A conserved Aeromonas salmonicida porin provides protective immunity to rainbow trout (original) (raw)
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Protein Expression and Purification, 2007
The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp. Methods and Results: The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli. Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila. Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations. Conclusions: The ompW-based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish. Significance and Impact of the Study: To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila. Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas. Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.
Fish & shellfish immunology, 2017
A virulent clonal population of Aeromonas hydrophila (VAh) is recognized as the etiological agent in outbreaks of motile aeromonas septicemia (MAS) in catfish aquaculture in the southeastern United States since 2009. Genomic subtraction revealed three outer membrane proteins present in VAh strain ML09-119 but not in low virulence reference A. hydrophila strains: major outer membrane protein OmpA1, TonB-dependent receptor (Tdr), and transferrin-binding protein A (TbpA). Here, the genes encoding ompA1, tdr, and tbpA were cloned from A. hydrophila ML09-119 and expressed in Escherichia coli. The purified recombinant OmpA1, Tdr, and TbpA proteins had estimated molecular weights of 37.26, 78.55, and 41.67 kDa, respectively. Catfish fingerlings vaccinated with OmpA1, Tdr, and TbpA emulsified with non-mineral oil adjuvant were protected against subsequent VAh strain ML09-119 infection with 98.59%, 95.59%, and 47.89% relative percent survival (RPS), respectively. Furthermore, the mean liver,...
Research in Microbiology, 2012
The gene coding for an outer membrane protein Omp48 of Aeromonas hydrophila isolated from an infected fish was cloned and sequenced. Analysis of nucleotide sequence showed the omp48 gene to be an adhesin encoding a protein of 426 amino acids with high identity to the omp48 gene of Aeromonas veronii, another fish pathogen. The gene belonged to the maltoporin group of porins and had high similarity to LamB porins of A. hydrophila, Aeromonas salmonicida and Vibrio parahaemolyticus. The expressed purified recombinant protein had an estimated molecular weight of 48 kDa. Further, rabbit hyperimmune sera against the recombinant protein reacted with A. hydrophila, Aeromonas sobria and A. veronii whole cell proteins at the region of 48 kDa, in western blotting. The recombinant protein was immunogenic in the fish Labeo rohita Hamilton. Fish immunized with recombinant protein, when challenged with virulent A. hydrophila and another bacterial fish pathogen, Edwardsiella tarda, showed relative percent survivals of 69 and 60, respectively. Our results suggest that Omp48 of A. hydrophila could be used as a potential vaccine candidate for protection not only against A. hydrophila infection, but also against the fish pathogen E. tarda.
Veterinary Immunology and Immunopathology, 2012
Aeromonas hydrophila is an important fish pathogen responsible for huge economic losses in aquaculture sector. The bacterial outer membrane proteins (OMPs), especially adhesins play a key role in the virulence of the bacteria and are considered potential vaccine candidates. We evaluated the immunogenicity of two important outer membrane proteins namely Aha1 and OmpW of A. hydrophila. These proteins were over-expressed in Escherichia coli, purified and used for the vaccination of common carp. Sequence analysis predicted that, Aha1 and OmpW are adhesins and antigenic. Common carp immunized with recombinant Aha1 and OmpW proteins showed significant antibody production and a relative percentage survival of 52 and 71 respectively indicating their protective efficacy against A. hydrophila infection.
Molecular Microbiology, 1992
Four outer-membrane proteins of Aeromonas hydrophila were purified and their N-terminal sequences and channel-forming properties were determined. Three could be matched with proteins from other species. One was a maltoporin, as its level increased when cells were grown in maltose-containing media, and the channel it formed was blocked by maltose. Another was like OmpF and OmpC of Escherichia coli, except that its channel fluctuated much more rapidly. The third protein, which was produced in low-phosphate medium, exhibited several properties of the general anion porin PhoE. The fourth showed no similarity to any known proteins. It had a unique N-terminus and it formed small sharply-defined cation-selective channels. Two other proteins which corresponded to OmpW of Vibrio cholerae and E. coli OmpA were partly characterized.
The A-layer protein of Aeromonas salmonicida: further characterization and a new isolation procedure
Journal of Fish Diseases, 1992
The predominant cell surliice protein (A-protein) of Aeromonas sahnonicida has been purified by a method utilizing a giycinc/hydroehloride cxlraetion from whole cells and HPLC/ion exehanger (DEAE) eolumns. This proeedure yielded two LPS-frec moleeules {a 40-and a ,SO-kDa form) both shown to eontain A-protein determinants. The former appears to be a digest produet of the latter, as a serine protease produeed by A. salmonicida was shown Ui prt^Kess the 5()-kDa term into a 4()-kDt\ molecule in vitro. The A-layer protein was shown to contain one isoform, although multiple isoclectrie forms appeared as preparative artifacts, probably due to deamidation. The A-layer protein and LPS are the most signifieant surfaee antigens reeognized by the Atlantie salmon B-lymphoeytes or antibodies. Immunoiogical studies of LPS-free and LPS-containing A-protein preparations were undertaken to test whether the two eomponents behave like antigcnie competitors or whether the LPS moiety eould adjuvant the antibody response against the A-protein. The latter was shown to be the ease.
Journal of Applied Microbiology, 2003
Aims: To clone, sequence and characterize the gene encoding the Omp48, a major outer membrane protein from Aeromonas veronii. Methods and Results: A genomic library of Aer. veronii was constructed and screened to detect omp48 gene sequences, but no positive clones were identified, even under low stringency conditions. The cloned gene probably was toxic to the host Escherichia coli strain, so the cloning of omp48 was achieved by inverse PCR. The nucleotide sequence of omp48 consisted of an open reading frame of 1278 base pairs. The predicted primary protein is composed of 426 amino acids, with a 25-amino-acid signal peptide and common Ala-X-Ala cleavage site. The mature protein is composed of 401 amino acids with a molecular mass of 44 256 Da. Conclusions: The omp48 gene from Aer. veronii was cloned, sequenced and characterized in detail. BLAST analysis of Omp48 protein showed sequence similarity (over 50%) to the LamB porin family from other pathogenic Gram-negative bacteria. Significance and Impact of the Study: Bacterial diseases are a major economic problem for the fish farming industry. Outer membrane proteins are potentially important vaccine components. The characterization of omp48 gene will allow further investigation of the potential of Omp48 as recombinant or DNA vaccine component to prevent Aer. veronii and related species infections in reared fish.
Carbohydrate-reactive, pore-forming outer membrane proteins of Aeromonas hydrophila
Infection and Immunity, 1994
Two outer membrane proteins of Aeromonas hydrophila A6, isolated in a one-step affinity chromatography process based on carbohydrate reactivity, were found to be pore-forming molecules in artificial planar bilayer membranes. These carbohydrate-reactive outer membrane proteins (CROMPs; M(r)s, 40,000 and 43,000) were subjected to amino acid analysis. The amino acid profiles for these two outer membrane proteins were almost identical. A partial protein sequence of a 14-amino-acid fragment of the 40,000-Da protein revealed homology with outer membrane porins of Escherichia coli and A. hydrophila. CROMPs were compared with carbohydrate-reactive porins also extracted from outer membranes of A. hydrophila A6. These porins were isolated by using standard porin purification techniques (insolubility in 2% sodium dodecyl sulfate, solubility in 0.4 M NaCl, and Sephacryl S-200 gel filtration), and then Synsorb H type 2 affinity chromatography was done. The physical and functional properties of t...
Novel antigens expressed by Aeromonas salmonicida grown in vivo
Infection and Immunity, 1993
Virulent and avirulent Aeromonas salmonicida strains grown inside intraperitoneal implants in Rainbow trout (Oncorhynchus mykiss) were examined for unique antigen expression. Western blots (immunoblots), performed with immune rabbit serum raised against in vivo-grown cells, revealed several unique antigens. With the exception of lipopolysaccharide (LPS), these novel antigens were destroyed after proteinase K treatment. The majority of these antigens were not induced in vitro in response to either iron limitation or anaerobiosis. In addition, electron microscopy demonstrated the presence of a putative capsule on in vivo-grown cells. Purification and fractionation of this carbohydrate material from cells grown in carbon-rich synthetic media resulted in the isolation and separation of an antigenically distinct LPS not seen with cells grown in standard media. Antiserum raised against in vivo-grown cells recognized both this LPS and the typical LPS of A. salmonicida apparent in in vitro-...