Identification of gyrB and rpoB gene mutations and differentially expressed proteins between a novobiocin-resistant Aeromonas hydrophila catfish vaccine strain and its virulent parent strain

• Published in: Veterinary microbiology 2013-10, Vol.166 (3-4), p.624-630
• Main Authors: Pridgeon, J.W, Yildirim-Aksoy, M, Klesius, P.H, Kojima, K, Mobley, J.A, Srivastava, K.K, Reddy, P.G
• Format: Article
• Language: English
• Subjects: adenosine triphosphate; Aeromonas hydrophila; Aeromonas hydrophila - drug effects; Aeromonas hydrophila - genetics; Aeromonas hydrophila - immunology; Aeromonas hydrophila - pathogenicity; Animals; Anti-Bacterial Agents - pharmacology; Bacterial Proteins - genetics; Bacterial Proteins - immunology; Bacterial Vaccines - genetics; Bacterial Vaccines - immunology; Blotting, Western; catfish; Catfishes - microbiology; DNA Gyrase - genetics; DNA Gyrase - immunology; DNA-Directed RNA Polymerases - genetics; Drug Resistance, Bacterial; Fish Diseases - microbiology; formate C-acetyltransferase; fructose-bisphosphate aldolase; Fructose-Bisphosphate Aldolase - genetics; gene expression regulation; gene overexpression; genes; glyceraldehyde-3-phosphate dehydrogenase; glycine hydroxymethyltransferase; Gram-Negative Bacterial Infections - microbiology; Gram-Negative Bacterial Infections - veterinary; H-transporting ATP synthase; Immunogen; Mass spectrometry; missense mutation; Mutation; Novobiocin - pharmacology; polyacrylamide gel electrophoresis; protein synthesis; proteins; Virulence factor; Western blotting
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2013.07.025

A total of 10 and 13 missense mutations were found in the deduced gyrB and rpoB proteins, respectively, between avirulent AH11NOVO vaccine strain and its virulent parent strain AH11P. SDS-PAGE revealed that six proteins bands were significantly over-expressed in AH11NOVO whereas five bands were significantly over-expressed in AH11P. Mass spectrometry identified seven proteins from the over-expressed AH11NOVO gel bands and five proteins from the over-expressed AH11P gel bands. QPCR confirmed that all 12 genes corresponding to the proteins identified by mass spectrometry were significantly over-expressed in AH11NOVO or AH11P. When AH11NOVO proteins were subjected to Western blot analysis, 13 protein bands exhibited significantly stronger reactivity with hyper-immune catfish sera. Fifteen proteins were identified from immunogenic protein bands, including six (formate acetyltransferase, chaperone htpG, transketolase, ATP synthase subunit alpha, asparagine-tRNA ligase, and serine hydroxymethyltransferase) that were over-expressed in AH11NOVO proteins and three (elongation factor G, class II fructose-bisphosphate aldolase, and a putative uncharacterized 23kDa protein) that were over-expressed in AH11P. In addition, the following six proteins were also identified from the immunogenic protein bands: pyruvate dehydrogenase E1 component, ATP synthase subunit beta, ribose-phosphate pyrophosphokinase, glyceraldehyde-3-phosphate dehydrogenase, 50S ribosomal L10, and 50S ribosomal L15. Our results might provide insights on how to develop novel efficacious vaccine against Aeromonas hydrophila infection.