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

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 reported for an increasing number of species, possibly indicating involvement of the T2S system in other important cellular processes. For example, alterations in outer membrane protein composition have been described for T2S mutants of V. cholerae, Aeromonas hydrophila, marine Vibrio sp. strain 60, and Shewanella oneidensis (30, 32, 63, 64). The levels of outer membrane porins OmpU, OmpT, and OmpS are decreased in T2S mutants of V. cholerae (63, 65), and likewise, disruption of T2S genes in A. hydrophila leads to diminished quantities of OmpF and OmpS (30). Similarly, the amounts of the c-type cytochromes MtrC and OmcA in the outer membranes of S. oneidensis T2S mutants are reduced (64). Furthermore, we have shown that inactivation of the T2S system in V. cholerae results in a reduced growth rate, compromised outer membrane integrity, and, as a consequence, induction of RpoE activity. In addition, our studies showed that V. cholerae T2S mutants are unable to survive the passage through the infant mouse gastrointestinal tract (65). Growth defects at low temperatures under laboratory conditions as well as in tap water and amoebae were also observed for T2S mutants of Legionella pneumophila (68).Interestingly, differential abundance of proteins involved in phosphate metabolism and iron uptake has been revealed by proteomic analysis of culture supernatants isolated from wild-type and T2S mutant strains of Pseudoaltermonas tunicata (22). Based on these results, it has been suggested that the T2S system might be involved in iron acquisition. Similarly, certain T2S mutants of Erwinia chrysanthemi exhibit defects indicative of changes in iron homeostasis (17). It has also been noted that the level of aconitate hydratase, an iron-sulfur cluster-containing enzyme, is reduced in L. pneumophila T2S mutants (16).In this study, in an attempt to explain the phenotypes associated with loss of T2S, we performed microarray gene expression profiling of wild-type and T2S-deficient strains. Our data revealed that inactivation of the T2S machinery results in a metabolic feedback loop leading to oxidative stress and changes in iron metabolism. By analyzing another V. cholerae mutant that shares a similar cell envelope phenotype while remaining competent for T2S, we show that the changes in iron homeostasis and oxidative stress are linked to cell envelope damage and extracytoplasmic stress.