Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.

Salmonella enterica serovar Typhi siderophore production is elevated and Fur inactivation causes cell filamentation and attenuation in macrophages.

Salmonella enterica serovars Typhi and Typhimurium are two closely related bacteria causing different types of infection in humans. Iron acquisition is considered essential for virulence. Siderophores are important iron chelators and production of enterobactin and salmochelins by these serovars was quantified. Overall, Salmonella Typhi produced higher levels of siderophores than Salmonella Typhimurium. The role of the global regulator Fur, involved in iron homeostasis, present and conserved in both these serovars, was then investigated. Deletion of the fur gene led to distinct phenotypes in these serovars. Defective growth in iron-rich and iron-limiting conditions and formation of filamentous cells was only observed in the S. Typhi fur mutant. Furthermore, Fur was required for optimal motility in both serovars, but motility was more reduced for the fur mutant of S. Typhi compared to S. Typhimurium. During interaction with human-cultured macrophages, Fur was more important for S. Typhi, as the fur mutant had severe defects in uptake and survival. Globally, these results demonstrate that Fur differentially affects the physiology and the virulence phenotypes of the two strains and is more critical for S. Typhi growth, morphology, motility and interaction with host cells than it is for S. Typhimurium.

Systematic Analysis of Two-Component Systems in Citrobacter rodentium Reveals Positive and Negative Roles in Virulence.

Citrobacter rodentium is a murine pathogen used to model intestinal infections caused by the human diarrheal pathogens enterohemorrhagic and enteropathogenic Escherichia coli During infection, bacteria use two-component systems (TCSs) to detect changing environmental cues within the host, allowing for rapid adaptation by altering the expression of specific genes. In this study, 26 TCSs were identified in C. rodentium, and quantitative PCR (qPCR) analysis showed that they are all expressed during murine infection. These TCSs were individually deleted, and the in vitro and in vivo effects were analyzed to determine the functional consequences. In vitro analyses only revealed minor differences, and surprisingly, type III secretion (T3S) was only affected in the ΔarcA strain. Murine infections identified 7 mutants with either attenuated or increased virulence. In agreement with the in vitro T3S assay, the ΔarcA strain was attenuated and defective in colonization and cell adherence. The ΔrcsB strain was among the most highly attenuated strains. The decrease in virulence of this strain may be associated with changes to the cell surface, as Congo red binding was altered, and qPCR revealed that expression of the wcaA gene, which has been implicated in colanic acid production in other bacteria, was drastically downregulated. The ΔuvrY strain exhibited increased virulence compared to the wild type, which was associated with a significant increase in bacterial burden within the mesenteric lymph nodes. The systematic analysis of virulence-associated TCSs and investigation of their functions during infection may open new avenues for drug development.

Regulation and production of Tcf, a cable-like fimbriae from Salmonella enterica serovar Typhi.

tcf (Typhi colonization factor) is one of the 12 putative chaperone/usher fimbrial clusters present in the Salmonella enterica serovar Typhi genome. We investigated the production, expression and regulation of tcf as well as its role during interaction with human cells. The tcf gene cluster was cloned and induced in Escherichia coli and S. Typhi, and the production of intertwined fibres similar to the Cbl (cable) pili of Burkholderia cepacia was observed on the bacterial surface by electron microscopy. In S. Typhi, tcf was expressed more after growth in M63 minimal medium than in standard Luria-Bertani medium. Analysis of the promoter region identified putative binding sites for the global regulators RcsB, ArgR and Fur. The expression of tcf was measured in isogenic strains lacking these global regulators. Under the conditions tested, the results showed that tcf expression was higher in the fur mutant and was regulated by iron concentration. Fur may regulate these fimbriae indirectly via the small RNAs RyhB1 and RyhB2. An isogenic mutant harbouring a deletion of the tcf cluster did not demonstrate any defect in adhesion or invasion of human epithelial cells, or in phagocytosis or survival in macrophages, when compared to the WT serovar Typhi strain. However, the tcf cluster contributed to adherence to human epithelial cells when introduced into E. coli. Thus, tcf genes encode functional fimbriae that can act as an adhesin and may contribute to colonization during typhoid fever.

Inhibition of outer membrane proteases of the omptin family by aprotinin.

Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by Escherichia coli OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen Citrobacter rodentium. Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also Yersinia pestis Pla and, to a lesser extent, E. coli OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys15 of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site.

The CpxRA two-component system is essential for Citrobacter rodentium virulence.

Citrobacter rodentium is a murine intestinal pathogen used as a model for the foodborne human pathogens enterohemorrhagic Escherichia coli and enteropathogenic E. coli. During infection, these pathogens use two-component signal transduction systems to detect and adapt to changing environmental conditions. In E. coli, the CpxRA two-component signal transduction system responds to envelope stress by modulating the expression of a myriad of genes. Quantitative real-time PCR showed that cpxRA was expressed in the colon of C57BL/6J mice infected with C. rodentium. To determine whether CpxRA plays a role during C. rodentium infection, a cpxRA deletion strain was generated and found to have a colonization defect during infection. This defect was independent of an altered growth rate or a defective type III secretion system, and single-copy chromosomal complementation of cpxRA restored virulence. The C. rodentium strains were then tested in C3H/HeJ mice, a lethal intestinal infection model. Mice infected with the ΔcpxRA strain survived infection, whereas mice infected with the wild-type or complemented strains succumbed to infection. Furthermore, we found that the cpxRA expression level was higher during early infection than at a later time point. Taken together, these data demonstrate that the CpxRA two-component signal transduction system is essential for the in vivo virulence of C. rodentium. In addition, these data suggest that fine-tuned cpxRA expression is important for infection. This is the first study that identifies a C. rodentium two-component transduction system required for pathogenesis. This study further indicates that CpxRA is an interesting target for therapeutics against enteric pathogens.

Role of the Salmonella enterica serovar Typhi Fur regulator and small RNAs RfrA and RfrB in iron homeostasis and interaction with host cells.

Iron is an essential element but can be toxic at high concentrations. Therefore, its acquisition and storage require tight control. Salmonella encodes the global regulator Fur (ferric uptake regulator) and the small regulatory non-coding RNAs (sRNAs) RfrA and RfrB, homologues of RyhB. The role of these iron homeostasis regulators was investigated in Salmonella enterica serovar Typhi (S. Typhi). Strains containing either single or combined deletions of these regulators were obtained. The mutants were tested for growth in low and high iron conditions, resistance to oxidative stress, expression and production of siderophores, and during interaction with host cells. The fur mutant showed a growth defect and was sensitive to hydrogen peroxide. The expression of the sRNAs was responsible for these defects. Siderophore expression by S. Typhi and both sRNAs were regulated by iron and by Fur. Fur contributed to invasion of epithelial cells, and was shown for the first time to play a role in phagocytosis and intracellular survival of S. Typhi in human macrophages. The sRNAs RfrA and RfrB were not required for interaction with epithelial cells, but both sRNAs were important for optimal intracellular replication in macrophages. In S. Typhi, Fur is a repressor of both sRNAs, and loss of either RfrA or RfrB resulted in distinct phenotypes, suggesting a non-redundant role for these regulatory RNAs.

So similar, yet so different: uncovering distinctive features in the genomes of Salmonella enterica serovars Typhimurium and Typhi.

Salmonella enterica represents a major human and animal pathogen. Many S. enterica genomes have been completed and many more genome sequencing projects are underway, constituting an excellent resource for comparative genome analysis studies leading to a better understanding of bacterial evolution and pathogenesis. Salmonella enterica serovar Typhimurium and Typhi are the best-characterized serovars, with the first being involved in localized gastroenteritis in many hosts and the latter causing a systemic human-specific disease. Here, we summarize the major genetic differences between the two different serovars. We detail the divergent repertoires of the virulence factors responsible for the pathogenesis of the organisms and that ultimately result in the distinct clinical outcomes of infection. This comparative genomic overview highlights hypotheses for future investigations on S. enterica pathogenesis and the basis of host specificity.