Nucleotide sequence of the gene,ompW, encoding a 22kDa immunogenic outer membrane protein ofVibrio cholerae

Abstract

Vibrio cholerae contains numerous outer membrane proteins, however, they vary in their abundance and immunogenicity (1, 2). Manning et al. (3) have previously described the molecular cloning of the gene for a 22kDa outer membrane protein which is produced in minor amounts under normal laboratory conditions. The function of this protein is at present unknown, but it is very immunogenic and may correspond to one of the major immunogenic proteins detected by Western blot analysis using convalescent human sera (4). The structural gene, ompW, for the 22kDa protein was previously localized by transposon mutagenesis of plasmid pPM440, a pBR322 clone of partially Sau3A cleaved V. cholerae DNA (3). Analysis of the various TN] 725 derivatives of pPM440 suggested that the 22kDa protein was synthesized as a precursor of about 24kDa, consistent with its outer membrane location. The nucleotide sequence determined here was derived using subclones of pPM440 in M13 mpl8 and mpl9, as well as by supercoil sequencing of the TN] 725 insertions using oligodeoxynucleotide primers (5' GCTGTCACGAGAA-CACCGTT 3' and 5' CTTACGGATGCCCGGAAA 3') specific for the two ends of the transposon. The nucleotide sequence is shown in Fig. 1. The translated open reading frame corresponds to a 23.474kDa precursor protein with a 21.447kDa mature form. The signal peptide and cleavage site agree well with the rules for such sequences, and the direction of transcription is consistent with the polarity effects of TN] 725 insertions close to the beginning of the gene. The potential transcriptional promoter and terminator sequences have been detected. Both the-35 and-10 regions are not ideal as might be expected from the low level of expression of the protein under laboratory conditions, perhaps implying that the gene is subject to regulation. The potential terminator structure detected after the termination codon is also relatively weak. This sequence provides the basis for further studies, in particular, for site-directed mutagenesis of the V. cholerae chromosome to construct isogenic pairs of strains for assessing the role of the protein in pathogenesis.

Isolation and Characterization of Vibrio tubiashii Outer Membrane Proteins and Determination of a toxR Homolog

“…2, a PCR product of (26) found that the expression of cholera toxin (CT), toxin-coregulated pilus (TcpA), OmpT, and OmpU of V. cholerae was affected by changes in osmolarity and amino acid concentration, while other environmental signals, such as temperature and pH, had more-pronounced effects on the expression of CT and TcpA than on the expression of OMPs. Unfortunately, among the OMPs characterized by NTAA and BLAST analyses in our study, protein homologs of OmpT, TcpA, and other OMPs reported by Provenzano et al (25), such as OmpA, OmpC, OmpF, and the highly immunogenic proteins OmpV, OmpW, and OmpX, were not positively identified in V. tubiashii by our approach (12,17). Therefore, further investigations using molecular approaches were conducted to help identify some of the OMPs by using PCR primers based on V. cholerae and other Vibrio species.…”

Compromised Outer Membrane Integrity in Vibrio cholerae Type II Secretion Mutants

“…The three bands representing proteins with molecular masses corresponding to 38, 26, and 22 kDa present in wild-type but not N⌬eps supernatants were excised and subjected to tandem mass spectrometry (at the Michigan Proteome Consortium). These proteins turned out to be outer membrane proteins, not extracellularly secreted proteins, and were identified as OmpU, OmpV (85), and OmpW (36,58). Their presence in the wild-type culture supernatant likely is due to the release of outer membrane fragments and/or vesicles during growth overnight, as immunoblotting revealed that OmpU could be removed from the culture supernatant by centrifugation at 170,000 ϫ g (data not shown).…”

Characterization of a 20-kDa pilus protein expressed by a diarrheogenic strain of non-O1/non-O139 Vibrio cholerae

“…A database search revealed that the amino-terminal sequence of the 20-kDa protein of 10325 pili is identical to that of a 22-kDa outer membrane protein (OmpW) of V. cholerae [23]. The OmpW protein, whose function is yet unknown, is poorly expressed in V. cholerae O1 strains [24].…”

“…A comparison of the data with those of other pilus proteins of V. cholerae (TcpA and MshA) revealed only weak homology, if any, with these protein sequences. However, a database search revealed complete (100%) identity between the amino-terminal sequence of the 10325 pilus protein and the corresponding sequence of a 22-kDa outer membrane protein (OmpW) of V. cholerae [23]. Each mouse was challenged with about 10 LD SH doses of 10325 (1.0U10 V CFU), O395 (2.7U10 U CFU) or Co270 (6.6U10 U CFU) in 0.1 ml with or without the antiserum or its Fab (IgG) preparation.…”