General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae.

The general secretion pathway (GSP) of Vibrio cholerae is required for secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane. In this study, we report the cloning and sequencing of a DNA fragment from V. cholerae, containing 12 open reading frames, epsC to -N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, Pseudomonas, and Xanthomonas spp. In addition to the two previously described genes, epsE and epsM (M. Sandkvist, V. Morales, and M. Bagdasarian, Gene 123: 81-86, 1993; L. J. Overbye, M. Sandkvist, and M. Bagdasarian, Gene 132:101-106, 1993), it is shown here that epsC, epsF, epsG, and epsL also encode proteins essential for GSP function. Mutations in the eps genes result in aberrant outer membrane protein profiles, which indicates that the GSP, or at least some of its components, is required not only for secretion of soluble proteins but also for proper outer membrane assembly. Several of the Eps proteins have been identified by use of the T7 polymerase-promoter system in Escherichia coli. One of them, a pilin-like protein, EpsG, was analyzed also in V. cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that in this organism it might be processed or otherwise modified by a prepilin peptidase. We believe that TcpJ prepilin peptidase, which processes the subunit of the toxin-coregulated pilus, TcpA, is not involved in this event. This is supported by the observations that apparent processing of EpsG occurs in a tcpJ mutant of V. cholerae and that, when coexpressed in E. coli, TcpJ cannot process EpsG although the PilD peptidase from Neisseria gonorrhoeae can.     

Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni. Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro. The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target.