- Email: [email protected]
Streptococcus pneumoniae as a genomics platform for broad-spectrum antibiotic discovery
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Streptococcus pneumoniae as a genomics platform for broadspectrum antibiotic discovery John I Glass*, Aimee E Belanger† and Gregory T Robertson‡ Streptococcus pneumoniae is a useful tool for the discovery of broad-spectrum antibiotics because of its genetic malleability and importance as a pathogen. Recent publications of complete chromosomal DNA sequences for S. pneumoniae facilitate rapid and effective use of genomics-based technology to identify essential genes encoding new targets for antibacterial drugs. These methods include computational comparative genomics, gene disruption studies to determine essentiality or identify essential genes, and gene expression analysis using microarrays and gel-based proteomics. We review how genomics has transformed the use of the pneumococcus for the pursuit of new antibiotics, and made it the best species for the identification and validation of new antibiotic targets.
bacterium being the most important cause of bacterial respiratory disease, and its utility for traditional microbiology and genomics-based experimentation. Pneumococcus attributes include: being Gram-positive, like many problematic pathogens; having the capacity to cause disease in mice; having exquisite drug sensitivity; and, most importantly, having a natural capacity to be transformed by exogenous DNA [2]. Thus, it may be genetically manipulated by allelic exchange. For example, polymerase chain reaction (PCR) amplicons containing different alleles of a gene, or even new genes flanked by resident DNA sequences, can be efficiently integrated into the chromosome [3].
Streptococcus pneumoniae genome sequences Addresses Infectious Diseases Research and Clinical Investigation, Eli Lilly and Company, Indianapolis, Indiana 46285, USA *e-mail: [email protected] † e-mail: [email protected] ‡ e-mail: [email protected] Current Opinion in Microbiology 2002, 5:338–342 1369-5274/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations CSP competence-stimulating peptide MPC murine peritoneal cavity PCR polymerase chain reaction STM signature-tagged mutagenesis
Introduction The bioterrorist threat and continuing battle in the hospitals against drug-resistant bacteria highlight the need for new antibiotics. The hope is that new science will beget those new drugs. Current antibiotics were largely discovered using approaches based on testing the capacity of chemical compounds and natural products to inhibit bacteria, preferably many different, if not all, species of bacteria. In 1995, the publication of the chromosomal sequence of Haemophilus influenzae marked an official beginning to the bacterial genomic era [1]. Although Escherichia coli is the most widely used species in the investigation of prokaryotic biology, perhaps the most used bacterium in genomics-based programs for antibiotic discovery is the Gram-positive bacterium Streptococcus pneumoniae, also known as the pneumococcus. Herein, we review how genomics has transformed the use of the pneumococcus for the pursuit of new antibiotics, and made it arguably the best species for the identification and validation of new antibiotic targets.
Why Streptococcus pneumoniae? The choice of S. pneumoniae as a genomics platform for discovery of broad-spectrum antibiotics is a result of the
A genomics-based strategy for antibiotic development requires genomic sequences as a cornerstone. By the mid- and late 1990s, at least eight different large-scale S. pneumoniae sequencing projects were started. Several of the studies discussed in this review based their analyses on unpublished partial sequence data resulting from these projects. In 2001, complete genomic sequences of the virulent capsule type 4 strain (TIGR4) [4••] and the sequence of R6 [5•], an avirulent laboratory strain derived from a virulent capsule type 2 strain D39, were published. Partial sequences of two other virulent strains were also published [6,7].
Identification of new targets for antibiotics using comparative genomics Genome sequences of bacterial pathogens contain ~2700 genes on average. Current antibiotics target less than 25 of those genes, so one of the primary uses of genomic sequences from bacterial pathogens was for the identification of new antibiotic targets. Good targets have three essential attributes: first, they must be essential for viability or required for infection and disease; second, they must be unique to bacteria or at least significantly different from orthologous genes in humans; and third, for broadspectrum antibiotics, the targets must be present in key pathogenic bacteria. Through comparative analysis of genomic sequences, one can winnow potential new targets from the gene complement of a bacterial species being used for preliminary target identification. Mushegian and Koonin [8] proposed that the subset of the genes present in both Mycoplasma genitalium, which has the smallest genome of any bacterium sequenced to date, and H. influenzae would comprise most of the genes essential for autonomous bacterial life. Later, as more sequences were completed, other studies refined this approach [9,10]. Similar analyses have been done for S. pneumoniae genes [7]. However, computational
Streptococcus pneumoniae as a genomics platform for broad-spectrum antibiotic discovery Glass, Belanger and Robertson
tools such as the TIGR Comprehensive Microbial Resource can more efficiently identify potential targets [11••]. In silico-derived, broad-spectrum antibacterial-drugtarget lists based on inclusion in all relevant pathogenic bacteria of humans contain 230–280 genes, depending on the set of organisms used in the comparison (Figure 1). Comparative genomics is reasonably effective for predicting genes of known function that are members of an essential core set. However, the capacity of computational methods to discern sequence patterns shared by genes of unknown function is less developed. Every potential target still needs to be experimentally validated (Figure 2). It is in this phase of target identification that S. pneumoniae is an especially good experimental platform.
Genetic approaches for target identification and validation Transposon mutagenesis has been widely used for global identification of non-essential genes in a range of bacteria. Although the S. pneumoniae chromosome readily accepts transposons, homologous-recombination-based approaches are the methods of choice because of the natural competence of the pneumococcus and because analysis of transposon insertion sites in other closely related bacteria shows a preference for hot spots [12,13]. Thus, although newly developed methods such as genome analysis and mapping by in vitro transposition (GAMBIT) [14] and the Marinerbased transposon, TnAraOut [15], have been very successful in identifying essential bacterial genes, it is unlikely that they will be extensively employed for S. pneumoniae. Homologous-recombination-based gene methods
The approach normally used to determine the essentiality of individual genes is insertional mutagenesis. For S. pneumoniae genes, this usually employs a plasmid carrying a DNA fragment homologous to an internal segment of the targeted gene, an antibiotic selection marker, and an origin of replication that is not functional in S. pneumoniae. Homologous recombination between the pneumococcal gene in the plasmid and the chromosomal gene results in a specific insertion into the genome. Plasmid-mediated antibiotic resistance is used to select recombinant bacteria, and only bacteria containing insertions in non-essential genes are recovered [16]. As with any insertional mutation approach, polar effects need to be considered. A recent improvement in the insertional mutagenesis approach inserts a S. pneumoniae tmRNA sequence into the gene to be disrupted that serves as a proteolysis tag. This eliminates the possibility of obtaining confusing results resulting from expression of a truncated protein encoded by the 5′ end of the gene that retains some function [17].
339
Figure 1
Good targets for antibacterial drugs
Homo sapiens 35–40K genes
S. pneumoniae R6 2046 genes O t h e r B A s D C E p G F e H c i e s
1923 genes conserved in both strains
S. pneumoniae TIGR4 2240 genes
Current Opinion in Microbiology
In silico target identification. Comparative genomics computational tools can determine genes conserved in medically relevant bacteria. The process is depicted here as a Venn diagram. Broad-spectrum antibiotic targets would ideally be conserved in at least the following species, in addition to S. pneumoniae: Mycoplasma pneumoniae (A), Chlamydia pneumoniae (B), H. influenzae (C), Streptococcus pyogenes (D), Staphylococcus aureus (E), Enterococcus faecalis (F), Bacillus anthracis (G) and Mycobacterium tuberculosis (H). The use of different sets of organisms and alternative methods for gene similarity analysis will result in different sets of conserved genes. Conserved bacterial genes without closely related homologs in the human gene set constitute potential broad-spectrum antibacterial targets.
competence of S. pneumoniae, such libraries can be used for allelic exchange to rapidly identify genes associated with specific phenotypes. An especially valuable application of this method is to identify genes encoding targets of potential drugs that show activity in whole-cell antibacterial assays. These ordered genomic libraries are generated under errorprone PCR conditions that rely on the premise that, during error-prone PCR, mutations are introduced into drugtarget-encoding genes that subsequently affect the capacity of a drug to bind to its cellular target. Amplicons containing drug targets can be identified from the library as those that yield drug-resistant colonies when transformed into S. pneumoniae. The specific gene within the amplicon that is associated with drug resistance can then be identified using bioinformatics, as well as genetic approaches (AE Belanger, unpublished data). This method can be readily adapted to other drug discovery applications. Signature-tagged mutagenesis
Ordered genomic libraries composed of PCR products that constitute a set of overlapping DNA fragments corresponding to the entire pneumococcus chromosome can be generated using oligonucleotide primers designed from the S. pneumoniae genome sequence. By exploiting the natural
Signature-tagged mutagenesis (STM) is used to identify genes that are vital for bacterial proliferation in vivo. Transposon-based STM has been effectively used in in vivo investigations of bacterial pathogenesis caused by bacteria other than S. pneumoniae (reviewed in [18]). For S. pneumoniae,
340
Techniques
Figure 2 (a)
~2000 S. pneumoniae genes
Whole-genome genedisruption-based essentialitiy determination in vitro or in vivo
(b)
In silico target identification
Identification of antibiotic target(s)
Generation of drug resistance: whole-genome mutagenesis using ordered libraries of PCR products
Gene-disruption-based essentialitiy determination of specific genes
Validated S. pneumoniae target(s)
Application of genomics to antibacterial target identification and validation. (a) The current standard practice is to identify a target either first through comparative genomics followed by experimental validation of gene product essentiality, or to identify and validate essentiality in a batch process using either random or ordered whole-genome mutagenesis. Once a target is validated, there are several approaches for finding inhibitors. (b) The targets of potential antibiotics identified using whole-cell assays must also be identified. Ordered libraries of PCR products made using error-prone PCR can be used to generate drug-resistant bacteria. These cells can be used to determine the gene(s) or the gene(s) producing a product affected by the drug.
Validation in other relevant bacteria Search for antibacterial compounds using target in high-throughput sceen or for rational drug design
Antibiotic compound found with whole cell assay Current Opinion in Microbiology
the STM method was modified to exploit the pneumococcal natural transformability using short, random genomic DNA fragments inserted in a suicide plasmid vector bearing a molecular tag to perform mutagenesis [19•,20].
allowing critical evaluation of cellular responses to a given agent or inhibitor and will allow researches to map previously uncharacterized pathways that may themselves lead to novel drug targets.
Antisense-RNA-mediated transcriptional attenuation
Use of gene expression technology to unravel novel quorum sensing pathways in pneumococcus
Antisense-RNA-based disruption of gene expression, which is being widely investigated for eukaryotic applications, is now being used to identify essential genes in prokaryotes. Rosenberg and colleagues [21] transformed Staphylococcus aureus with a plasmid library containing short (200–800 base pairs) staphylococcal DNA fragments behind a tetracycline-regulated promoter. Analysis of colonies that grew without tetracycline but not in the presence of tetracycline led to the identification of ~150 essential S. aureus genes. Furthermore, because this is a regulatable system for expression of antisense RNAs, titration of antisense RNA expression can be used to identify important non-essential genes whose inhibition gives rise to small, slow-growing colonies. This approach is amenable to in vitro and in vivo studies. Presumably, it would be equally effective for analysis of S. pneumoniae.
Gene expression, proteomics and drug targets One immediate application of S. pneumoniae genome sequence data is in the development of new tools for studying complex genome-wide expression profiles and the rapid identification of proteins observed using proteomic-based approaches (for recent reviews, see [22,23]). Such approaches hold tremendous potential to aid in the development of novel therapeutic agents by
Several studies have already taken advantage of DNA microarrays to aid in the understanding of complex regulatory pathways in S. pneumoniae; one of the first high-density microarray studies in bacteria was done using pneumococcal DNA arrays [24]. Later studies evaluated transcriptional changes associated with entrance of S. pneumoniae into a specialized state in which the bacterium becomes naturally competent for DNA uptake, a process implicated in virulence by STM (see above and [19•]). Genetic competence in S. pneumoniae is a transient physiologic state resulting from a quorum-sensing regulatory circuit that is responsive to a secreted peptide pheromone [25] and transduced through a second regulatory protein [26]. Using a partial gene array containing ~100 known or predicted competence genes, Peterson and colleagues [27••] demonstrated temporal expression of early (regulatory) and late (processing) competence genes after addition of competence-stimulating peptide (CSP) [27••]. Using a less direct approach, a second group [28••] screened a nearly complete gene array for genes that were induced or repressed after entrance into genetic competence. The results of this study were comparable to those reported by Peterson [27••] and, in total, these two studies nearly doubled the number of genes that were previously
Streptococcus pneumoniae as a genomics platform for broad-spectrum antibiotic discovery Glass, Belanger and Robertson
known to be responsive to CSP. Gene array technology also played an integral role in the recent identification of an additional pneumococcal quorum-sensing system believed to function in antimicrobial peptide production and immunity [29]. Taken together, these studies illustrate the capacity of array technology to deconvolute the response of an organism to an unknown regulator, inhibitor or stimulus. Gel-based proteomics and drug discovery
One advantage of the proteomic approach over traditional nucleic-acid-based approaches is that it allows visualization of not only changes in gene expression under a given condition, but post-transcriptional changes also (for example, protein modifications or alterations in protein stability). Moreover, proteomics may be more readily accessible to the general research field, as no commercial source for S. pneumoniae gene arrays is presently available. Proteomicsbased approaches have been previously employed to visualize protein changes accompanying entrance into the competent state [30], to identify surface-associated immunogenic proteins [31], and to evaluate alterations in protein profiles between spontaneous phase variants of S. pneumoniae that differ in their tissue tropism and virulence [32]. This technology has also been employed to map the proteomic differences between non-isogenic clinical isolates expressing susceptibility or atypical M-type resistance to erythromycin [33].
Application of proteomics and expression technology to pneumococcal pathogenesis Using a simple in vivo cultivation system and RNA dot blot analysis, Orihuela and colleagues [34•] established a positive correlation between the expression of select pneumococcal virulence-associated genes (that is, ply and capA3) and environmental factors encountered in the murine peritoneal cavity (MPC). Moreover, the protein profile of MPC-cultivated bacteria was significantly altered, compared to that of the in vitro-grown control. Although the cultivation system employed did not allow direct host–pathogen interactions and excluded larger solutes from free diffusion, this simple study illustrates an example of how proteomics technologies and array technologies can contribute to our current understanding of this complex pathogen. This union of these technologies can provide important information about the interface of the pneumococcus with its host, which in turn may lead to new antimicrobial approaches.
Conclusions Because of its importance as a human pathogen and its tractability in traditional approaches to testing potential antibiotics, S. pneumoniae has always been an important tool in the development of antibacterial drugs. The pre-genomic era produced drugs that inhibit slightly more than 1% of ~2000 S. pneumoniae gene products. New antibiotics will require use of new targets, and improved target identification and validation is the real promise of a genomics-based approach. Access to genomic sequences for S. pneumoniae
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has changed the way the organism was used to evaluate potential broad-spectrum antibiotics. The complete chromosomal sequences and improvements in computational tools for comparative genomics make this a more effective step in identifying new targets. Methods to experimentally validate new targets using mutagenesis have also evolved to utilize genomics and the natural transformability of the pneumococcus. As we have reviewed, in the seven years since the H. influenzae genome sequencing christened the genomic era, pneumococcal geneticists have rapidly assimilated the advances of genomics to enhance the utility of this bacterium for antibiotic discovery. The next advances will probably be more applied uses of technologies like antisense RNA, gene expression analysis and proteomics to guide target identification and drug hunting.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM et al.: Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 1995, 269:496-512.
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Avery OT, MacLeod C, McCarty M: Studies on the chemical nature of the substance inducing transformation of the pneumococcal types. J Exp Med 1944, 79:137-158.
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4. ••
Tettelin H, Nelson KE, Paulsen IT, Eisen JA, Read TD, Peterson S, Heidelberg J, DeBoy R, Haft D, Dodson RJ et al.: Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 2001, 293:498-506. This paper, and supplementary web data available at http://www.sciencemag. org/cgi/content/full/293/5529/498/DC1, illustrates a comprehensive view of S. pneumoniae systems biology. It integrates previously published determinations of virulence-associated pneumococcus genes as determined by STM and traditional approaches into a genomic context and nomenclature. 5. •
Hoskins J, Alborn WE Jr, Arnold J, Blaszczak LC, Burgett S, DeHoff BS, Estrem ST, Fritz L, Fu DJ, Fuller W et al.: Genome of the bacterium Streptococcus pneumoniae strain R6. J Bacteriol 2001, 183:5709-5717. The website for the S. pneumoniae strain R6 sequencing project (http://www.streppneumoniae.com/) serves as a curated forum for pneumococcal gene annotation where pneumococcal biologists can have edited comments about gene annotation posted to add depth to the notes for genes, and to keep the annotation current. 6.
Streptococcus pneumoniae Genome Diversity Project on World Wide Web URL: http://genome.microbio.uab.edu/strep
7.
Dopazo J, Mendoza A, Herrero J, Caldara F, Humbert Y, Friedli L, Guerrier M, Grand-Schenk E, Gandin C, Francesco MD et al.: Annotated draft genomic sequence from a Streptococcus pneumoniae type 19F clinical isolate. Microb Drug Resist 2001, 7:99-125.
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Mushegian AR, Koonin EV: A minimal gene set for cellular life derived by comparison of complete bacterial genomes. Proc Natl Acad Sci USA 1996, 93:10268-10273.
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Arigoni F, Talabot F, Peitsch M, Edgerton M, Meldrum E, Allet E, Fish R, Jamotte T, Curchod M, Loferer H: A genome-based approach for the identification of essential bacterial genes. Nat Biotechnol 1998, 16:851-856.
10. Bruccoleri R, Dougherty T, Davison D: Concordance analysis of microbial genomes. Nucleic Acids Res 1998, 26:4482-4486. 11. TIGR Comphrensive Microbial Resource on World Wide Web URL: •• http://www.tigr.org/tigr-scripts/CMR2/CMRHomePage.spl The suite of tools and microbial genome sequences provided on this webpage can be easily and effectively used for computational comparative genomics.
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Techniques
12. Hallet B, Rezsohazy R, Mahillon J, Delcour J: IS231A insertion specificity: consensus sequence and DNA bending at the target site. Mol Microbiol 1994, 14:131-139. 13. Renault P, Nogrette JF, Galleron N, Godon JJ, Ehrlich SD: Specificity of insertion of Tn1545 transposon family in Lactococcus lactis subsp. lactis. Dev Biol Stand 1995, 85:535-541. 14. Akerley B, Rubin E, Novick V, Amaya K, Judson N, Mekalanos J: A genome-scale analysis for identification of genes required for growth or survival of Haemophilus influenzae. Proc Natl Acad Sci USA 2002, 99:966-971. 15. Judson N, Mekalanos J: TnAraOut, a transposon-based approach to identify and characterize essential bacterial genes. Nat Biotechnol 2000, 18:740-745. 16. Lee MS, Seok C, Morrison DA: Insertion-duplication mutagenesis in Streptococcus pneumoniae: targeting fragment length is a critical parameter in use as a random insertion tool. Appl Environ Microbiol 1998, 64:4796-4802. 17.
Molnos J, Lange R, Amrein K: An improved vector system for insertional gene inactivation inspired by the tmRNA-tagging system of S. pneumoniae. J Microbiol Methods 2000, 42:197-201.
18. Tang C, Bakshi S, Sun YH: Identification of bacterial genes required for in vivo survival. J Pharm Pharmacol 2001, 53:1575-1579. 19. Lau G, Haataja S, Lonetto M, Kensit S, Marra A, Bryant A, McDevitt D, • Morrison D, Holden D: A functional genomic analysis of type 3 Streptococcus pneumoniae virulence. Mol Microbiol 2001, 40:555-571. STM was used to identify S. pneumoniae virulence-associated genes in murine pneumonia and bacteremia models. Data are presented that indicate pneumococcal competence for genetic transformation has a role in virulence. 20. Polissi A, Pontiggia A, Feger G, Altieri M, Mottl H, Ferrari L, Simon D: Large-scale identification of virulence genes from Streptococcus pneumoniae. Infect Immun 1998, 66:5620-5629. 21. Ji Y, Zhang B, Van Horn S, Warren P, Woodnutt G, Burnham M, Rosenberg M: Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA. Science 2001, 293:2266-2269. 22. Harrington CA, Rosenow C, Retief J: Monitoring gene expression using DNA microarrays. Curr Opin Microbiol 2000, 3:285-291. 23. VanBogelen RA, Schiller EE, Thomas JD, Neidhardt FC: Diagnosis of cellular states of microbial organisms using proteomics. Electrophoresis 1999, 20:2149-2159. 24. de Saizieu A, Certa U, Warrington J, Gray C, Keck W, Mous J: Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays. Nat Biotechnol 1998, 16:45-48. 25. Pestova EV, Havarstein LS, Morrison DA: Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system. Mol Microbiol 1996, 21:853-862.
26. Lee MS, Morrison DA: Identification of a new regulator in Streptococcus pneumoniae linking quorum sensing to competence for genetic transformation. J Bacteriol 1999, 181:5004-5016. 27. ••
Peterson S, Cline RT, Tettelin H, Sharov V, Morrison DA: Gene expression analysis of the Streptococcus pneumoniae competence regulons by use of DNA microarrays. J Bacteriol 2000, 182:6192-6202. This study, and [28••], employed DNA microarrays to quantitatively evaluate the induction and timing of competence gene expression in pneumococcus, and describes several previously uncharacterized genetic loci that respond to the CSP quorum sensing signal. These studies illustrate the capacity of DNA microarrays to contribute to the understanding of complex biological pathways and the response of an organism to an uncharacterized inhibitor or stimulus. 28. Rimini R, Birger J, Feeger G, Roberts T, de Francesco M, Gozzi A, •• Faggioni F, Domenici E, Wallace D, Frandssen N et al.: Global analysis of transcription kinetics during competence development in Streptococcus pneumoniae using high density DNA arrays. Mol Microbiol 2000, 36:1279-1292. See annotation to [27••]. 29. de Saizieu A, Gardes C, Flint N, Wagner C, Kamber M, Mitchell TJ, Keck W, Amrein KE, Lange R: Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide. J Bacteriol 2000, 182:4696-4703. 30. Morrison DA, Baker MF: Competence for genetic transformation in pneumococcus depends on synthesis of a small set of proteins. Nature 1979, 282:215-217. 31. Overweg K, Kerr A, Sluijter M, Jackson MH, Mitchell TJ, de Jong AP, de Groot R, Hermans PW: The putative proteinase maturation protein A of Streptococcus pneumoniae is a conserved surface protein with potential to elicit protective immune responses. Infect Immun 2000, 68:4180-41488. 32. Overweg K, Pericone CD, Verhoef GG, Weiser JN, Meiring HD, de Jong AP, de Groot R, Hermans PW: Differential protein expression in phenotypic variants of Streptococcus pneumoniae. Infect Immun 2000, 58:4604-4610. 33. Cash P, Argo E, Ford L, Lawrie L, McKenzie H: A proteomic analysis of erythromycin resistance in Streptococcus pneumoniae. Electrophoresis 1999, 20:2259-2268. 34. Orihuela CJ, Janssen R, Robb CW, Watson DA, Niesel DW: • Peritoneal culture alters Streptococcus pneumoniae protein profiles and virulence properties. Infect Immun 2000, 68:6082-6086. This paper describes the use of gel-based proteomics and transcriptional analyses to evaluate changes in gene expression resulting from environmental factors encountered during growth of pneumococcus in the murine peritoneal cavity. These authors report changes in virulence factor expression and adherence to respiratory epithelium, following exposure to environmental signals encountered in the host. This study may serve as a paradigm for additional comprehensive approaches that should aid in the understanding of the pneumococcal host–pathogen interaction.
Streptococcus pneumoniae as a genomics platform for broadspectrum antibiotic discovery John I Glass*, Aimee E Belanger† and Gregory T Robertson‡ Streptococcus pneumoniae is a useful tool for the discovery of broad-spectrum antibiotics because of its genetic malleability and importance as a pathogen. Recent publications of complete chromosomal DNA sequences for S. pneumoniae facilitate rapid and effective use of genomics-based technology to identify essential genes encoding new targets for antibacterial drugs. These methods include computational comparative genomics, gene disruption studies to determine essentiality or identify essential genes, and gene expression analysis using microarrays and gel-based proteomics. We review how genomics has transformed the use of the pneumococcus for the pursuit of new antibiotics, and made it the best species for the identification and validation of new antibiotic targets.
bacterium being the most important cause of bacterial respiratory disease, and its utility for traditional microbiology and genomics-based experimentation. Pneumococcus attributes include: being Gram-positive, like many problematic pathogens; having the capacity to cause disease in mice; having exquisite drug sensitivity; and, most importantly, having a natural capacity to be transformed by exogenous DNA [2]. Thus, it may be genetically manipulated by allelic exchange. For example, polymerase chain reaction (PCR) amplicons containing different alleles of a gene, or even new genes flanked by resident DNA sequences, can be efficiently integrated into the chromosome [3].
Streptococcus pneumoniae genome sequences Addresses Infectious Diseases Research and Clinical Investigation, Eli Lilly and Company, Indianapolis, Indiana 46285, USA *e-mail: [email protected] † e-mail: [email protected] ‡ e-mail: [email protected] Current Opinion in Microbiology 2002, 5:338–342 1369-5274/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations CSP competence-stimulating peptide MPC murine peritoneal cavity PCR polymerase chain reaction STM signature-tagged mutagenesis
Introduction The bioterrorist threat and continuing battle in the hospitals against drug-resistant bacteria highlight the need for new antibiotics. The hope is that new science will beget those new drugs. Current antibiotics were largely discovered using approaches based on testing the capacity of chemical compounds and natural products to inhibit bacteria, preferably many different, if not all, species of bacteria. In 1995, the publication of the chromosomal sequence of Haemophilus influenzae marked an official beginning to the bacterial genomic era [1]. Although Escherichia coli is the most widely used species in the investigation of prokaryotic biology, perhaps the most used bacterium in genomics-based programs for antibiotic discovery is the Gram-positive bacterium Streptococcus pneumoniae, also known as the pneumococcus. Herein, we review how genomics has transformed the use of the pneumococcus for the pursuit of new antibiotics, and made it arguably the best species for the identification and validation of new antibiotic targets.
Why Streptococcus pneumoniae? The choice of S. pneumoniae as a genomics platform for discovery of broad-spectrum antibiotics is a result of the
A genomics-based strategy for antibiotic development requires genomic sequences as a cornerstone. By the mid- and late 1990s, at least eight different large-scale S. pneumoniae sequencing projects were started. Several of the studies discussed in this review based their analyses on unpublished partial sequence data resulting from these projects. In 2001, complete genomic sequences of the virulent capsule type 4 strain (TIGR4) [4••] and the sequence of R6 [5•], an avirulent laboratory strain derived from a virulent capsule type 2 strain D39, were published. Partial sequences of two other virulent strains were also published [6,7].
Identification of new targets for antibiotics using comparative genomics Genome sequences of bacterial pathogens contain ~2700 genes on average. Current antibiotics target less than 25 of those genes, so one of the primary uses of genomic sequences from bacterial pathogens was for the identification of new antibiotic targets. Good targets have three essential attributes: first, they must be essential for viability or required for infection and disease; second, they must be unique to bacteria or at least significantly different from orthologous genes in humans; and third, for broadspectrum antibiotics, the targets must be present in key pathogenic bacteria. Through comparative analysis of genomic sequences, one can winnow potential new targets from the gene complement of a bacterial species being used for preliminary target identification. Mushegian and Koonin [8] proposed that the subset of the genes present in both Mycoplasma genitalium, which has the smallest genome of any bacterium sequenced to date, and H. influenzae would comprise most of the genes essential for autonomous bacterial life. Later, as more sequences were completed, other studies refined this approach [9,10]. Similar analyses have been done for S. pneumoniae genes [7]. However, computational
Streptococcus pneumoniae as a genomics platform for broad-spectrum antibiotic discovery Glass, Belanger and Robertson
tools such as the TIGR Comprehensive Microbial Resource can more efficiently identify potential targets [11••]. In silico-derived, broad-spectrum antibacterial-drugtarget lists based on inclusion in all relevant pathogenic bacteria of humans contain 230–280 genes, depending on the set of organisms used in the comparison (Figure 1). Comparative genomics is reasonably effective for predicting genes of known function that are members of an essential core set. However, the capacity of computational methods to discern sequence patterns shared by genes of unknown function is less developed. Every potential target still needs to be experimentally validated (Figure 2). It is in this phase of target identification that S. pneumoniae is an especially good experimental platform.
Genetic approaches for target identification and validation Transposon mutagenesis has been widely used for global identification of non-essential genes in a range of bacteria. Although the S. pneumoniae chromosome readily accepts transposons, homologous-recombination-based approaches are the methods of choice because of the natural competence of the pneumococcus and because analysis of transposon insertion sites in other closely related bacteria shows a preference for hot spots [12,13]. Thus, although newly developed methods such as genome analysis and mapping by in vitro transposition (GAMBIT) [14] and the Marinerbased transposon, TnAraOut [15], have been very successful in identifying essential bacterial genes, it is unlikely that they will be extensively employed for S. pneumoniae. Homologous-recombination-based gene methods
The approach normally used to determine the essentiality of individual genes is insertional mutagenesis. For S. pneumoniae genes, this usually employs a plasmid carrying a DNA fragment homologous to an internal segment of the targeted gene, an antibiotic selection marker, and an origin of replication that is not functional in S. pneumoniae. Homologous recombination between the pneumococcal gene in the plasmid and the chromosomal gene results in a specific insertion into the genome. Plasmid-mediated antibiotic resistance is used to select recombinant bacteria, and only bacteria containing insertions in non-essential genes are recovered [16]. As with any insertional mutation approach, polar effects need to be considered. A recent improvement in the insertional mutagenesis approach inserts a S. pneumoniae tmRNA sequence into the gene to be disrupted that serves as a proteolysis tag. This eliminates the possibility of obtaining confusing results resulting from expression of a truncated protein encoded by the 5′ end of the gene that retains some function [17].
339
Figure 1
Good targets for antibacterial drugs
Homo sapiens 35–40K genes
S. pneumoniae R6 2046 genes O t h e r B A s D C E p G F e H c i e s
1923 genes conserved in both strains
S. pneumoniae TIGR4 2240 genes
Current Opinion in Microbiology
In silico target identification. Comparative genomics computational tools can determine genes conserved in medically relevant bacteria. The process is depicted here as a Venn diagram. Broad-spectrum antibiotic targets would ideally be conserved in at least the following species, in addition to S. pneumoniae: Mycoplasma pneumoniae (A), Chlamydia pneumoniae (B), H. influenzae (C), Streptococcus pyogenes (D), Staphylococcus aureus (E), Enterococcus faecalis (F), Bacillus anthracis (G) and Mycobacterium tuberculosis (H). The use of different sets of organisms and alternative methods for gene similarity analysis will result in different sets of conserved genes. Conserved bacterial genes without closely related homologs in the human gene set constitute potential broad-spectrum antibacterial targets.
competence of S. pneumoniae, such libraries can be used for allelic exchange to rapidly identify genes associated with specific phenotypes. An especially valuable application of this method is to identify genes encoding targets of potential drugs that show activity in whole-cell antibacterial assays. These ordered genomic libraries are generated under errorprone PCR conditions that rely on the premise that, during error-prone PCR, mutations are introduced into drugtarget-encoding genes that subsequently affect the capacity of a drug to bind to its cellular target. Amplicons containing drug targets can be identified from the library as those that yield drug-resistant colonies when transformed into S. pneumoniae. The specific gene within the amplicon that is associated with drug resistance can then be identified using bioinformatics, as well as genetic approaches (AE Belanger, unpublished data). This method can be readily adapted to other drug discovery applications. Signature-tagged mutagenesis
Ordered genomic libraries composed of PCR products that constitute a set of overlapping DNA fragments corresponding to the entire pneumococcus chromosome can be generated using oligonucleotide primers designed from the S. pneumoniae genome sequence. By exploiting the natural
Signature-tagged mutagenesis (STM) is used to identify genes that are vital for bacterial proliferation in vivo. Transposon-based STM has been effectively used in in vivo investigations of bacterial pathogenesis caused by bacteria other than S. pneumoniae (reviewed in [18]). For S. pneumoniae,
340
Techniques
Figure 2 (a)
~2000 S. pneumoniae genes
Whole-genome genedisruption-based essentialitiy determination in vitro or in vivo
(b)
In silico target identification
Identification of antibiotic target(s)
Generation of drug resistance: whole-genome mutagenesis using ordered libraries of PCR products
Gene-disruption-based essentialitiy determination of specific genes
Validated S. pneumoniae target(s)
Application of genomics to antibacterial target identification and validation. (a) The current standard practice is to identify a target either first through comparative genomics followed by experimental validation of gene product essentiality, or to identify and validate essentiality in a batch process using either random or ordered whole-genome mutagenesis. Once a target is validated, there are several approaches for finding inhibitors. (b) The targets of potential antibiotics identified using whole-cell assays must also be identified. Ordered libraries of PCR products made using error-prone PCR can be used to generate drug-resistant bacteria. These cells can be used to determine the gene(s) or the gene(s) producing a product affected by the drug.
Validation in other relevant bacteria Search for antibacterial compounds using target in high-throughput sceen or for rational drug design
Antibiotic compound found with whole cell assay Current Opinion in Microbiology
the STM method was modified to exploit the pneumococcal natural transformability using short, random genomic DNA fragments inserted in a suicide plasmid vector bearing a molecular tag to perform mutagenesis [19•,20].
allowing critical evaluation of cellular responses to a given agent or inhibitor and will allow researches to map previously uncharacterized pathways that may themselves lead to novel drug targets.
Antisense-RNA-mediated transcriptional attenuation
Use of gene expression technology to unravel novel quorum sensing pathways in pneumococcus
Antisense-RNA-based disruption of gene expression, which is being widely investigated for eukaryotic applications, is now being used to identify essential genes in prokaryotes. Rosenberg and colleagues [21] transformed Staphylococcus aureus with a plasmid library containing short (200–800 base pairs) staphylococcal DNA fragments behind a tetracycline-regulated promoter. Analysis of colonies that grew without tetracycline but not in the presence of tetracycline led to the identification of ~150 essential S. aureus genes. Furthermore, because this is a regulatable system for expression of antisense RNAs, titration of antisense RNA expression can be used to identify important non-essential genes whose inhibition gives rise to small, slow-growing colonies. This approach is amenable to in vitro and in vivo studies. Presumably, it would be equally effective for analysis of S. pneumoniae.
Gene expression, proteomics and drug targets One immediate application of S. pneumoniae genome sequence data is in the development of new tools for studying complex genome-wide expression profiles and the rapid identification of proteins observed using proteomic-based approaches (for recent reviews, see [22,23]). Such approaches hold tremendous potential to aid in the development of novel therapeutic agents by
Several studies have already taken advantage of DNA microarrays to aid in the understanding of complex regulatory pathways in S. pneumoniae; one of the first high-density microarray studies in bacteria was done using pneumococcal DNA arrays [24]. Later studies evaluated transcriptional changes associated with entrance of S. pneumoniae into a specialized state in which the bacterium becomes naturally competent for DNA uptake, a process implicated in virulence by STM (see above and [19•]). Genetic competence in S. pneumoniae is a transient physiologic state resulting from a quorum-sensing regulatory circuit that is responsive to a secreted peptide pheromone [25] and transduced through a second regulatory protein [26]. Using a partial gene array containing ~100 known or predicted competence genes, Peterson and colleagues [27••] demonstrated temporal expression of early (regulatory) and late (processing) competence genes after addition of competence-stimulating peptide (CSP) [27••]. Using a less direct approach, a second group [28••] screened a nearly complete gene array for genes that were induced or repressed after entrance into genetic competence. The results of this study were comparable to those reported by Peterson [27••] and, in total, these two studies nearly doubled the number of genes that were previously
Streptococcus pneumoniae as a genomics platform for broad-spectrum antibiotic discovery Glass, Belanger and Robertson
known to be responsive to CSP. Gene array technology also played an integral role in the recent identification of an additional pneumococcal quorum-sensing system believed to function in antimicrobial peptide production and immunity [29]. Taken together, these studies illustrate the capacity of array technology to deconvolute the response of an organism to an unknown regulator, inhibitor or stimulus. Gel-based proteomics and drug discovery
One advantage of the proteomic approach over traditional nucleic-acid-based approaches is that it allows visualization of not only changes in gene expression under a given condition, but post-transcriptional changes also (for example, protein modifications or alterations in protein stability). Moreover, proteomics may be more readily accessible to the general research field, as no commercial source for S. pneumoniae gene arrays is presently available. Proteomicsbased approaches have been previously employed to visualize protein changes accompanying entrance into the competent state [30], to identify surface-associated immunogenic proteins [31], and to evaluate alterations in protein profiles between spontaneous phase variants of S. pneumoniae that differ in their tissue tropism and virulence [32]. This technology has also been employed to map the proteomic differences between non-isogenic clinical isolates expressing susceptibility or atypical M-type resistance to erythromycin [33].
Application of proteomics and expression technology to pneumococcal pathogenesis Using a simple in vivo cultivation system and RNA dot blot analysis, Orihuela and colleagues [34•] established a positive correlation between the expression of select pneumococcal virulence-associated genes (that is, ply and capA3) and environmental factors encountered in the murine peritoneal cavity (MPC). Moreover, the protein profile of MPC-cultivated bacteria was significantly altered, compared to that of the in vitro-grown control. Although the cultivation system employed did not allow direct host–pathogen interactions and excluded larger solutes from free diffusion, this simple study illustrates an example of how proteomics technologies and array technologies can contribute to our current understanding of this complex pathogen. This union of these technologies can provide important information about the interface of the pneumococcus with its host, which in turn may lead to new antimicrobial approaches.
Conclusions Because of its importance as a human pathogen and its tractability in traditional approaches to testing potential antibiotics, S. pneumoniae has always been an important tool in the development of antibacterial drugs. The pre-genomic era produced drugs that inhibit slightly more than 1% of ~2000 S. pneumoniae gene products. New antibiotics will require use of new targets, and improved target identification and validation is the real promise of a genomics-based approach. Access to genomic sequences for S. pneumoniae
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has changed the way the organism was used to evaluate potential broad-spectrum antibiotics. The complete chromosomal sequences and improvements in computational tools for comparative genomics make this a more effective step in identifying new targets. Methods to experimentally validate new targets using mutagenesis have also evolved to utilize genomics and the natural transformability of the pneumococcus. As we have reviewed, in the seven years since the H. influenzae genome sequencing christened the genomic era, pneumococcal geneticists have rapidly assimilated the advances of genomics to enhance the utility of this bacterium for antibiotic discovery. The next advances will probably be more applied uses of technologies like antisense RNA, gene expression analysis and proteomics to guide target identification and drug hunting.
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