Compromised Outer Membrane Integrity in Vibrio cholerae Type II Secretion Mutants

Abstract

The type II secretion (T2S) system of Vibrio cholerae is a multiprotein complex that spans the cell envelope and secretes proteins important for pathogenesis as well as survival in different environments. Here we report that, in addition to the loss of extracellular secretion, removal or inhibition of expression of the T2S genes, epsC-N, results in growth defects and a broad range of alterations in the outer membrane that interfere with its barrier function. Specifically, the sensitivity to membrane-perturbing agents such as bile salts and the antimicrobial peptide polymyxin B is increased, and periplasmic constituents leak out into the culture medium. As a consequence, the E stress response is induced. Furthermore, due to the defects caused by inactivation of the T2S system, the ⌬eps deletion mutant of V. cholerae strain N16961 is incapable of surviving the passage through the infant mouse gastrointestinal tract. The growth defect and leaky outer membrane phenotypes are suppressed when the culture medium is supplemented with 5% glucose or sucrose, although the eps mutants remain sensitive to membrane-damaging agents. This suggests that the sugars do not restore the integrity of the outer membrane in the eps mutant strains per se but may provide osmoprotective functions.Gram-negative bacteria possess highly sophisticated and organized cell envelopes that consist of inner and outer membranes separated by the periplasmic compartment and the peptidoglycan layer. The outer membrane is made up of a lipopolysaccharide (LPS)-phospholipid asymmetric bilayer and functions as a barrier preventing entry of toxic substances, including antibiotics, dyes, and detergents (60, 72). At the same time, the outer membrane allows nutrient acquisition and transport of molecules in and out of the cell. Several dedicated transport systems have evolved for this purpose, and at least six pathways are required for extracellular protein secretion alone (43,67). One such system is the type II secretion (T2S) system, which has been identified in a wide variety of Proteobacteria, including many pathogens (for reviews, see references 12 and 74). Many of the proteins secreted by the T2S pathway, such as proteases, lipases, cellulases, pectinases, phospholipases, lipases, and toxins, contribute to virulence. These secreted proteins are synthesized with signal peptides and are first transported into the periplasmic compartment by Sec-or Tat-dependent processes and then cross the outer membrane through the T2S machinery (66, 89).In recent years, much attention has been paid to the interactions between individual components and the mechanism by which the multiprotein T2S complex is assembled. The present model for T2S machines includes a secretion pore in the outer membrane, a multiprotein subcomplex localized in the cytoplasmic membrane, a pseudopilus that spans the periplasmic compartment, and an ATPase in the cytoplasm (38). Besides the interactions within the T2S complex, it appears that T2S components also interact with other cell envelope con...

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

“…The resulting PCR amplification products were purified, subcloned into pPCR-Script TM ampicillin resistance SK(ϩ) (Stratagene), and then cloned into the appropriate vector as indicated. Introduction of plasmids and suicide vectors into V. cholerae strains was performed by conjugation with Escherichia coli S17.1 or triparental conjugation as described previously (29,47,48).…”

“…Cholera is a life-threatening diarrheal disease that predominantly occurs in developing countries of Asia, Africa, and South America (24). The T2S system, extracellular protein secretion (Eps), of V. cholerae is responsible for secretion of five known proteins, including cholera toxin, chitinase (ChiA-1), chitin-binding protein (GbpA), hemagglutinin protease (HAP), and lipase (25)(26)(27)(28)(29)(30).…”

Calcium Is Essential for the Major Pseudopilin in the Type 2 Secretion System

“…Functional Secretion Studies of GspG Mutants-The ⌬gspG strain and the complementing pMMB67EH-gspG plasmid were constructed as described previously (33). Mutations were introduced in gspG gene with QuikChange II site-directed mutagenesis kit (Stratagene) using pMMB-gspG as a template.…”

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.…”