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Category: Genomics and Bioinformatics; Bacterial Pathogenesis
This landmark volume details progress in the fast-changing world of bacterial genomics. The availability of genome sequences pervades every aspect of bacteriology. Bacteriologists now can examine the genomic sequence for every significant bacterial pathogen of humans, plants, and animals. With chapters from more than forty scientists from around the world, Bacterial Pathogenomics explains the scientific advances that have resulted from the application of bacterial genome sequencing to the study of how bacterial pathogens have evolved and how these bacteria cause disease.
The first half of the book reviews the impact of genomics on our understanding of selected groups of pathogens, including Escherichia coli, mycobacteria, Neisseria, staphylococci, spirochetes, Campylobacter, plant pathogens, and Photorhabdus. The remaining chapters cover themes that cut across taxonomic boundaries, such as genomic signatures of intracellularity, the impact of shared genomic tools and datasets, pathogenomics of bacterial biothreat agents, the impact of phages on the evolution of bacterial pathogenicity, gram-positive protein secretion, cell wall biosynthesis, and intracellular pathogens. Bacterial Pathogenomics will prove indispensable in the library of any bacteriology research group and will act as a key text for anyone studying bacterial genomics or the molecular basis of bacterial infection.
Electronic Only, 453 pages, full-color insert, illustrations, index.
This chapter provides an introduction to pathogenomics. Any bacterial genome-sequencing project comprises a number of steps and follows the 80:20 principle: that is, 80%, or more, of the work gets done in 20% of the time, while finishing the job consumes a disproportionate amount of effort. The first stage in any bacterial genome project is to choose one or more strains to sequence. The next stage in a genome-sequencing project is to grow enough bacterial cells to yield sufficient genomic DNA for the creation of a random shotgun library in Escherichia coli. Shotgun sequencing continues until an acceptable coverage of the genome has been achieved. The final stage in obtaining a bacterial genome sequence is finishing, during which all gaps are closed and all ambiguities resolved. A number of interesting features can be identified rapidly: repetitive sequences and homopolymeric tracts; noncoding RNA genes (tRNAs, rRNAs, small regulatory RNAs); recently acquired DNA; leading and lagging strands and the origin of replication. An interesting sideline will be the metagenomics of the bacteriophage community, given both the numerical preponderance of phages in the biosphere and their well-established role in the evolution and dissemination of bacterial virulence factors.
This chapter discusses the insights into pathogenicity and the wider processes of Escherichia coli genome evolution that have resulted from the sequencing of the E. coli K-12 genome, and, more recently, those from a number of pathogenic E. coli strains, including several from the related “genus” Shigella. It also discusses the available resources for E. coli genomics and the progress that has been made in recent years toward a complete understanding of E. coli biology. Genes that are acquired by horizontal transfer subsequently undergo a process of amelioration as their base composition becomes acclimatized to the new host. Programs such as BLAST can be used to identify unexpected similarities between genes in phylogenetically distinct species, although it should be noted that BLAST results do not necessarily correspond to those from more robust phylogenetic analyses. Interestingly, strains classified as group B2 are rarely found as human commensals, and the group includes many strains with extraintestinal pathogenic E. coli (ExPEC) virulence determinants. As with enterohemorrhagic E. coli (EHEC) O157:H7, Shigella flexneri serotype 2a was an obvious target for whole-genome sequencing because of its importance as a pathogen. The processes of E. coli genome evolution are clear from the genome sequences already available.
This chapter focuses on insights gained from genome exploration and functional analyses. It provides an overview of the evolution and of the infection strategies used by pathogenic mycobacteria and how this information may be useful for developing new insights into disease control. The importance of molecular characterization of Mycobacterium tuberculosis strains was also shown in a recent microarray analysis of a large number of epidemiologically well-defined M. tuberculosis isolates from San Francisco. One of the key findings of the sequence analysis was that M. tuberculosis differs radically from other bacteria in that approximately 10% of its coding capacity is dedicated to genes encoding proteins that are involved in lipid metabolism, both biosynthetic activities and lipolytic functions. The genes encoding the ATP binding cassette (ABC) transporters occupy about 2.5% of the genome of M. tuberculosis. These multi-subunit permeases, which transport various molecules like ions, amino acids, peptides, antibiotics, polysaccharides, and proteins across biological membranes, are classified as importers and exporters. The current bacillus Calmette-Guérin (BCG) vaccine is efficient in the protection against tuberculosis (TB) meningitis, but it often provides only minor protection against pulmonary TB, the prevalent form in adults. The availability of genome sequences from several mycobacterial species provides the principal resource for researchers to explore their pathogenic life cycle and evolution.
This chapter primarily focuses on the studies that have made use of the two published Neisseria meningitidis genome sequences. Potential silent cassettes were identified for mafB and fhaB, suggesting that the proteins encoded by the expression loci of the genes can undergo antigenic variation by a similar mechanism to pilE. Nitric oxide (NO) is released from macrophages in response to N. meningitidis lipopolysaccharide (LPS), and it is an intermediate in the denitrification of nitrite to nitrous oxide. Host neutrophils and macrophages commonly use superoxide radicals to eradicate bacteria after invasion of the host. Radicals generate strand breaks in DNA and RNA and affect fatty acid structure in the cell membranes. Three penicillin-binding proteins (PBPs) were identified experimentally in the N. gonorrhoeae and were considered to be reasonably well established. The genes encoding the RTX-like proteins are scattered across the chromosome and are present in different allelic versions in different strains. One of the most extensively investigated regulatory proteins of the Neisseria spp. is MtrR, which was first identified as a regulator of the expression of the MtrCDE and Far AB efflux pumps in N. gonorrhoeae. Through hybridization of chromosomal DNA, rather than cDNA, the presence of sequences within a strain of interest can be determined.
Staphylococci that can cause disease in humans are Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. saprophyticus, S. hominis, S. warneri, S. lugdunensis, S. schleiferi subsp. schleiferi, S. capitis subsp. ureolyticus, and S. simulans. This chapter briefly discusses the often complex biology of the organism before the genomic insights determined from the sequence. One of the most valuable tools that can arise from whole genome sequences are whole-genome microarrays. A study on core variable genes revealed new information about the S. aureus genome and its evolution. S. haemolyticus are generally more resistant to antiobiotics than other staphylococci, and it could be that they accumulate more of these genes than other species. As the methods for classifying strains into dominant types become more widespread, further epidemiological patterns and associations between certain lineages, toxins, and the infections they cause are being considered. Unlike the genomic islands in other staphylococci that are often associated with virulence, the arginine catabolism mobile element (ACME) island is associated with drug resistance. Each of the sequenced staphylococcal genomes carries genes common to all other sequenced staphylococci, genes specific to particular species, genes specific to particular lineages, and mobile genetic elements (MGEs).
This chapter reports on three pathogenic spirochetes: Treponema pallidum, causative agent of syphilis, and other treponemes; Borrelia burgdorferi, causative agent of Lyme disease, and related species; and Leptospira species, responsible for leptospirosis. The spirochetes are quite different from many of the other well-studied pathogens that are principally found in the proteobacteria and gram-positive groups. Treponema paraluiscuniculi is not pathogenic for humans and causes veneral spirochetosis in rabbits. Differences in the chromosome sequence can be used for molecular diagnostic testing of treponemal subspecies and strains and for epidemiological studies. The genome sequence information enables new approaches to be developed to determine the function of genes and their possible role in pathogenesis. To identify antigens important in the human immune response to syphilis, the serum antibody reactivity of patients with syphilis was examined with 908 T. pallidum proteins by using the same techniques as those for the rabbit sera. The genus Borrelia comprises spirochetes with loose wavelike coils that survive exclusively by transmission back and forth between vertebrate and arthropod hosts. The chromosome of B. burgdorferi encodes most of the housekeeping genes required for in vitro survival and growth, as demonstrated by strain B. burgdorferi B313, which has lost most of the plasmids. Leptospira is found in many animal species, and this broad reservoir provides a continuous source for infection, making this one of the most widespread zoonotic infections.
Members of the genus Campylobacter are defined as fastidious, microaerophilic, oxidase positive, nonfermentative, gram-negative bacteria. All predicted proteins from the five Campylobacter genomes were compared with data from other published microbial genomes by BLASTP. The chromosomes of all five Campylobacter strains in this comparative study were examined for the presence or absence of clustered regularly interspaced short palindromic repeats (CRISPR) elements in intergenic regions. In this study, a strain is considered CRISPR positive when it contains two or more direct repeats of a 21-bp or larger DNA segment separated by unique spacer sequences of a similar size. The Campylobacter species strains analyzed in this study have the Sec-dependent and Sec-independent protein export pathways for the secretion of proteins across the inner or periplasmic membrane. Of the 580 open reading frames (ORFs) conserved between the Campylobacter and Helicobacter species included in this study, 27 ORFs involved in flagellar biosynthesis and function were conserved between Campylobacter and Helicobacter. A research group applied in vitro mariner-based transposition system to identify genes involved in motility in C. jejuni 81-176. They followed up this work to show that flagellar genes were regulated by σ54, but not σ28. Although some proteomic studies have been conducted for C. jejuni, these were limited to differential analysis of specific mutations, planktonic versus biofilm growth, and on NCTC 11168 stocks with different amounts of passaging.
This chapter highlights some intriguing patterns of genome change identified from genome-sequencing projects and molecular evolution studies. Trends include striking similarities between intracellular mutualists and parasites that may reflect common pressures of an intracellular lifestyle, as well as key differences that highlight distinct genomic consequences of their host associations. Bacteria form a variety of associations with host cells. At one end of the spectrum, the replication and spread of intracellular parasites impose a fitness cost to hosts. At the other end of the symbiotic spectrum, certain intracellular bacterial groups form exclusively mutualistic associations with hosts. Analysis of partial genome regions indicates that, compared with its Buchnera relatives, the exceptionally small genome of Buchnera associated with the aphid Cinara cedri shows more extensive loss of metabolic than informational functions. Consistent with their infectious lifestyles, obligately intracellular parasites typically encode numerous mechanisms to invade host tissue and cells and to escape the host immune system. To date, genome-wide rate comparisons strongly suggest a consistent, genome-wide rate increase in intracellular species, as expected under the influences of increased mutation and/or genetic drift. The availability of two or more genomes for a particular group offers a window into the evolution of genome architecture, or changes in the order and strand orientation of shared loci. The rapid growth of genomics has elucidated processes that shape wide variation in genome size, gene content, patterns of DNA sequence evolution, and levels of genome fluidity in the bacterial world.
This chapter discusses the relevance of model host pathogenesis as a third general approach to studying microbial virulence, in which infections are studied in the context of nonvertebrate whole animal hosts. The chapter explains the combination of four key steps: (i) the development of the model host-pathogen system, (ii) the development of genomic tools in both the pathogen and host, (iii) the distribution and use of these tools by the greater research community beyond the laboratories involved in their initial development, and (iv) the collection and ultimate integration of experimental data from a wide variety of research groups made possible by the widespread use of a common resource and its accompanying Web-accessible public database. Pseudomonas aeruginosa PA14 was chosen for the construction of a non-redundant mutant library because it is remarkably virulent in the greatest number of model hosts tested, as well as in a number of murine systems of infection. There are four major advantages to construction of a non-redundant mutant library approach as opposed to the traditional approach of screening a random collection of strains for avirulent or attenuated mutants. A microarray experiment in a Caenhorhabidits elegans mutant defective in a newly defined host defense response gene identified candidate downstream genes important in the response to pathogens.
The idea of "pathogenomics" encompasses every aspect of microbial pathogenesis. Before genome sequences for biothreat pathogens became available, the types of whole-genome approaches applied today were unimaginable to investigators. This chapter therefore revolves around a period of transition in the study of pathogen biology in general and of biothreat agents in particular, much of it stemming from the recent biodefense funding impetus. In discussing the genome biology of classical biothreat pathogens, several common themes emerge. The chapter provides an overview of these ideas before discussing the specific pathogenomics of each agent. The study of genomics emphasizes the similarities of the major themes in biology and allows advances that occur in one field to be quickly transferred to others. The enormity of the impact of horizontal gene transfer (HGT) on the genome content of bacterial species is gradually coming to light. In a postgenomic study, comparative proteomics of the secretomes performed with two-dimensional gel electrophoresis between fully virulent Bacillus anthracis, pXO1+/pXO2+ and pXO1-/pXO2- was performed. The pathobiology of B. anthracis, stimulated by the genome sequence, is a highly active field. As knowledge of biothreat pathogenesis grows, so does the possibility of identifying common pathways in the host. For instance, many of the bacteria described in the chapter invade macrophages at some point when causing disease.
An interesting facet of Streptococcus pyogenes and Staphylococcus aureus, is their double role as commensals and pathogens. In this context, certain aspects of bacterial pathogenicity can be interpreted as a recall of antipredation strategies that bacteria evolved against phagocytosis by protozoan grazers. Many bacteria contain multiple genomes of bacterial viruses in their chromosomes. Prophage DNA can constitute a sizable part of the total bacterial DNA. When genomes from closely related bacteria were compared in a dot plot analysis, prophage sequences frequently accounted for a substantial, if not major, part of the differences between the genomes. Prophages can be present in many different forms, ranging from inducible prophages via prophages showing deletions, insertion, and rearrangements, to prophage remnants that lost most of the phage genome. In prophages from gram-negative bacteria, “extra” genes were identified near both prophage DNA ends. Prophages seem to be only transient passengers on the bacterial chromosomes, at least when seen on an evolutionary timescale. In an appealing model, the emergence of new, unusually virulent subclones of M3 strains is explained by the sequential acquisition of prophages, suggesting bacterial pathogenicity evolution in the fast lane. The virulence genes have certainly not evolved in phages but are the result of close bacterial interaction with the eukaryotic cell. Phages are perhaps only the handy gene carriers efficiently shuttling genes around in the bacterial world.
This chapter briefly reviews the Sec pathway and its modification in gram-positive bacteria and discusses second independent protein targeting pathways. Some gram-positive bacteria have evolved protein secretion machines dedicated to the assembly of macro-molecular structures such as flagella, type 4 pili, or Flp pili, all of which can be viewed by light or electron microscopy as elongated organelles that protrude from the cell surface. The chapter describes the general principles of protein secretion in gram-positive bacteria along with a few examples. The factors that catalyze protein secretion can be predicted in silico and identified in genomes of all gram-positive bacteria. Sec-mediated protein secretion has been best studied in Escherichia coli and, more recently, in Bacillus subtilis. S-layer proteins are deposited on the surface of gram-positive bacteria to establish specific interactions with cell wall polymers. Many other proteins decorate the cell wall envelope of gram-positive bacteria but are not covalently attached to the envelope. The major subunit of flagella, flagellin, is exported through the hollow conduct made by the basal body across the membrane and hook. Rational vaccine design was achieved by interrogating conserved antigens (secreted or surface displayed) for protective immunity, which led to the identification of multiple surface proteins of group B streptococci as candidates.
This chapter focuses only on the major structural polysaccharide components of bacterial cell walls, so to present a coherent commentary on the impact of genomics on the understanding of cell wall biosynthesis. The chapter is organized to relate a commentary on the basic processes of cell wall biosynthesis derived from decades of chemical analyses and classical genetics studies in the pregenomic era. Recently, researchers have made tremendous progress in imaging of bacterial cell walls by cryoelectron microscopy and atomic force microscopy, which has provided new detailed insights into cell wall organization in both gram-positive and gram-negative bacteria. The construction of the murein sacculus is essentially a two-stage process. In the first, a disaccharide-peptide monomer unit is assembled by using UDP-linked and then polyprenyl phosphate-linked intermediates. Next, transglycosylases (TGs) catalyze the polymerization of the glycan chains and transpeptidases (TPs); the penicillin-binding proteins (PBPs) cross-link the peptide cross-bridges between glycan chains and thus incorporate nascent material into the existing PG sacculus framework. An architecturally similar but less complex cell wall core structure is conserved across the Corynebacterineae, which presented the possibility that comparative genomics might scout new routes toward the understanding of the construction of these fascinating structures. In this era of rapidly emerging multidrug resistance, the efforts to understand bacterial pathogens through study of their cell wall biosynthesis, in identification of novel targets, by defining modes of action of current drugs, and by investigating the development of resistance, must keep pace with the rapidly evolving adversaries.
This chapter discusses the insights gained from genomics of intracellular pathogens using Listeria monocytogenes as an example. The genus comprises six species, two of which are pathogenic, L. monocytogenes for humans and animals, and L. ivanovii, mainly for ruminants. L. monocytogenes is an environmental bacterium that lives on decomposing plants, but the acquisition of virulence factors, most probably by horizontal gene transfer, allows L. monocytogenes to also infect humans and many other mammalian hosts. The newly studied surface proteins are present in all L. monocytogenes strains investigated. Further studies should focus on strain-specific surface proteins because diversity among these may account for strain differences in virulence and in niche adaptation. Williams and colleagues introduced in-frame deletions into 15 of 16 response regulator genes and characterized the resulting mutants. In this study, the deletion of the individual response regulator genes had only minor effects on in vitro and in vivo growth of the bacteria, except for DegU. A coupled bioinformatics/microarray approach applied to identify sigma B-regulated genes confirmed the overlap between the PrfA and the sigma B regulon. In this study, SigB-dependent promoter sequences were searched in the L. monocytogenes EGDe genome sequence. The pronounced diversity among LPXTG proteins, identified by whole-genome comparisons, was further substantiated by this comparative genomics study using DNA/DNA array hybridization.
This chapter provides an overview of how genome sequence data are changing our understanding of the mechanisms and evolution of plant pathogenesis, and discusses the opportunities and challenges genome data present for future research. The majority of plant pathogenic bacteria contain large numbers of genomic islands, and it is clear that in many cases they play an essential role in disease. Genes encoding the P. syringae phytotoxins coronatine and syringomycin reside on PIs, as do their recently discovered counterparts in E. carotovora subsp. atroseptica SCR1043. Pathogenicity and virulence factors have received the greatest focus in the genomic analysis of plant pathogens. Protein secretion systems are essential for pathogenesis in most plant pathogenic bacteria and have been extensively studied, particularly in proteobacterial pathogens. The phytohormones indole-3-acetic acid (IAA or auxin) is produced by many phytopathogenic bacteria. IAA production has been shown to contribute to in planta and epiphytic growth, virulence, and the regulation of syringomycin in P. syringae. Genome analyses of plant pathogens have highlighted three aspects of physiological adaptation to life on plants: specialization, innovation, and flexibility. A largely uninvestigated element in understanding the content and function of plant pathogen genomes, particularly the genomes of facultative plant pathogens, rests on understanding what plant pathogenic bacteria do when not causing disease.
This chapter addresses questions related to the genomics of Photorhabdus, compares the Photorhabdus genome with Escherichia coli to identify putative pathogenicity islands, and compares different Photorhabdus species in order to identify islands that are putatively specific to different species with different life cycles. At present, we have full genome sequences from both an exclusive insect pathogen, P. luminescens TT01, and an insect pathogen that has also been recovered from human wounds, P. asymbiotica. Comparisons between these two very different species should therefore also shed light on the evolution of vertebrate pathogenicity in the Photorhabdus genus. Comparison of Photorhabdus virulence cassette (PVC) loci of different Photorhabdusstrains shows that different strains possess different complements of units. In order to clarify the relationship between the Photorhabdus insect-related (PIR) AB toxins and the protein labeled as a JHE in the Colorado potato beetle, we cloned plu4093 and plu4092 from TT01 and tested them for injectable activity against Galleria. Similarly, comparisons of the tca loci of different Photorhabdus species show that three different deletions have led to the three independent losses of tca components in P. temperata, P. asymbiotica, and P. luminescens TT01. DNA-based microarrays have been used in the comparative genomics of different Photorhabdus species and strains in order to attempt to identify regions involved in the specificity of the Photorhabdus nematode interaction. The authors have compared the genome of the insect pathogen with that of the human pathogen in an attempt to understand what makes Photorhabdus asymbiotica so pathogenic.
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