Location: Aquatic Animal Health ResearchTitle: 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 Author
|Wei Pridgeon, Yuping|
|Mobley, James A.|
Submitted to: Veterinary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/22/2013
Publication Date: 9/1/2013
Publication URL: http://handle.nal.usda.gov/10113/58421
Citation: Wei Pridgeon, Y., Yildirim-Aksoy, M., Klesius, P.H., Kojima, K., Mobley, J., Srivastava, K., Reddy, P. 2013. 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. Veterinary Microbiology. 166:624-630. Interpretive Summary: To understand the molecular basis of novobiocin resistance in live attenuated vaccine strain AH11NOVO, multiple approaches were used in this study. Genomic DNA sequencing results revealed that mutations at the target site of novobiocin as well as other genes might be the reason why the vaccine strain is resistant to novobiocin. In addition, transcriptional and translational regulation of antibiotic-resistance related genes might also have contributed to the resistance of the vaccine strain to novobiocin. Furthermore, overexpression of immunogens in the vaccine strains might have contributed to its efficacy as a vaccine.
Technical Abstract: Sequence comparison between the full-length 2412 bp DNA gyrase subunit B (gyrB) gene of a novobiocin resistant Aeromonas hydrophila AH11NOVO vaccine strain and that of its virulent parent strain AH11P revealed 10 missense mutations. Similarly, sequence comparison between the full-length 4092 bp RNA polymerase beta subunit (rpoB) gene of AH11NOVO and that of AH11P revealed 13 missense mutations. SDS-PAGE and LC-ESI-MS/MS revealed a total of 23 proteins presumably over-expressed in AH11NOVO, with putative functions in the following four major categories: 1) resistance to antibiotics or response to stress (7/23=30%); 2) immunogens (6/23=26%); 3) virulence factors (5/23=22%); and 4) metabolism (5/23=22%). Quantitative PCR revealed that the transcriptional levels of the majority (19/23=83%) genes corresponding to the proteins identified by LC-ESI-MS/MS were over-expressed at least 10-fold higher in AH11NOVO compared to that in AH11P. Western blot and LC-ESI-MS/MS revealed a total of 55 AH11 proteins recognized by either preimmune or hyperimmune catfish sera, including 9 of the 23 proteins identified earlier. The nine proteins included the following six putative immunogens that was identified earlier: 1) pyruvate dehydrogenase E1 component; 2) phosphate aetyltransferase; 3) chaperone htpG; 4) transketolase; 5) serine hydroxymethyltransferase; and 6) fructose-biphosphate aldolase. In addition, the following three proteins that were not previously known as immunogenic were identified by both LC-ESI-MS/MS: 1) acetate kinase; 2) periplasmic nitrate reductase; and 3) cysteine synthase. Our results will improve our understanding of the molecular basis of novobiocin resistance and how AH11NOVO works as a vaccine.