Extensive genomic diversity and selective conservation of virulence determinants in enterohemorrhagic Escherichia coli strains of O157 and non O157 serotypes

Background: Enterohemorrhagic Escherichia coli (EHEC) O157 causes severe food-borne illness in humans. The chromosome of O157 consists of 4.1 Mb backbone sequences shared by benign E. coli K-12, and 1.4 Mb O157-specific sequences encoding many virulence determinants, such as Shiga toxin genes (stx genes) and the locus of enterocyte effacement (LEE). Non-O157 EHECs belonging to distinct clonal lineages from O157 also cause similar illness in humans. According to the "parallel" evolution model, they have independently acquired the major virulence determinants, the stx genes and LEE. However, the genomic differences between O157 and non-O157 EHECs have not yet been systematically analyzed. Results: Using microarray and whole genome PCR scanning analyses, we performed a whole genome comparison of 20 EHEC strains of O26, O111, and O103 serotypes with O157. In non-O157 EHEC strains, although genome sizes were similar with or rather larger than O157 and the backbone regions were well conserved, O157-specific regions were very poorly conserved. Around only 20% of the O157- specific genes were fully conserved in each non-O157 serotype. However, the non-O157 EHECs contained a significant number of virulence genes that are found on prophages and plasmids in O157, and also multiple prophages similar to, but significantly divergent from, those in O157. Conclusion: Although O157 and non-O157 EHECs have independently acquired a huge amount of serotype- or strain-specific genes by lateral gene transfer, they share an unexpectedly large number of virulence genes. Independent infections of similar but distinct bacteriophages carrying these virulence determinants are deeply involved in the evolution of O157 and non-O157 EHECs

The Shiga toxin 2 production level in enterohemorrhagic Escherichia coli O157:H7 is correlated with the subtypes of toxin-encoding phage

Enterohemorrhagic E. coli (EHEC) causes diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome. Their major virulence factor is Shiga toxin (Stx), which is encoded by bacteriophages. Of the two types of Stx, the production of Stx2, particularly that of Stx2a (a subtype of Stx2), is a major risk factor for severe EHEC infections, but the Stx2 production level is highly variable between strains. Here, we define four major and two minor subtypes of Stx2a-encoding phages according to their replication proteins. The subtypes are correlated with Stx2a titers produced by the host O157 strains, suggesting a critical role of the phage subtype in determining the Stx2a production level. We further show that one of the two subclades in the clade 8, a proposed hyper-virulent lineage of O157, carries the Stx2 phage subtype that confers the highest Stx2 production to the host strain. The presence of this subclade may explain the proposed high virulence potential of clade 8. These results provide novel insights into the variation in virulence among O157 strains and highlight the role of phage variation in determining the production level of the virulence factors that phages encode

The Lifestyle of the Segmented Filamentous Bacterium: A Non-Culturable Gut-Associated Immunostimulating Microbe Inferred by Whole-Genome Sequencing

Numerous microbes inhabit the mammalian intestinal track and strongly impact host physiology; however, our understanding of this ecosystem remains limited owing to the high complexity of the microbial community and the presence of numerous non-culturable microbes. Segmented filamentous bacteria (SFBs), which are clostridia-related Gram-positive bacteria, are among such non-culturable populations and are well known for their unique morphology and tight attachment to intestinal epithelial cells. Recent studies have revealed that SFBs play crucial roles in the post-natal maturation of gut immune function, especially the induction of Th17 lymphocytes. Here, we report the complete genome sequence of mouse SFBs. The genome, which comprises a single circular chromosome of 1 620 005 bp, lacks genes for the biosynthesis of almost all amino acids, vitamins/cofactors and nucleotides, but contains a full set of genes for sporulation/germination and, unexpectedly, for chemotaxis/flagella-based motility. These findings suggest a triphasic lifestyle of the SFB, which comprises two types of vegetative (swimming and epicellular parasitic) phases and a dormant (spore) phase. Furthermore, SFBs encode four types of flagellin, three of which are recognized by Toll-like receptor 5 and could elicit the innate immune response. Our results reveal the non-culturability, lifestyle and immunostimulation mechanisms of SFBs and provide a genetic basis for the future development of the SFB cultivation and gene-manipulation techniques

Global population structure, genomic diversity and carbohydrate fermentation characteristics of clonal complex 119 (CC119), an understudied Shiga toxin-producing E. coli (STEC) lineage including O165:H25 and O172:H25.

peer reviewedAmong Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified

NleC, a Type III Secretion Protease, Compromises NF-κB Activation by Targeting p65/RelA

The NF-κB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen-associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-κB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely related bacteria responsible for major food-borne diseases worldwide. Via a needle-like protein complex called the type three secretion system (T3SS), these pathogens deliver virulence factors directly to host cells and modify cellular functions, including by suppressing the inflammatory response. Using gain- and loss-of-function screenings, we identified two bacterial effectors, NleC and NleE, that down-regulate the NF-κB signal upon being injected into a host cell via the T3SS. A recent report showed that NleE inhibits NF-κB activation, although an NleE-deficient pathogen was still immune-suppressive, indicating that other anti-inflammatory effectors are involved. In agreement, our present results showed that NleC was also required to inhibit inflammation. We found that NleC is a zinc protease that disrupts NF-κB activation by the direct cleavage of NF-κB's p65 subunit in the cytoplasm, thereby decreasing the available p65 and reducing the total nuclear entry of active p65. More importantly, we showed that a mutant EPEC/EHEC lacking both NleC and NleE (ΔnleC ΔnleE) caused greater inflammatory response than bacteria carrying ΔnleC or ΔnleE alone. This effect was similar to that of a T3SS-defective mutant. In conclusion, we found that NleC is an anti-inflammatory bacterial zinc protease, and that the cooperative function of NleE and NleC disrupts the NF-κB pathway and accounts for most of the immune suppression caused by EHEC/EPEC

Wide Distribution of O157-Antigen Biosynthesis Gene Clusters in Escherichia coli

Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci

Genome evolution and plasticity of <em>Serratia marcescens</em>:an important multidrug resistant nosocomial pathogen

Serratia marcescens is an important nosocomial pathogen that can cause an array of infections, most notably of the urinary tract and bloodstream. Naturally, it is found in many environmental niches, and is capable of infecting plants and animals. The emergence and spread of multidrug-resistant strains producing extended-spectrum or metallo beta-lactamases now pose a threat to public health worldwide. Here we report the complete genome sequences of two carefully selected S. marcescens strains, a multidrug-resistant clinical isolate (strain SM39) and an insect isolate (strain Db11). Our comparative analyses reveal the core genome of S. marcescens and define the potential metabolic capacity, virulence, and multidrug resistance of this species. We show a remarkable intraspecies genetic diversity, both at the sequence level and with regards genome flexibility, which may reflect the diversity of niches inhabited by members of this species. A broader analysis with other Serratia species identifies a set of approximately 3,000 genes that characterize the genus. Within this apparent genetic diversity, we identified many genes implicated in the high virulence potential and antibiotic resistance of SM39, including the metallo beta-lactamase and multiple other drug resistance determinants carried on plasmid pSMC1. We further show that pSMC1 is most closely related to plasmids circulating in Pseudomonas species. Our data will provide a valuable basis for future studies on S. marcescens and new insights into the genetic mechanisms that underlie the emergence of pathogens highly resistant to multiple antimicrobial agents

The Defective Prophage Pool of Escherichia coli O157: Prophage–Prophage Interactions Potentiate Horizontal Transfer of Virulence Determinants

Bacteriophages are major genetic factors promoting horizontal gene transfer (HGT) between bacteria. Their roles in dynamic bacterial genome evolution have been increasingly highlighted by the fact that many sequenced bacterial genomes contain multiple prophages carrying a wide range of genes. Enterohemorrhagic Escherichia coli O157 is the most striking case. A sequenced strain (O157 Sakai) possesses 18 prophages (Sp1–Sp18) that encode numerous genes related to O157 virulence, including those for two potent cytotoxins, Shiga toxins (Stx) 1 and 2. However, most of these prophages appeared to contain multiple genetic defects. To understand whether these defective prophages have the potential to act as mobile genetic elements to spread virulence determinants, we looked closely at the Sp1–Sp18 sequences, defined the genetic defects of each Sp, and then systematically analyzed all Sps for their biological activities. We show that many of the defective prophages, including the Stx1 phage, are inducible and released from O157 cells as particulate DNA. In fact, some prophages can even be transferred to other E. coli strains. We also show that new Stx1 phages are generated by recombination between the Stx1 and Stx2 phage genomes. The results indicate that these defective prophages are not simply genetic remnants generated in the course of O157 evolution, but rather genetic elements with a high potential for disseminating virulence-related genes and other genetic traits to other bacteria. We speculate that recombination and various other types of inter-prophage interactions in the O157 prophage pool potentiate such activities. Our data provide new insights into the potential activities of the defective prophages embedded in bacterial genomes and lead to the formulation of a novel concept of inter-prophage interactions in defective prophage communities