Dual Zinc Transporter Systems in Vibrio cholerae Promote Competitive Advantages over Gut Microbiome

Abstract

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...

Identification and Characterization of a Putative Manganese Export Protein in Vibrio cholerae

“…The bacteria must be able to control the intracellular concentration of Mn and other metal ions in each environment and adapt quickly to changes. There is significant available information about the homeostasis of iron (40,41) and, to a lesser extent, of zinc (42) in V. cholerae. In contrast, there is little information on the biology of manganese in V. cholerae.…”

Bacterial Strategies to Maintain Zinc Metallostasis at the Host-Pathogen Interface

“…When the intracellular zinc (40,41); MT, metallothionein; CDF, cation diffusion facilitator transporter; RND, resistance-nodulation-cell division transporter. Zn (yellow circles) homeostasis is governed by a pair of Zn-sensing regulators, an uptake regulator (exemplified by Zur or AdcR) and efflux regulator (by ZntR or pneumococcal SczA).…”

“…More recently, other transporters have been shown to function in Zn uptake in S. enterica and Vibrio cholera (40,41). As discussed above, host strategies to limit bacterial infection exploit both zinc toxicity, perhaps best understood for intracellular niches (3,42), and severe zinc limitation (starvation) (43).…”

OxyR2 Modulates OxyR1 Activity and Vibrio cholerae Oxidative Stress Response

“…The streptomycin-treated adult mouse model was used as previously described (23,32). Briefly, 5-week-old CD-1 mice were provided with drinking water containing 0.5% (wt/vol) streptomycin and 0.5% sucrose.…”