Physiological Response Of Aquatic Pathogen Non-O1/O139 Vibrio Cholerae Under Starvation And Expression Analysis Of RpoS Gene In Response To Environmental Stress

Non-O1/O139 Vibrio cholerae is widely distributed in various aquatic environments,including marine and freshwater environments,which is a pathogen of various aquatic organisms causing mass mortalities.Like other bacterial species,non-O1/O139 V.cholerae is usually exposed to nutrient-poor waters,and it can survive for a long time and cause disease during starvation by sequential changes in cell physiology,morphology and gene expression.RpoS is widely considered to be the general stress response factor of many other Vibrio species,and the rpoS gene is an important gene for the survival of bacteria under stress conditions.In this study,the changes in the morphology,survival,the expression of stress resistance-related genes of non-O1/O139 V cholerae GXFL1-4 under starvation stress were investigated,and the expression levels of rpoS under environment stress were detected.This study will contribute to understand the regulatory mechanisms of non-O1/O139 V.cholerae in favoring survival under starvation stress.The main research results of the paper are as follows:1.The determination of physiological and biochemical phenotypes,including survivability,motility,morphological changes,extracellular enzyme activity,virulence and metabolism-associated genes and pathogenicity of non-O1/O139 V.cholerae GXFL1-4 were carried out after starvation stress for six months.The results demonstrated that non-O1/O139 V.cholerae GXFL1-4 was still culturable,the cell counts kept declining to 4.63 ± 0.10 Log CFU/mL,and the motility of non-O1/O139 V cholerae decreased after 6 months of starvation.Scanning electron microscopy revealed that the starved cells exhibited shortened rod,shrinking and rougher surface.Furthermore,detection of virulence factors and virulenceassociated genes in starved cells showed that the starved strain still produced caseinase,gelatinase,lipase,lecithinase,amylase and β-hemolysin,and possessed metalloproteinase（mp）,outer membrane protein（ompU）,hemolysin（hlyA）,glucose isomerase（galE）,DNA binding reactor（phoB）and glycosyltransferase for podosome polysaccharide biosynthesis（vpsL）.In addition,comparison of pathogenicity between 6-months starved and wild-type of non-O1/O139 V.cholerae showed that starved cells presented a degree of virulence similar to that of wild-type cells.The LD50 of starved and wild-type cells to M.rosenbergii was 7.589×106 CFU/mL,1.881×107 CFU/mL,respectively,suggesting non-O1/O139 V.cholerae can undergo a rapid adaptation to nutrients deficiency and express virulent to aquatic animals.2.To investigate the molecular-level responses of non-O1/O139 V.cholerae against starvation stress,transcriptome analyses of starved and wild-type non-O1/O139 V.cholerae cells were conducted to determine stress resistance-related genes and pathways.The transcriptome analysis results revealed that there were a total of 371 differentially expressed genes（DEGs）,including 191 up-regulated genes and 180 down-regulated genes.Among DEGs,genes responsible for resistance to environmental stress such as sigma factors（rpoS,rpoD,rpoN,rpoE）,and the genes responsible for flagellar assembly such as flgO,flgI and flgB were downregulated significantly in the starved cells.In addition,GO analysis of the DEGs demonstrated that significantly enriched entries involving resistance to starvation stress were signaling,ion transmembrane transport,etc.KEGG enrichment analysis showed that the DEGs were mainly enriched the environmental adaptation related subcategories,such astwocomponent system,flagellar assembly,bacterial chemotaxis,and citrate cycle.These results showed that non-O1/O139 V.cholerae can survive for long-terrm under starvation stress by regulating a series of stress resistance,flagellar assembly,material metabolism and energy related genes.3.To further determine the functional roles of rpoS in survival of non-O1/O139 V.cholerae under starvation stress,the changes in expression levels of rpoS gene under temperature,osmotic pressure,starvation,pH stresses were determined by quantitative realtime PCR（qRT-PCR）.The results showed that rpoS gene was significantly up-regulated under starvation stress for 28 d.The rpoS gene of non-O1/OV.cholerae was significantly up-regulated under acidic stress,but was inhibited under alkaline stress.In addition,the rpoS gene was significantly upregulated high salinity.Furthermore,the results showed that the rpoS gene was involved in response to high temperature stress.These results indicated that rpoS gene played an important role in the survival of non-O1/O139 V.cholerae in various stressful conditions.In this study,the physiological response and gene expression changes of the aquatic pathogen non-O1/O139 V.cholerae under starvation were investigated.The results revealed that non-O1/O139 V.cholerae can survive for long-term and cause diseases under starvation stress by dwarfing of cell structure,regulating rpoS and other stress resistance-related genes.These results will help the development of intervention strategies to control non-O1/O139 V.cholerae infection in aquaculture.