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ABSTRACTEscherichia colistrains expressing the K1 capsule are a major cause of sepsis and meningitis in human neonates. The development of these diseases is dependent on the expression of a range of virulence factors, many of which remain uncharacterized. Here, we show that all but 1 of 34 E. coliK1 neonatal isolates carried clbAand clbP, genes contained within the pkspathogenicity island and required for the synthesis of colibactin, a polyketide-peptide genotoxin that causes genomic instability in eukaryotic cells by induction of double-strand breaks in DNA. Inactivation of clbAand clbPin E. coliA192PP, a virulent strain of serotype O18:K1 that colonizes the gastrointestinal tract and translocates to the blood compartment with very high frequency in experimental infection of the neonatal rat, significantly reduced the capacity of A192PP to colonize the gut, engender double-strand breaks in DNA, and cause invasive, lethal disease. Mutation of clbA, which encodes a pleiotropic enzyme also involved in siderophore synthesis, impacted virulence to a greater extent than mutation of clbP, encoding an enzyme specific to colibactin synthesis. Restoration of colibactin gene function by complementation reestablished the fully virulent phenotype. We conclude that colibactin contributes to the capacity of E. coliK1 to colonize the neonatal gastrointestinal tract and to cause invasive disease in the susceptible neonate.

ABSTRACTThe formation of an incapacitating biofilm on Caenorhabditis elegansby Yersinia pseudotuberculosisrepresents a tractable model for investigating the genetic basis for host-pathogen interplay during the biofilm-mediated infection of a living surface. Previously we established a role for quorum sensing (QS) and the master motility regulator, FlhDC, in biofilm formation by Y. pseudotuberculosison C. elegans. To obtain further genome-wide insights, we used transcriptomic analysis to obtain comparative information on C. elegansin the presence and absence of biofilm and on wild-type Y. pseudotuberculosisand Y. pseudotuberculosisQS mutants. Infection of C. eleganswith the wild-type Y. pseudotuberculosisresulted in the differential regulation of numerous genes, including a distinct subset of nematode C-lectin (clec) and fatty acid desaturase (fat) genes. Evaluation of the corresponding C. elegansclec-49and fat-3deletion mutants showed delayed biofilm formation and abolished biofilm formation, respectively. Transcriptomic analysis of Y. pseudotuberculosisrevealed that genes located in both of the histidine utilization (hut) operons were upregulated in both QS and flhDCmutants. In addition, mutation of the regulatory gene hutCresulted in the loss of biofilm, increased expression of flhDC, and enhanced swimming motility. These data are consistent with the existence of a regulatory cascade in which the Hut pathway links QS and flhDC. This work also indicates that biofilm formation by Y. pseudotuberculosison C. elegansis an interactive process during which the initial attachment/recognition of Yersiniato/by C. elegansis followed by bacterial growth and biofilm formation.

ABSTRACTClostridium difficileis an important nosocomial pathogen and the leading cause of antibiotic-associated diarrhea. Multilocus sequence typing indicates that C. difficilestrains belong to five distinct genetic clades encompassing several PCR ribotypes (RT). Since their emergence in 2003, hypervirulent RT027 strains have been a major focus of research; in contrast, our current understanding of RT017-mediated disease pathogenesis lags far behind. In this study, we aimed to characterize host immunity to CF5 and M68, two genetically well-defined RT017 strains. Both strains engaged with host Toll-like receptor 2/6 (TLR2/6), TLR2-CD14, and TLR5 to similar extents in a model cell line. Despite this, CF5 mediated significantly greater dendritic cell (DC) interleukin-12 (IL-12), IL-27, and IL-10 immunity than M68. Both strains elicited similar IL-1β mRNA levels, and yet only M68 caused a marked increase in secretory IL-1β. A CF5 cocultured-DC cytokine milieu drove an equipotent Th1 and Th17 response, while M68 promoted greater Th17 immunity. Human gastrointestinal ex vivocytokine responses to both strains were characterized. Taken together, our data suggest that C. difficilestrains mediate overlapping and yet distinct mucosal and DC/T cell immunity. Finally, toxin-driven IL-1β release supports the hypothesis that this cytokine axis is a likely target for therapeutic intervention for C. difficileinfection.

ABSTRACTClostridium difficilepersists in hospitals by exploiting an infection cycle that is dependent on humans shedding highly resistant and infectious spores. Here we show that human virulent C. difficilecan asymptomatically colonize the intestines of immunocompetent mice, establishing a carrier state that persists for many months. C. difficilecarrier mice consistently shed low levels of spores but, surprisingly, do not transmit infection to cohabiting mice. However, antibiotic treatment of carriers triggers a highly contagious supershedder state, characterized by a dramatic reduction in the intestinal microbiota species diversity, C. difficileovergrowth, and excretion of high levels of spores. Stopping antibiotic treatment normally leads to recovery of the intestinal microbiota species diversity and suppresses C. difficilelevels, although some mice persist in the supershedding state for extended periods. Spore-mediated transmission to immunocompetent mice treated with antibiotics results in self-limiting mucosal inflammation of the large intestine. In contrast, transmission to mice whose innate immune responses are compromised (Myd88−/−) leads to a severe intestinal disease that is often fatal. Thus, mice can be used to investigate distinct stages of the C. difficileinfection cycle and can serve as a valuable surrogate for studying the spore-mediated transmission and interactions between C. difficileand the host and its microbiota, and the results obtained should guide infection control measures.

ABSTRACTHemolysins have been found to possess a variety of functions in bacteria, including a role in virulence. Helicobacter pyloridemonstrates hemolytic activity when cultured on unlysed blood agar plates which is increased under iron-limiting conditions. However, the role of an H. pylorihemolysin in virulence is unclear. Scrutiny of the H. pylori26695 genome sequence suggests the presence of at least two distinct hemolysins, HP1086 and HP1490, in this strain. Previous studies have shown that the in vitro hemolytic activity of H. pyloriis reduced when it is coincubated with dextran 5000, suggesting the presence of a pore-forming cytolysin. HP1086 has homology to pore-forming cytolysins (TlyA) from other bacterial species, and the introduction of the clonedH. pylori tlyAgene into a nonhemolyticEscherichia colistrain conferred hemolytic activity. AnH. pylori tlyAdefined mutant showed reduced in vitro hemolytic activity, which appears to be due to pore formation, as the hemolytic activity of the wild-type strain is reduced to the same level as the tlyAmutant by the addition of dextran 5000. The mutant also showed reduced adhesion to human gastric adenocarcinoma cells and failed to colonize the gastric mucosa of mice. These data clearly suggest a role in virulence for H. pyloriTlyA, contrary to the suggestion that hemolytic activity is an in vitro phenomenon for this pathogen.

ABSTRACTInfection of the mucous layer of the human stomach byHelicobacter pylorirequires the bacterium to be motile and presumably chemotactic. Previous studies have shown that fully functional flagella are essential for motility and colonization, but the role of chemotaxis remains unclear. The two-component regulatory system CheA/CheY has been shown to play a major role in chemotaxis in other enteric bacteria. Scrutiny of the 26695 genome sequence suggests that H. pylorihas two CheY response regulators: one a separate protein (CheY1) and the other (CheY2) fused to the histidine kinase sensor CheA. Defined deletion mutations were introduced intocheY1, cheY2, and cheAin H. pyloristrains N6 and SS1. Video tracking revealed that the wild-type H. pyloristrain moves in short runs with frequent direction changes, in contrast to movement ofcheY2, cheAY2, and cheAY2 cheY1mutants, whose motion was more linear. The cheY1mutant demonstrated a different motility phenotype of rapid tumbling. All mutants had impaired swarming and greatly reduced chemotactic responses to hog gastric mucin. Neither cheY1nor cheAY2mutants were able to colonize mice, but they generated a significant antibody response, suggesting that despite impaired chemotaxis, these mutants were able to survive in the stomach long enough to induce an immune response before being removed by gastric flow. Additionally, we demonstrated that cheY1failed to colonize gnotobiotic piglets. This study demonstrates the importance of the roles ofcheY1, cheY2, and cheAin motility and virulence of H. pylori.

Eimeria tenellacan cause the disease coccidiosis in chickens. The direct and often detrimental impact of this parasite on chicken health, welfare, and productivity is well recognized; however, less is known about the secondary effects that infection may have on other gut pathogens.