Aeromonas hydrophila, a gram negative bacterium is a major fish pathogen and causes major economic losses to aquaculture industry. Outer membrane proteins play a significant role in its survival during different envir...Aeromonas hydrophila, a gram negative bacterium is a major fish pathogen and causes major economic losses to aquaculture industry. Outer membrane proteins play a significant role in its survival during different environmental conditions and bacterial pathogenesis. The outer membrane protein R (OmpR) is a member of the two-component regulatory system of Aeromonas hydrophila which differentially regulates the expression of OmpF or OmpC depending on the osmolarity conditions. Role of OmpR has been demonstrated in its virulence in other infectious bacteria and it is found to be a potential drug target/vaccine candidate. However, the OmpR of A. hydrophila has not been characterized. In the present study, we report recombinant expression, purification of the OmpR of A. hydrophila strain Ah17 in salt inducible E. coli GJ1158 cells. Leaky expression of rOmpR was confirmed by Western blot analysis using anti-6 × His antibody. The histidine tagged recombinant OmpR (rOmpR) (~29 kDa) was purified using Ni-NTA affinity chromatography from the soluble fraction of induced E. coli cells. The rOmpR was found to be highly immunogenic with end point titres of greater than 1:80,000. The anti-rOmpR antisera were capable of agglutinating live A. hydrophila cells, thus showing vaccine potential of the rOmpR.展开更多
Bacterial canker disease caused by Pseudomonas syringae pv.actinidiae(Psa)is the most devastating disease in kiwifruit cultivation.The EnvZ/OmpR two-component system(TCS)has been confirmed to regulate virulence and me...Bacterial canker disease caused by Pseudomonas syringae pv.actinidiae(Psa)is the most devastating disease in kiwifruit cultivation.The EnvZ/OmpR two-component system(TCS)has been confirmed to regulate virulence and mediate environmental stress responses in Gram-negative bacteria.However,the functional role of EnvZ/OmpR in Psa has not been fully clarified.In this study,we constructed markerless ompR,envZ,and ompR-envZ mutants,and ompR complementation and overexpression strains using homologous recombination.The deletion of ompR or envZ tremendously reduced the swimming and swarming motility of Psa,as well as tolerance to osmotic stress,while overexpression of ompR impaired its virulence against kiwifruit but enhanced exopolysaccharide production.EnvZ negatively regulated hrpR/S expression in both King’s B and minimal medium,whereas OmpR regulated hrpR/S expression negatively in King’s B and positively in minimal medium.However,OmpR did not regulate the expression of genes gacA,algU,lpxC,fur,and fleQ,which are associated with known virulence functions,despite its binding to their promoters.Additionally,based on bioinformatic prediction,two new OmpR regulons(envC and tolQ)related to virulence were identified in Psa.Meanwhile,OmpR directly bound to the promoters of envC and tolQ,and negatively regulated their expression in minimal medium.These findings enrich our understanding of the OmpR-mediated regulatory network and its roles in the pathogenesis of P.syringae.展开更多
文摘Aeromonas hydrophila, a gram negative bacterium is a major fish pathogen and causes major economic losses to aquaculture industry. Outer membrane proteins play a significant role in its survival during different environmental conditions and bacterial pathogenesis. The outer membrane protein R (OmpR) is a member of the two-component regulatory system of Aeromonas hydrophila which differentially regulates the expression of OmpF or OmpC depending on the osmolarity conditions. Role of OmpR has been demonstrated in its virulence in other infectious bacteria and it is found to be a potential drug target/vaccine candidate. However, the OmpR of A. hydrophila has not been characterized. In the present study, we report recombinant expression, purification of the OmpR of A. hydrophila strain Ah17 in salt inducible E. coli GJ1158 cells. Leaky expression of rOmpR was confirmed by Western blot analysis using anti-6 × His antibody. The histidine tagged recombinant OmpR (rOmpR) (~29 kDa) was purified using Ni-NTA affinity chromatography from the soluble fraction of induced E. coli cells. The rOmpR was found to be highly immunogenic with end point titres of greater than 1:80,000. The anti-rOmpR antisera were capable of agglutinating live A. hydrophila cells, thus showing vaccine potential of the rOmpR.
基金supported by the National Key R&D Program of China(2022YFD1400200)the National Natural Science Foundation of China(32072378)+1 种基金the Undergraduate Innovation and Entrepreneurship Training Program of Anhui Agricultural University(S202310364173)the Development Fund for Talent Personnel of Anhui Agricultural University(rc342216).
文摘Bacterial canker disease caused by Pseudomonas syringae pv.actinidiae(Psa)is the most devastating disease in kiwifruit cultivation.The EnvZ/OmpR two-component system(TCS)has been confirmed to regulate virulence and mediate environmental stress responses in Gram-negative bacteria.However,the functional role of EnvZ/OmpR in Psa has not been fully clarified.In this study,we constructed markerless ompR,envZ,and ompR-envZ mutants,and ompR complementation and overexpression strains using homologous recombination.The deletion of ompR or envZ tremendously reduced the swimming and swarming motility of Psa,as well as tolerance to osmotic stress,while overexpression of ompR impaired its virulence against kiwifruit but enhanced exopolysaccharide production.EnvZ negatively regulated hrpR/S expression in both King’s B and minimal medium,whereas OmpR regulated hrpR/S expression negatively in King’s B and positively in minimal medium.However,OmpR did not regulate the expression of genes gacA,algU,lpxC,fur,and fleQ,which are associated with known virulence functions,despite its binding to their promoters.Additionally,based on bioinformatic prediction,two new OmpR regulons(envC and tolQ)related to virulence were identified in Psa.Meanwhile,OmpR directly bound to the promoters of envC and tolQ,and negatively regulated their expression in minimal medium.These findings enrich our understanding of the OmpR-mediated regulatory network and its roles in the pathogenesis of P.syringae.
