Study On The Mechanism Of Acid Resistance,Pathogenicity And Regulation Of Periplasmic Protein HdeB On Edwardsiella Piscicida

Edwardsiella piscicida is considered a broad-host aquatic pathogenic bacterium,capable of infecting a variety of aquaculture economic fish,causing huge economic losses to our aquaculture industry,and an in-depth understanding of E.piscicida resistance and pathogenicity mechanisms is essential.E.piscicida usually has to face a variety of environmental stresses before and after surviving in vitro and infesting the host,among which acid stress is the most common one.E.piscicida is a Gram-negative bacterium with a bilayered membrane structure,with the periplasmic space located between the bacterial inner and outer membranes.When the pH in the environment decreases,H+can enter directly into the periplasmic space and cause severe acid damage to the proteins in the periplasm.HdeB is an essential acid-tolerant protein in the periplasmic space of Gram-negative bacteria and plays an importance role in bacterial resistance to acid stress pressure.hdeB normally forms the manipulator hdeAB with hdeA,but analysis of the E.piscicida genome revealed that only the HdeB homolog（ETAE₂870）and not the manipulator hdeAB are present in this bacterium,which may lead to a very different function of HdeB in E.piscicida than in other pathogenic bacteria.Therefore,it is critical to understand the mechanisms of resistance and pathogenicity of E.piscicida HdeB for accurate prevention and control of Edwardsiella disease.The function of hdeB in acid resistance and pathogenicity of E.piscicida was further investigated by constructing an hdeB in-frame deletion strain（ΔhdeB）and its back-complement（ΔhdeBC）.hdeB deletion and overexpression were found to enhance the acid tolerance of E.piscicida at pH=2.5,3 and 4.However,the addition of the protease inhibitor phenylmethylsulfonyl fluorid（PMSF）at pH=2.5 significantly decreased the acid tolerance of ΔhdeB.The above results suggest that HdeB plays an important role in the resistance of E.piscicida to acid stress and also imply that the acid resistance function of HdeB may be related to the protease.HdeB is an important acid-tolerant protein in the bacterial periplasmic space that prevents misfolding of periplasmic proteins in acidic environments.To investigate the effect of E.piscicida HdeB on periplasmic proteins,we extracted WT and ΔhdeB under normal conditions,acidic conditions,and acidic conditions with the addition of PMSF to compare periplasmic proteins.It was found that the periplasmic protein of ΔhdeB was significantly reduced under normal conditions compared to WT;after acid stimulation,both WT and ΔhdeB periplasmic protein showed a reduction in periplasmic protein compared to normal conditions,with more reduction in ΔhdeB periplasmic protein than WT;after the addition of PMSF based on acidic conditions,the amount of periplasmic protein of WT and ΔhdeB was restored,and it is noteworthy that The amount of ΔhdeB periplasmic protein was not significantly different from that of WT.The results suggest that HdeB can play a role in protecting periplasmic proteins,and this protective effect may be related to protease and protease inhibitory proteins.Analysis of the E.piscicida genome revealed a serine protease inhibitory protein,Ecotin,which is present in the periplasmic space and can inhibit a wide range of trypsin-like and serine peptidases.HdeB was found to bind to Ecotin in both normal and acidic conditions by protein pendant assays.These results suggest that HdeB is able to protect periplasmic proteins from excessive degradation in acidic environments.To investigate whether hdeB was associated with the virulence of E.piscicida,the biofilm-forming ability of the wild strains WT,ΔhdeB and ΔhdeBC was first compared,and it was found that the absence of hdeB did not affect the biofilm-forming ability of the bacteria under normal and acidic conditions.Subsequently,the ability of WT,ΔhdeB andΔhdeBC to proliferate in mouse macrophages RAW264.7 was examined,and it was found that ΔhdeB was slightly stronger than the wild strain in intracellular proliferation,but not significantly.Finally,the ability of WT,ΔhdeB and ΔhdeBC to infiltrate tilapia tissues was examined,and the results showed that the ability of ΔhdeB to infiltrate tilapia kidney and spleen was slightly enhanced compared with the wild strain,but not significantly.To explore the mechanism of HdeB regulation,we analyzed the hdeB promoter region sequence to identify potential CpxR transcriptional regulatory binding sites.hdeB promoter reporter strain assays showed that CpxR significantly inhibited hdeB promoter activity.EMSA experiments showed that CpxR could directly bind to the hdeB promoter region.tyrR is considered a classical regulatory protein of hdeB,and this study found that TyrR was able to inhibit hdeB promoter activity under acidic conditions,but had no effect under normal conditions.In addition,we also analyzed the regulatory relationship of an unknown regulatory factor ETAE₂871 downstream of hdeB on hdeB,however,the results showed that hdeB was not regulated by ETAE₂871 under both normal and acidic conditions.The above experimental results suggest that CpxR can bind to the hdeB promoter and repress its transcriptional activity;TyrR can repress hdeB promoter activity under acidic conditions,but the exact mechanism needs to be further explored.In summary,our study indicates that HdeB plays an important role in the resistance of E.piscicida to acidic adversity environments.