Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae.

BACKGROUND: Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37. CONCLUSION/SIGNIFICANCE: Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids.

EpsE is a cytoplasmic component of the type II secretion system in Vibrio cholerae. Through ATP hydrolysis and an interaction with the cytoplasmic membrane protein EpsL, EpsE supports secretion of cholera toxin across the outer membrane. In this study, we have determined the effect of the cytoplasmic domain of EpsL (cyto-EpsL) and purified phospholipids on the ATPase activity of EpsE. Acidic phospholipids, specifically cardiolipin, bound the copurified EpsE/cyto-EpsL complex and stimulated its ATPase activity 30-130-fold, whereas the activity of EpsE alone was unaffected. Removal of the last 11 residues (residues 243-253) from cyto-EpsL prevented cardiolipin binding as well as stimulation of the ATPase activity of EpsE. Further mutagenesis of the C-terminal region of the EpsL cytoplasmic domain adjacent to the predicted transmembrane helix suggested that this region participates in fine tuning the interaction of EpsE with the cytoplasmic membrane and influences the oligomerization state of EpsE thereby stimulating its ATPase activity and promoting extracellular secretion in V. cholerae.

Characterization and metabolic function of a peroxisomal sarcosine and pipecolate oxidase from Arabidopsis.

Sarcosine oxidase (SOX) is known as a peroxisomal enzyme in mammals and as a sarcosine-inducible enzyme in soil bacteria. Its presence in plants was unsuspected until the Arabidopsis genome was found to encode a protein (AtSOX) with approximately 33% sequence identity to mammalian and bacterial SOXs. When overexpressed in Escherichia coli, AtSOX enhanced growth on sarcosine as sole nitrogen source, showing that it has SOX activity in vivo, and the recombinant protein catalyzed the oxidation of sarcosine to glycine, formaldehyde, and H(2) O(2) in vitro. AtSOX also attacked other N-methyl amino acids and, like mammalian SOXs, catalyzed the oxidation of l-pipecolate to Delta(1)-piperideine-6-carboxylate. Like bacterial monomeric SOXs, AtSOX was active as a monomer, contained FAD covalently bound to a cysteine residue near the C terminus, and was not stimulated by tetrahydrofolate. Although AtSOX lacks a typical peroxisome-targeting signal, in vitro assays established that it is imported into peroxisomes. Quantitation of mRNA showed that AtSOX is expressed at a low level throughout the plant and is not sarcosine-inducible. Consistent with a low level of AtSOX expression, Arabidopsis plantlets slowly metabolized supplied [(14)C]sarcosine to glycine and serine. Gas chromatography-mass spectrometry analysis revealed low levels of pipecolate but almost no sarcosine in wild type Arabidopsis and showed that pipecolate but not sarcosine accumulated 6-fold when AtSOX expression was suppressed by RNA interference. Moreover, the pipecolate catabolite alpha-aminoadipate decreased 30-fold in RNA interference plants. These data indicate that pipecolate is the endogenous substrate for SOX in plants and that plants can utilize exogenous sarcosine opportunistically, sarcosine being a common soil metabolite.