum of pathogens. 33 VagH The vagH mutant, in addition to a growth defect in MDM, showed lower Yop secretion as well as a slightly delayed cytotoxicity towards HeLa cells. It also displayed the most severe virulence attenuation. VagH is homologous to HemK in E. coli shown to be an S-adenosylmethionine (SAM)–dependent N5-methyltransferase transferring a methyl group to Release Factors 1 and 2 (RF1 and RF2) [53, 55]. This protein and its function in Y. pseudotuberculosis are discussed in more detail in Paper II. Homologues to vagH have been implicated in several virulence studies since ours. In EPEC, the homologue is upregulated in infected patients [235], and in Porphyromonas gingivalis, one of the causative agents of adult periodontitis, the gene was also found to be induced in epithelial cell cultures by Park and coworkers [236]. The expression of the vagH homologue in S. pneumoniae has in two different studies been shown to be induced in vivo [237, 238], and in one case the operon containing the vagH homologue was also shown to be important for virulence [238], but in the other study a deletion of the gene did not affect virulence [237]. However, the implications of induced expression of vagH homologues in other pathogens makes this gene interesting for further evaluation; see Papers II, III, and IV. Phenotypic Characterization of the vagH Mutant (Paper II) We decided to study the vagH mutant further and to evaluate the potential of VagH as an antimicrobial drug target. HemK was first misannotated as protoporphyrinogen oxidase [239] and later hypothesized to be a DNA methyltransferase [240] before its correct function as a glutamine methyltransferase was established [53, 55]. Glutamine transmethylation is very unusual; only one additional case is known in bacteria: the methylation of the ribosomal protein L3 [241, 242]. The targets of the MTase activity of HemK in E. coli are RF1 and RF2 [53, 55]. These proteins are class I release factors involved in translational termination. When a translating ribosome encounters a stop codon on an mRNA, the release factors are recruited to decode the stop codon and induce the release of the synthesized peptide. The importance of the methylation is different in E. coli K-12 compared to most other bacteria due to a threonine in position 246 of RF2 in K-12; this amino acid is an alanine or a serine in other organisms. The threonine renders RF2 more dependent on the methylation for efficient function [243]; that is also why a hemK mutant has a reduced growth rate that can be rescued by a mutation changing the threonine to an alanine [53, 55]. In line with this, the vagH mutant grows as wild-type in rich media (Paper I). 34 VagH Is a SAM-Dependent Methyltransferase We first wanted to establish whether VagH possessed the same MTase activity as HemK. We set up an assay developed by Colson et al. [244] using purified VagH or HemK together with total protein lysates prepared from different strains and incubated this together with radio-labeled SAM ([3H]-SAM). The incorporation of [3H]-CH3 was measured by scintillation counting or by autoradiography. From these experiments (Figures 1–3 in Paper II), it was clear that VagH also is a SAM-dependent methyltransferase transmethylating the same targets as HemK. The results also showed that the targets are saturated in the wild-type strain and also that the RF2 of Y. pseudotuberculosis migrates differently compared to the E. coli homologue in a SDS-PAGE gel (Figure 3, Paper II). The difference in theoretical molecular weight between these two proteins is only 42 Da, and the difference in apparent molecular weight is not easily explained due to differences in sequence or methylation status. We have tried to solve this issue by careful masspectrometry analysis (Åke Engström, IMBIM) but not been successful; another strategy could be to create hybrids between the E. coli and Y. pseudotuberculosis proteins to identify the domain responsible for this difference. Microarray and 2D-Gel Analysis and a Link to the T3SS Now that we had established the function of VagH, we wanted to study the effects of a mutation in vagH on a large scale, genome, and proteome. To achieve this, we performed microarray and 2D-gel proteome analysis comparing the vagH mutant and the wild-type strain. The results from these analyses are presented in Tables 1 and 2 in Paper II. In both assays, very few genes/proteins showed differential expression levels in the mutant compared to the wild-type. In a microarray analysis of the hemK mutant in E. coli, 260 genes were differentially expressed [55], but in our case only 22 genes were found to have different expression levels. This might reflect the differences in position 246 in RF2 of the two organisms and the growth reduction of the hemK mutant. Of the 22 genes with different expression levels, 6 ribosomal genes were downregulated in the vagH mutant, indicating that the balance of ribosomal proteins is altered in the mutant. The proteome analysis prompted us to analyze an iron-binding protein 2.8-fold downregulated in the mutant. Iron acquisition is important for Yersinia in vivo [245], and therefore this was an interesting finding. However, when this gene was deleted, no difference in virulence between the mutant and wild-type was observed in the mouse model of infection. YopD, a part of the T3SS, was upregulated twofold in the mutant; this was interesting as we, in Paper I, found 35 the Yop secretion of a vagH mutant to be downregulated. YopD is involved in both negative regulation of the T3SS [195] and the process of translocation of the toxins into the eukaryotic target cell [196]. As we here grew the bacteria under noninducing conditions (37°C, 2.5 mM calcium), the data are not necessarily conflicting; an upregulation of a negative regulator is in agreement with the results of the previous experiments. To establish whether the effects on the T3SS of VagH were dependent on its MTase activity, we overexpressed either wild-type or enzymatically inactive VagH in the vagH mutant and analyzed Yop secretion. The enzymatically inactive variant of VagH could not transcomplement the vagH mutant with respect to Yop secretion, but the wild-type VagH could. The effect of VagH on the T3SS is discussed in more detail in Paper III. The vagH Mutant Behaves like a T3SS Strain in the Mouse Model of Infection and in the Interaction with Macrophages We were also interested in doing a more careful virulence study of the vagH mutant and analyzing where the mutant was cleared from the mice and whether it was capable of reaching deeper tissues (liver, spleen). We infected mice orally and followed the infection over time by dissecting PP and spleens. The vagH mutant behaved like a Y. pseudotuberculosis strain cured of the virulence plasmid (encoding the T3SS) incapable of reaching deeper tissues and also unable to proliferate in PP (Figure 4, Paper II). When mice were infected intraperitoneally, the vagH mutant was severely attenuated with an ID50 value 500-fold higher than wild-type (ID50 wild-type; 3.2x103 cfu; vagH; 1.6x106 cfu), again demonstrating the importance of vagH in virulence. The effects on Yop secretion together with the fact that the mutant behaves like a T3SS null strain in the animal model lead to the conclusion that the virulence attenuation observed is due to the lack of a properly functioning T3SS. To rule out other possibilities contributing to the virulence attenuation, we also investigated the vagH mutant and its response to eukaryotic cells such as J774.1 cells (mouse macrophage cell line) and HeLa cells (epithelial cell line). Y. pseudotuberculosis is capable of intracellular growth in macrophages. The survival in macrophages is not dependent on the T3SS [208], but it is important for virulence [219]. To study a virulence feature not dependent on the T3SS, we analyzed the ability of the vagH mutant to replicate inside macrophages and found it to be similar to the wild-type strain (Figure 6a, Paper II). In contrast to this, when the mutant is grown in presence of macrophages allowing for T3SS induction and extracellular growth, the mutant did not proliferate as well as the wild- 36 type (Figure 6b, Paper II). If cultivated in the presence of HeLa cells, the mutant and wildtype again showed similar phenotypes. HeLa cells are nonprofessional phagocytes in contrast to macrophages. The vagH mutant does possess a functional T3SS, because it is able to translocate YopE into HeLa cells, causing cytotoxicity (Paper I); it can therefore inhibit phagocytosis by the epithelial cells. In the interaction with macrophages, on the other hand, the reduction of Yop secretion might be enough for these phagocytes to kill the bacteria. The results in this paper strongly suggest that the attenuated virulence phenotype of the vagH mutant is mainly due to the effects the deletion of vagH has on the T3SS. We can also conclude that VagH is a SAM-dependent MTase targeting RF1 and RF2. VagH and the connection to the T3SS in more detail (Paper III) As we had found a link between VagH and the T3SS we wanted to dissect this connection further. The first step we took was to identify at what level the effects of vagH mutation had on the T3SS. We did this by insertion of a transcriptional fusion of yopE to luxAB under the native yopE promoter. This creates a fusion where we can estimate the transcriptional activity of the yopE promoter by measuring the light activity of LuxAB. Fig. 6. A schematic overview of the yopE:luxAB fusion mRNA. If an inhibitor binds to the 5´-UTR of the yopE:luxAB transcript this will decrease the amount of YopE produced compared to LuxAB if the inhibitor is not present. 37 We can also use this construct to measure the translational efficiency of YopE over LuxAB, see Figure 6 for an overview, as the proteins have individual ribosomal binding sites and thereby independent translation. When performing this analysis we found that the vagH mutant had lower output from the yopE promoter, and also decreased efficiency of translation of yopE over luxAB, compared to the wild-type strain (see Paper III, Figure 1). The transcription could be restored to wild-type levels by an additional deletion of the negative regulator yopD, this deletion also restores the translational efficiency of YopE. In a single yopD mutant strain the efficiency of translation of YopE is increased indicating, in line with previous reports [193, 202, 203], that YopD inhibits translation of yopE. A deletion of lcrG, however, does not restore either transcription or translation in a vagH mutant strain although a single lcrG mutant has a more efficient translation of yopE compared to wild-type. LcrG and YopD belong to two different classes of regulators; it is believed that LcrG exerts its regulatory function by inhibiting premature secretion of the negative regulators [205]. The finding that a vagH mutant is affected differently by a mutation in lcrG and yopD is interesting as it discriminates between these two classes of regulatory mutants. Further studies of these two double mutants (vagHlcrG and vagHyopD) will probably elucidate the regulatory functions of both LcrG and YopD. LcrF is the central positive regulator of the T3SS in Yersinia, and in addition to its previously described role as a transcriptional activator we have found evidence that it also plays a role in translational regulation of YopE expression. Over expression of LcrF from an inducible promoter increases the efficiency of translation of yopE in all strains tested (vagH, lcrG, yopD, vagHlcrG, vagHyopD, and wild-type), see Figure 3, Paper III. This could be due to a direct effect of LcrF or an indirect effect through the increased expression of a protein under the control of LcrF. We reasoned if the latter was the case this would be a protein encoded for on the virulence plasmid and we therefore over expressed all of the proteins encoded for by the yscB-yscL operon. No positive effects of over expression of these proteins on the translational efficiency of YopE were observed. However, over expression of YscH and YscI reduced the induction of the T3SS. We also investigated potential effects of LcrV, LcrG, YscU but found non of these proteins to influence the YopE translation. Thus, to the best of our knowledge, it is a direct effect of increased amounts of LcrF that improves the efficiency of translation of YopE. We also found evidence of YopD acting as a negative regulator of LcrF translation. In a vagH mutant, the level of LcrF is reduced although no effect is seen on the transcription of lcrF (measured by a lcrF:luxAB fusion). This suggests that the translation of LcrF is 38 reduced in the vagH mutant, this reduction can be suppressed by a deletion of yopD arguing for a role of YopD in translational regulation of lcrF similar to its role in regulation of yopE translation. The finding that YopD regulates the expression of LcrF connects the negative regulatory loop with the positive loop. Another way to increase the amount of LcrF in a vagH mutant is to over express the ribosomal recycling factor (RRF) (see Figure 4, Paper III). Over expression of RRF probably increases the amount of translation competent ribosomes in the bacteria as it is involved in ribosome recycling. It is plausible that a vagH mutant has less free ribosomes due to lowered efficiency of translational termination. A hemK mutant of E. coli shows increased amounts of tmRNA tagged proteins, an indication of stalled ribosomes [55]. These results suggest that translation of LcrF is particularly sensitive to the amount of available free ribosomes perhaps due to a weak ribosomal binding site or blockage of this site. Over expression of RRF also increases the expression of YopE, possibly both by increasing transcription and translation and this is achieved by the increased amount of LcrF. Screening for VagH Inhibitors (Paper IV) Now that we had elucidated the function of VagH, we set up a high throughput screen searching for molecules inhibiting the VagH enzymatic activity. A small molecule library containing 9,400 molecules was screened using the assay described in Paper II with the exception that a potential inhibitor was added to each reaction mixture. In the screen, we identified five compounds that inhibited the in vitro activity of VagH. Two molecules (compounds 2 and 3, Figure 1, Paper IV) belong to the same chemical class. Compound 1 was also identified as its 2-(2-naphtyl)-acetic acid salt indicating that the screening assay is robust and reproducible. Two additional molecules belonging to the same class as 2 and 3 were also present in the library. These were retested but again failed to inhibit the reaction; this result, and the fact that our assay picked up similar molecules, validates the screening method. For these compounds to be interesting as possible antimicrobial agents, they have to be able to enter the bacteria and inhibit the VagH enzymatic activity inside the bacteria. To evaluate this, we took advantage of the effect that the MTase activity of VagH has on Yop expression (see Papers I, II, and III). Bacteria were grown in the presence of compounds 1- 39 5 under conditions known to induce the T3SS (37°C and depletion of calcium). To evaluate the induction of the T3SS we monitored the expression of one of the Yops, YopB (Figure 2, Paper IV). Only compounds 2 and 3 had an effect on the expression of YopB. We do not know the reason why the other compounds now failed to inhibit VagH; possible explanations include that the compounds do not enter the bacteria, or that they are hydrolyzed in the broth or inside the bacteria and thereby inactivated. The compounds that failed to inhibit the reaction in vivo were discarded from further analysis, and we instead continued to study the class of compounds represented by compounds 2 and 3 in more detail. The antimicrobial compound should inhibit survival only in vivo and not in vitro, because a deletion of vagH has this phenotype; therefore the compounds should not inhibit growth of Y. pseudotuberculosis in rich media. To evaluate this, bacteria were grown in the presence of compounds 2 and 3, and the OD600 was monitored over time. The compounds did not inhibit or slow the growth of Y. pseudotuberculosis in rich media (Figure 3, Paper III). Next we wanted to expand the number of molecules in our analysis from this class of compounds; we therefore investigated the inhibitory properties of 13 analogues to compound 3. The results from this are summarized in Figure 4, Paper IV. The most promising, compounds 6 and 15, were subjected to a more careful analysis of their in vivo inhibitory effects and were found to inhibit Yop expression at 20 μM and 40 μM, which is lower compared to compound 3 (~80 μM). We also examined the effects of the compounds in a cellular-based assay where HeLa cells were infected with an lcrG mutant strain of Y. pseudotuberculosis. Before the addition to the cells, the bacteria were grown in the presence of various concentrations of the compounds; thereafter changes in the morphology of the HeLa cells were monitored. We infected the HeLa cells with an lcrG mutant strain, as we have found that a double vagHlcrG mutant is incapable of inducing a cytotoxic response in the HeLa cells, a phenotype mainly associated mainly with the deletion of vagH. In this assay, the compounds inhibited the translocation of the Yops, delaying the cytotoxic response (Figure 5, Paper III), which confirms their in vivo inhibitory effects. We also tried to set up a more purified system based on His-tagged, purified RF2 to establish MICs (Minimal inhibitory concentration) of the compounds. Unfortunately, most of the compounds failed to inhibit this reaction; only compound 4 without in vivo inhibitory effects could inhibit the VagH enzymatic activity in the purer system, and we were, therefore, unable to further evaluate the inhibitory properties of the compounds. 40 Conclusions We have here outlined a strategy to identify novel antimicrobial compounds by a bioinformatics and HTS approach. The main findings in this thesis are: • We have by a bioinformatics approach identified five virulence associated genes (vags) in Y. pseudotuberculosis. These vags are conserved in a wide spectrum of human pathogens making them all possible antimicrobial drug targets. • One of the vags, vagH, has been studied in more detail for its potential as a drug target. • VagH is an S-adenosyl-methionine dependent methyltransferase targeting RF1 and RF2. In Y. pseudotuberculosis the strongest phenotypes of a vagH mutant are: • o Virulence attenuation. o Down regulated T3SS. The effect on the T3SS is likely the cause of the virulence attenuation. The down regulation is due to lower production of the positive T3SS activator LcrF. This can be increased by a deletion of yopD, the amount of LcrF can also be increased both in the wild-type and the vagH mutant by over expression of RRF. The analysis of the vagH mutant has elucidated some new regulatory aspects of the T3SS. • o YopD regulates translation of LcrF. o LcrF expression is sensitive to the amount of free ribosomes. We set up an in vitro assay system where a library of small molecules was screened in a high throughput manner for inhibitory effects on the VagH methyltransferase activity o Five out of 9, 400 compounds screened were found to inhibit VagH. o Two of these five also had an effect in bacterial culture on VagH as measured by the expression of T3S-effectors (YopB and YopE) o Evaluation of structural analogues to one of the compounds decreased the concentration necessary for inhibition in the bacterial system. o • The compounds could also inhibit T3SS dependent cytotoxicity. We have identified a novel class of compounds with antimicrobial properties. 41 Tack!!! Åren på molekylär biologen blev många och roliga, detta till stor del på grund alla som jobbar eller har jobbat på avdelningen. Först ut i raden av personer som ska tackas är min handledare, Hasse, du har varit en toppen handledare under dessa år. Tack för att du trott på mig och låtit mig jobba på men funnits där med support när det gått trögt eller med entusiasm när det gått bra. Ibland är jobbet mindre viktigt än annars, det har du förstått och det är jag väldigt tacksam för. Tillslut så handledde du mig även praktiskt i labbet, bättre sent än aldrig! Tack också till Rolle N som däremot tidigt fick visa runt i labbet och svara på 1 miljon frågor. Britt-Marie Kihlberg för att du fick den första artikeln skriven och publicerad, Åke Forsberg för samarbete, Mikael Elofsson för detsamma! Janne för delat skrivrum och även för många insikter i hur det fungerar här i huset, och dessutom en hel radda cyklar. Det har varit tomt efter dig! Ann-Catrin och Elin, roligt med nya tillskott (det har ju bara gått två år…). Ann-Catrin, tack också för det arbete du gjort (nästan alltid med ett leende…) och all hjälp med smått och gott i labbet. Kaffehörnan - alltid där och oftast med kaffe om inte Rolle R har kaffevecka ☺ Cathrin P, för att man känner sig pigg när man ser dig på morgonen, Maria F för att du försöker få tiden att växa, jobba på. Tack också till alla glada deltagare i Journal Clubs och gruppmöten: Margareta, Sara C, Lisandro (lycka till!), Linda, Barbara, Medisa, Anthony (från Apan ☺), Moa (tack för samarbete), Jeanette, Matt, Vicky, Debbie och alla andra från nu och förr. Katrin, syjuntor, VM-tips och torsdagsluncher, jag kan till och med (nästan) förlåta att du håller på Färjestad. Myggflickorna Jenny och Maria, roligt med parasiter, tack för musvaktandet. Christer – tack för hjälpen med mössen och för att du alltid är så nice. Det är ni andra Borrelia/Chlamydia män också (Pär och Leslie). StorPia, Lill-Pia, Pia Charlotte jag får byta namn så att jag också kan få ett jobb… och alla ni andra på Innate, speciellt Henrik för hjälp med molekyler och sånt som jag inte förstår mig på. Petra, du är så glad och söt, dessutom är den mest omtänksamma vän man kan tänka sig, tack för all hjälp i och utanför labbet! Martina, du har bidragit mycket till denna avhandling! Jag tänker inte bara på typsnitt och slavarbete utan också för att du är så fin. Saknar dig mycket! Magnus, du är också fin, engagerad och entusiastisk dessutom, det är alltid roligt att locka fram 42 blodådrorna med lite Ebba Grön… David E, med stort tålamod med mig i fiskeribranschen och även som extra fru, jag ställer gärna upp igen! Miss J, du är starkare än somliga (de flesta) tror, glöm inte det och kom även ihåg att du är lysande på många sätt (dock inte på att passa tiden…) Sofia, snäll och godhjärtad som få, du ställer upp i alla väder vad det än gäller. Johan, hård utanpå men mycket mjuk inuti, en av de argaste men trognaste MoDo supportrarna. Christina, det hade absolut inte varit lika kul utan dig, skidåkning, Malaysia osv. Fredrik, Maria och Viktor, det är sååå roligt att ni flyttat till Malmö! Goda Grannar: Gunnarn, du tillför sarkasm (gillas!) och lite kultur i vårt lilla gäng, Charlotte, vårt rättesnöre som är väldigt generös och har varit mycket bra att ha under skrivandet, Niklas och Maria mina nyaste grannar och tappra medkämpar i pendlarbranschen även om jag tycker ni gav upp lite lätt ☺. Anna, vi får aldrig glömma Riksgränsen, ja, vissa saker kan vi glömma…Erik, kalas, Kicki du är också kalas. Petra, Tom, Peter S, Sven, Håkan, Linda, Tobias tack för allt roligt. Annica, Anna, och Mia för att ni gjorde det roligt att springa omkring i overall och för alla mysiga träffar sedan dess, roligt att alla blir fler. Malin, du var också söt i overall och dessutom en mycket rolig lägenhetsdelare.. Katta, du är verkligen en frisk fläkt och råbra ☺. Carro, du är en toppenvän, jag lovar att komma till Genéve snart och hälsa på dig och Matte även om jag är lite besviken på att ni lagt ner mitt hotell i Solna… Och så familjen. Det har minst sagt hänt mycket de senaste åren och med en del saker är det svårt att se meningen. Jag är så glad för er; Alexander det lilla solskenet, Mamma du finns alltid där, Eva syster-yster och min estetiska konsult, du är fabulös! Henrik, längtar tills vi ska bo ihop för alltid! Du är min bäste vän och kärleken (L). 43 References 1. McDevitt, D. and M. Rosenberg, Exploiting genomics to discover new antibiotics. Trends Microbiol, 2001. 9(12): p. 611-7. 2. Pucci, M.J., Use of genomics to select antibacterial targets. Biochem Pharmacol, 2006. 71(7): p. 1066-72. 3. Walsh, C., Molecular mechanisms that confer antibacterial drug resistance. Nature, 2000. 406(6797): p. 775-81. 4. Jin, D.J. and C.A. 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