Bacterial Pathogenomics

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 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.

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.

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.

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.

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 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 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.