Zinc is an essential trace metal required for numerous cellular processes in all forms of life. In order to maintain zinc homeostasis, bacteria have developed several transport systems to regulate its uptake. In this study, we investigated zinc transport systems in the enteric pathogen Vibrio cholerae, the causative agent of cholera. Bioinformatic analysis predicts that two gene clusters, VC2081 to VC2083 (annotated as zinc utilization genes znuABC) and VC2551 to VC2555 (annotated as zinc-regulated genes zrgABCDE), are regulated by the putative zinc uptake regulator Zur. Using promoter reporter and biochemical assays, we confirmed that Zur represses znuABC and zrgABCDE promoters in a Zn 2؉ -dependent manner. Under Zn 2؉ -limiting conditions, we found that mutations in either the znuABC or zrgABCDE gene cluster affect bacterial growth, with znuABC mutants displaying a more severe growth defect, suggesting that both ZnuABC and ZrgABCDE are involved in Zn 2؉ uptake and that ZnuABC plays the predominant role. Furthermore, we reveal that ZnuABC and ZrgABCDE are important for V. cholerae colonization in both infant and adult mouse models, particularly in the presence of other intestinal microbiota. Collectively, our studies indicate that these two zinc transporter systems play vital roles in maintaining zinc homeostasis during V. cholerae growth and pathogenesis. Metal ions are required for many crucial biological processes and are necessary for the survival of living organisms, including bacteria (1). For example, zinc is an essential cofactor for enzymatic reactions, DNA synthesis, and gene expression (2). One study has shown that over 3% of Escherichia coli proteins contain zinc (3). Bacteria have therefore evolved sophisticated systems to control their intracellular zinc concentrations in response to zinc fluctuations in the environment. One system utilized by nearly all bacteria is ZnuABC, a high-affinity zinc uptake system belonging to the ATP binding cassette (ABC) transporter family (4). Three proteins constitute this system: ZnuA, a periplasmic Zn 2ϩ binding protein that captures and delivers zinc to ZnuB, which serves as an inner membrane channel, and ZnuC, an ATPase that provides the energy needed for zinc transport (4). On the other hand, zinc levels in bacteria need to be tightly regulated, as excess zinc has deleterious effects on cells, such as prevention of Mn 2ϩ intracellular accumulation (5) and inhibition of enzymes (6). Zinc transport genes are generally controlled by Zur, a member of the Fur protein family of metal-dependent transcriptional regulators (7). Under zinc-replete conditions, Zur binds free Zn 2ϩ . The Zur-Zn complex then binds to the promoter of znuABC, thus blocking the binding of RNA polymerase (8). Under zinc-deficient conditions, the zinc binding sites of Zur are unoccupied, leading to the destabilization of Zur and the inability to bind and repress znuABC transcription, thus allowing zinc acquisition. In some bacteria, in addition to repressing Zn 2ϩ uptake transporters, Zur can a...

dVibrio cholerae is the causative agent of the diarrheal disease cholera. The ability of V. cholerae to colonize and cause disease requires the intricately regulated expression of a number of virulence factors during infection. One of the signals sensed by V. cholerae is the presence of oxygen-limiting conditions in the gut. It has been shown that the virulence activator AphB plays a key role in sensing low oxygen concentrations and inducing the transcription of another key virulence activator, TcpP. In this study, we used a bacterial two-hybrid system to further examine the effect of oxygen on different virulence regulators. We found that anoxic conditions enhanced the interaction between TcpP and ToxR, identified as the first positive regulator of V. cholerae virulence genes. We further demonstrated that the TcpP-ToxR interaction was dependent on the primary periplasmic protein disulfide formation enzyme DsbA and cysteine residues in the periplasmic domains of both ToxR and TcpP. Furthermore, we showed that in V. cholerae, an interaction between TcpP and ToxR is important for virulence gene induction. Under anaerobic growth conditions, we detected ToxR-TcpP heterodimers, which were abolished in the presence of the reducing agent dithiothreitol. Our results suggest that V. cholerae may sense intestinal anoxic signals by multiple components to activate virulence.

Typhoid fever remains a public health threat in many countries. A positive result in traditional culture is a gold-standard for typhoid diagnosis, but this method is time consuming and not sensitive enough for detection of samples containing a low copy number of the target organism. The availability of the loop-mediated isothermal amplification (LAMP) assay, which offers high speed and simplicity in detection of specific targets, has vastly improved the diagnosis of numerous infectious diseases. However, little research efforts have been made on utilizing this approach for diagnosis of Salmonella enterica serovar Typhi by targeting a single and specific gene. In this study, a LAMP assay for rapid detection of S. Typhi based on a novel marker gene, termed STY2879-LAMP, was established and evaluated with real-time PCR (RT-PCR). The specificity tests showed that STY2879 could be amplified in all S. Typhi strains isolated in different years and regions in China, whereas no amplification was observable in non-typhoidal strains covering 34 Salmonella serotypes and other pathogens causing febrile illness. The detection limit of STY2879-LAMP for S. Typhi was 15 copies/reaction in reference plasmids, 200 CFU/g with simple heat-treatment of DNA extracted from simulated stool samples and 20 CFU/ml with DNA extracted from simulated blood samples, which was 10 fold more sensitive than the parallel RT-PCR control experiment. Furthermore, the sensitivity of STY2879-LAMP and RT-PCR combining the traditional culture enrichment method for simulated stool and blood spiked with lower S. Typhi count during the 10 h enrichment time was also determined. In comparison with LAMP, the positive reaction time for RT-PCR required additional 2-3 h enrichment time for either simulated stool or blood specimens. Therefore, STY2879-LAMP is of practical value in the clinical settings and has a good potential for application in developing regions due to its easy-to-use protocol.

Receptor recognition is a key step in the initiation of phage infection. Previously, we found that VP3, the T7 family phage of the Vibrio cholerae serogroup O1 biotype El Tor, can adsorb to the core oligosaccharide (OS) of lipopolysaccharides of V. cholerae. However, some wild-type strains of V. cholerae possessing the intact OS gene cluster still have VP3 binding but are resistant to VP3 infection. Moreover, an OS gene deletion mutant still exhibits weak VP3 binding, suggesting multiple factors are possibly involved in VP3 binding to V. cholerae. Here, we report that the outer-membrane protein TolC of V. cholerae is involved in the host adsorption of VP3. We observed that TolC directly interacts with the VP3 tail fiber protein gp44 and its C-terminal domains, and also found that three amino acid residues in the outside loops of TolC, at positions 78, 290 and 291, are critical for binding to gp44. Among VP3-resistant wild type V. cholerae strains, frequent amino acid residue mutations were observed in the loops around the sites 78, 290 and 291 which were predicted to be exposed to the cell surface. These findings reveal a co-receptor-binding mechanism for VP3 infection of V. cholerae and that both outer membrane TolC and OS are necessary for successful VP3 infection of V. cholerae. We conclude that mutations on the outside loops of the receptor may confer V. cholerae strains with VP3 phage-resistance, enabling these strains to survive in environments containing VP3 or related phages.