A 25-kDa 
β-Lactam-induced Outer Membrane Protein of Vibrio cholerae

Deb, A; Bhattacharyya, Debasish; Das, Jyotirmoy

doi:10.1074/jbc.270.7.2914

Cited by 6 publications

(4 citation statements)

References 27 publications

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“…The sizes of proteins were estimated using commercially available (Genei, Bangalore, India, or Invitrogen) protein molecular weight standards. Contamination in the outer membrane preparation due to inner membrane was always Ͻ5% as determined by cytochrome assay, as described previously (13).…”

Section: Methodsmentioning

confidence: 99%

“…V. cholerae C6709 and its isogenic mutant SHK17 grown under AKI conditions were harvested and sonicated, and the resulting cell lysate was separated into subcellular fractions as described previously (13). The outer membrane fraction was solubilized, and outer membrane proteins, together with whole-cell lysate proteins, were analyzed by SDS-12.5% polyacrylamide gel electrophoresis (PAGE), followed by staining with Coomassie blue or by Western blot analysis.…”

Section: Methodsmentioning

confidence: 99%

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Mutation in the relA Gene of Vibrio cholerae Affects In Vitro and In Vivo Expression of Virulence Factors

2003

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The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing. In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (relA VCH ) was cloned and sequenced. The relA VCH gene encodes a 738-aminoacid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli. A ⌬relA::kan allele was generated by replacing ϳ31% of the open reading frame of wild-type relA of V. cholerae El Tor strain C6709 with a kanamycin resistance gene. The V. cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation. Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions. In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results. The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V. cholerae. Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17. Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V. cholerae. In SHK17, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT.Vibrio cholerae is a facultative anaerobic gram-negative bacterium and the causative agent of the severe diarrheal disease cholera. In addition to residing temporarily in the intestinal lumen of humans during the diseased state, V. cholerae has its natural niche in the aquatic environment, residing in the freeliving aquatic flora found in estuarine areas and in association with crustaceans and mollusks (25). The strains of V. cholerae that cause epidemic cholera belong to serogroups O1 and O139 (3, 4, 28, 41, 50). The O1 serogroup is again divided into two biotypes, classical and El Tor (28). Strains other than O1 and O139 are known as non-O1/non-O139 vibrios.A pathogen in its natural environment and host-associated state is subjected to a plethora of stresses, such as fluctuations in pH, salinity, osmolarity, oxygen tension, temperature, and nutritional availability. These offer selective pressure to a bacterium, eliciting various adaptive responses for its survival. The adaptive response to nutritional stre...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mutation in the relA Gene of Vibrio cholerae Affects In Vitro and In Vivo Expression of Virulence Factors

2003

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“…Unlike other gramnegative organisms, V. cholerae cells are equally sensitive to penicillin and ampicillin and in general are more sensitive than Escherichia coli to most of the beta-lactam antibiotics, resulting in adaptive mutation and beta-lactam-resistant cells' emerging at a high frequency (31). A beta-lactam-induced 25-kDa protein which might be responsible for conferring resistance to beta-lactam antibiotics by interfering with OmpU, through which the beta-lactam antibiotics normally enter the cell, has recently been identified in the outer membrane of beta-lactamresistant cells (5).…”

mentioning

confidence: 99%

Porins of Vibrio cholerae: purification and characterization of OmpU

et al. 1996

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Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been purified to homogeneity. The synthesis of all the three proteins is regulated by the osmolarity of the growth medium. The pore-forming ability of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded. The 27-kDa protein, OmpX, though osmoregulated, is not a porin. OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl. OmpU is an acidic protein and is a homotrimer of 38-kDa monomeric units. Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit. Removal of Ca2+ irreversibly disrupts the structure and pore-forming ability of the protein. The pore size of OmpU is 1.6 nm, and the specific activity of the OmpU channel is two- to threefold higher than that of Escherichia coli porin OmpF, synthesis of which resembles that of OmpU with respect to the osmolarity of the growth medium. The pore size of OmpT, which is analogous to OmpC of E. coli, is smaller than that of OmpU. Southern blot hybridization of V. cholerae genomic DNA digested with several restriction endonucleases with nick-translated E. coli ompF as the probe revealed no nucleotide sequence homology between the ompU and ompF genes. OmpU is also not antigenically related to OmpF. Anti-OmpF antiserum, however, cross-reacted with the 45-kDa V. cholerae outer membrane protein, OmpS, the synthesis of which is regulated by the presence of maltose in the growth medium. OmpU hemagglutinated with rabbit and human blood. This toxR-regulated protein is one of the possible virulence determinants in V. cholerae (V. L. Miller and J. J. Mekalanos, J. Bacteriol. 170:2575-2583, 1988).

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“…The outer membrane protein profile of control and aPDT‐treated V. harveyi cells were evaluated following standard protocols ( Laemmli, 1970 ; Sanderma and Stroming, 1972 ). The outer membrane protein was extracted according to Sabri and colleagues (2000 ) and Deb and colleagues (1995 ) with slight modifications. The control and aPDT‐treated cells of V. harveyi were washed copiously with sterile saline and resuspended in PBS containing 5 mM phenylmethylsulfonyl fluoride (PMSF).…”

Section: Methodsmentioning

confidence: 99%

Reducing Vibrio load in Artemia nauplii using antimicrobial photodynamic therapy: a promising strategy to reduce antibiotic application in shrimp larviculture

Asok

Arshad

Corso

et al. 2011

Microbial Biotechnology

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SummaryWe propose antimicrobial photodynamic therapy (aPDT) as an alternative strategy to reduce the use of antibiotics in shrimp larviculture systems. The growth of a multiple antibiotic resistant Vibrio harveyi strain was effectively controlled by treating the cells with Rose Bengal and photosensitizing for 30 min using a halogen lamp. This resulted in the death of > 50% of the cells within the first 10 min of exposure and the 50% reduction in the cell wall integrity after 30 min could be attributed to the destruction of outer membrane protein of V. harveyi by reactive oxygen intermediates produced during the photosensitization. Further, mesocosm experiments with V. harveyi and Artemia nauplii demonstrated that in 30 min, the aPDT could kill 78.9% and 91.2% of heterotrophic bacterial and Vibrio population respectively. In conclusion, the study demonstrated that aPDT with its rapid action and as yet unreported resistance development possibilities could be a propitious strategy to reduce the use of antibiotics in shrimp larviculture systems and thereby, avoid their hazardous effects on human health and the ecosystem at large.

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A 25-kDa β-Lactam-induced Outer Membrane Protein of Vibrio cholerae

Cited by 6 publications

References 27 publications

Mutation in the relA Gene of Vibrio cholerae Affects In Vitro and In Vivo Expression of Virulence Factors

Mutation in the relA Gene of Vibrio cholerae Affects In Vitro and In Vivo Expression of Virulence Factors

Porins of Vibrio cholerae: purification and characterization of OmpU

Reducing Vibrio load in Artemia nauplii using antimicrobial photodynamic therapy: a promising strategy to reduce antibiotic application in shrimp larviculture

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