Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis inVibrio cholerae

Abstract

The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.Vibrio cholerae, a rod-shaped, highly motile, gram-negative bacterium, is the causative agent of the life threatening diarrheal disease cholera (59). The type II secretion (T2S) system plays an important role in the pathogenesis of V. cholerae by secreting cholera toxin (63), which is largely responsible for the symptoms of the disease (33). The T2S system is widespread and well conserved in gram-negative bacteria inhabiting a variety of ecological niches and likely contributes to environmental survival as well as to virulence (11,21). In V. cholerae, secretion via the T2S machinery is supported by a transenvelope complex of 12 Eps proteins (EpsC to EpsN) and the type 4 prepilin peptidase PilD (VcpD) (25,44,63). Transport of exoproteins by the T2S system occurs via a two-step process. The first step, which is either Sec or Tat dependent, requires recognition of the N-terminal signal peptide of the exoproteins and translocation through the inner membrane to the periplasm. Then the folded proteins engage the T2S machinery and are subsequently exported across the outer membrane to the extracellular milieu (23,29).Besides periplasmic accumulation of exoproteins, additional phenotypes of T2S mutants are reporte...

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

“…Approximately half (84) of the proteins were common to both strains, whereas 19 proteins were identified solely in the supernatant isolated from wild-type V. cholerae, and 84 protein species were unique to the N⌬eps strain (supplemental Tables S1-S3). The greater number of protein species detected in the culture supernatant derived from the T2S mutant was consistent with our previous observations that inactivation of the T2S pathway results in a leaky cell envelope where periplasmic and cytoplasmic proteins are nonspecifically released to the culture medium (supplemental Table S3) (29,58).…”

“…This results in induction of extracytoplasmic and oxidative stress responses and in alterations in iron homeostasis (29,58). As a consequence, culture supernatants isolated from the T2S mutants display radically different protein profiles in comparison with wild-type strains with both the number of protein species present as well as their levels being elevated (29).…”

“…We considered the following as putative T2S substrates: 1) those that were only present in the wild-type supernatant; 2) those that contained predicted N-terminal signal peptides and/or were predicted to be extracellularly secreted, and 3) those whose gene expression was not reduced in the T2S mutants (58). Proteins that met these criteria are detailed in Table 1.…”

Regulation of bacterial virulence gene expression by cell envelope stress responses

“…73,76 Aside from its export role, some studies have also implicated this T2SS in maintaining cell envelope integrity. 77,78 Removal of the eps genes compromises the OM, which is characterized by leakage of periplasmic components, the reduction of specific OM proteins, increased sensitivity to membrane-perturbing agents, and activation of the RpoE response. 77,78 Activation of RpoE is consistent with removal of the T2SS compromising the cell envelope and might result, at least in part, from T2SS substrates accumulating in the periplasm.…”

“…77,78 Removal of the eps genes compromises the OM, which is characterized by leakage of periplasmic components, the reduction of specific OM proteins, increased sensitivity to membrane-perturbing agents, and activation of the RpoE response. 77,78 Activation of RpoE is consistent with removal of the T2SS compromising the cell envelope and might result, at least in part, from T2SS substrates accumulating in the periplasm. However, a particularly interesting and recent finding is that one of the promoters driving expression of the epsC-N operon is RpoE-dependent.…”