Suppressor Mutations in Type II Secretion Mutants of Vibrio cholerae: Inactivation of the VesC Protease.

The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including Vibrio cholerae Disruption of the T2SS genes in V. cholerae results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six V. cholerae T2SS mutants with deletions or insertions in either the epsG, epsL, or epsM genes and identified secondary mutations in all mutants. Two of the six T2SS mutants contain distinct mutations in the gene encoding the T2SS-secreted protease VesC. Other mutations were found in genes coding for V. cholerae cell envelope proteins. Subsequent sequence analysis of the vesC gene in 92 additional T2SS mutant isolates identified another 19 unique mutations including insertions or deletions, sequence duplications, and single-nucleotide changes resulting in amino acid substitutions in the VesC protein. Analysis of VesC variants and the X-ray crystallographic structure of wild-type VesC suggested that all mutations lead to loss of VesC production and/or function. One possible mechanism by which V. cholerae T2SS mutagenesis can be tolerated is through selection of vesC-inactivating mutations, which may, in part, suppress cell envelope damage, establishing permissive conditions for the disruption of the T2SS. Other mutations may have been acquired in genes encoding essential cell envelope proteins to prevent proteolysis by VesC.IMPORTANCE Genome-wide transposon mutagenesis has identified the genes encoding the T2SS in Vibrio cholerae as essential for viability, but the reason for this is unclear. Mutants with deletions or insertions in these genes can be isolated, suggesting that they have acquired secondary mutations that suppress their growth defect. Through whole-genome sequencing and phenotypic analysis of T2SS mutants, we show that one means by which the growth defect can be suppressed is through mutations in the gene encoding the T2SS substrate VesC. VesC homologues are present in other Vibrio species and close relatives, and this may be why inactivation of the T2SS in species such as Vibrio vulnificus, Vibrio sp. strain 60, and Aeromonas hydrophila also results in a pleiotropic effect on their outer membrane assembly and integrity.

Measuring In Vitro ATPase Activity for Enzymatic Characterization.

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Zinc coordination is essential for the function and activity of the type II secretion ATPase EpsE.

The type II secretion system Eps in Vibrio cholerae promotes the extracellular transport of cholera toxin and several hydrolytic enzymes and is a major virulence system in many Gram-negative pathogens which is structurally related to the type IV pilus system. The cytoplasmic ATPase EpsE provides the energy for exoprotein secretion through ATP hydrolysis. EpsE contains a unique metal-binding domain that coordinates zinc through a tetracysteine motif (CXXCX29 CXXC), which is also present in type IV pilus assembly but not retraction ATPases. Deletion of the entire domain or substitution of any of the cysteine residues that coordinate zinc completely abrogates secretion in an EpsE-deficient strain and has a dominant negative effect on secretion in the presence of wild-type EpsE. Consistent with the in vivo data, chemical depletion of zinc from purified EpsE hexamers results in loss of in vitro ATPase activity. In contrast, exchanging the residues between the two dicysteines with those from the homologous ATPase XcpR from Pseudomonas aeruginosa does not have a significant impact on EpsE. These results indicate that, although the individual residues in the metal-binding domain are generally interchangeable, zinc coordination is essential for the activity and function of EpsE.

The Type II secretion system delivers matrix proteins for biofilm formation by Vibrio cholerae.

Gram-negative bacteria have evolved several highly dedicated pathways for extracellular protein secretion, including the type II secretion (T2S) system. Since substrates secreted via the T2S system include both virulence factors and degradative enzymes, this secretion system is considered a major survival mechanism for pathogenic and environmental species. Previous analyses revealed that the T2S system mediates the export of ≥ 20 proteins in Vibrio cholerae, a human pathogen that is indigenous to the marine environment. Here we demonstrate a new role in biofilm formation for the V. cholerae T2S system, since wild-type V. cholerae was found to secrete the biofilm matrix proteins RbmC, RbmA, and Bap1 into the culture supernatant, while an isogenic T2S mutant could not. In agreement with this finding, the level of biofilm formation in a static microtiter assay was diminished in T2S mutants. Moreover, inactivation of the T2S system in a rugose V. cholerae strain prevented the development of colony corrugation and pellicle formation at the air-liquid interface. In contrast, extracellular secretion of the exopolysaccharide VPS, an essential component of the biofilm matrix, remained unaffected in the T2S mutants. Our results indicate that the T2S system provides a mechanism for the delivery of extracellular matrix proteins known to be important for biofilm formation by V. cholerae. Because the T2S system contributes to the pathogenicity of V. cholerae by secreting proteins such as cholera toxin and biofilm matrix proteins, elucidation of the molecular mechanism of T2S has the potential to lead to the development of novel preventions and therapies.

Flow cytometry-based enrichment for cell shape mutants identifies multiple genes that influence Helicobacter pylori morphology.

The helical cell shape of Helicobacter pylori is highly conserved and contributes to its ability to swim through and colonize the viscous gastric mucus layer. A multi-faceted peptidoglycan (PG) modification programme involving four recently characterized peptidases and two accessory proteins is essential for maintaining H. pylori's helicity. To expedite identification of additional shape-determining genes, we employed flow cytometry with fluorescence-activated cell sorting (FACS) to enrich a transposon library for bacterial cells with altered light scattering profiles that correlate with perturbed cell morphology. After a single round of sorting, 15% of our clones exhibited a stable cell shape defect, reflecting 37-fold enrichment. Sorted clones with straight rod morphology contained insertions in known PG peptidases, as well as an insertion in csd6, which we demonstrated has ld-carboxypeptidase activity and cleaves monomeric tetrapeptides in the PG sacculus, yielding tripeptides. Other mutants had only slight changes in helicity due to insertions in genes encoding MviN/MurJ, a protein possibly involved in initiating PG synthesis, and the hypothetical protein HPG27_782. Our findings demonstrate FACS robustly detects perturbations of bacterial cell shape and identify additional PG peptide modifications associated with helical cell shape in H. pylori.