Responses To Environmental Stresses And Regulation Of Virulence-associated Phenotypes Mediated By RelA And SpoT In Vibrio Alginolyticus

Vibrio alginolyticus occurs ubiquitously in various reservoirs including oceans and estuaries around the world.As a halophilic,gram-negative opportunistic pathogen,V.alginolyticus has been reported for not only infecting fish,shrimp,shellfish and other aquatic animals,but also causing human diseases like otitis and diarrhea.Initially,flagella and other organelles would accelerate bacterial attachment to invade the host.Exopolysaccharides（EPSs）and other matrices are subsequently produced to promote biofilm formation,which help them confront harsh environmental conditions and the host immune systems after colonization.Various sensing systems existed in bacteria are often used to quickly monitor and adjust the complex physiological processes.Guanine nucleotide tetraphosphate/pentaphosphate（collectively called（p）ppGpp）,as a key second messenger,plays a significant role in regulating the complex biological activities.So far,there is no report about the intricate assessment of（p）ppGpp synthetase genes in combating stress adaptation and virulence regulation in V.alginolyticus.In this study,we constructed（p）ppGpp-synthetase deletion mutants（ΔrelA,ΔrelAΔspo T）and complemented strains（ΔrelA-p Rel A,ΔrelAΔspo T-p Spo T）of a wild pathogenic V.alginolyticus strain HN08155,respectively.Subsquently,we determined their alteration of growth properties under different stress conditions,virulent-related phenotypes and estimation of 50%lethal dose(LD₅₀)by comparison with the wild type（WT）.Our results declared that the putative roles of（p）ppGpp for environmental adaption and virulence regulation in V.alginolyticus,which providing a theoretical basis for revealing the pathogenic mechanism and formulating preventive measures.The main features and results have been summarized as follows:（1）Sequence analysis of（p）ppGpp synthetase genes in V.alginolyticus HN08155.The whole genome sequencing results showed that the open reading frames（ORFs）of relA and spo T correlated to 2220 bp and 2121 bp,respectively.Amino acid sequence alignment analysis in Rel A and Spo T protein showed high degree of similarity（Rel A:87%-97%amino acid identity,Spo T:91%-97%amino acid identity）in some Vibrios.Both Rel A and Spo T protein revealed the presence of three conserved domains:SYNTH domain,TGS domain and ACT domain,whereas,Spo T contained one more HD domain than Rel A at the N-terminus.Besides,the Spo T possessed three highly conserved residues in HD domain,which suggested that the nucleotide-binding pocket was genetically edited for（p）ppGpp hydrolysis.However,Spo T depicted the conserved RXKD motif in SYNTH domain and EXDD motif existed in the Rel A,confirming the GTP as a preferred substrate in synthesizing（p）ppGpp upon stress.Sequence analysis results indicated that（p）ppGpp in V.alginolyticus was controlled by relatively conserved（p）ppGpp synthetase Rel A and synthetase/hydrolase Spo T,respectively.（2）Studies on growth characteristics of different strains upon amino acids starvation and antibiotic mupirocin.Experimental results showed that compared with the WT and complemented strains,the growth ability of theΔrelA andΔrelAΔspo T was impaired in the modified M9 minimal medium（without amino acids solution）.However,the growth ability of theΔrelA andΔrelAΔspo T could be restored to the normal level by supplementing the modified M9 minimal medium with 13 essential amino acids solution,simultaneously.Furthermore,ΔrelA andΔrelAΔspo T did not show resistance to mupirocin as the WT and the complemented strains when 3μg/m L mupirocin was additionally into the 2216E medium.These results indicated that relA appeared to play the prominent role in stress resistance to amino acids starvation and mupirocin in V.alginolyticus.（3）Studies on growth characteristics of different strains upon different stress conditions.Experimental results showed that compared with the WT,ΔrelA and complemented strains,ΔrelAΔspo T was less resistant to bile salts,3%ethanol or heat shock（37°C and 42°C）.Interestingly,the results also showed that theΔrelAΔspo T grew more rapidly under lower doses（0.5%,1%）of ethanol or cold stress（22°C）,which resulted in earlier attaining of stationary phase than other strains.These results indicated that spo T rather than relA played a critical role in response to bile salts,ethanol,oxygen and temperature stress,and different regulatory functions mediated by（p）ppGpp existed in V.alginolyticus under different stress conditions.（4）Studies on virulent-related phenotypes and estimation of LD₅₀ of different strains.Experimental results showed that compared with the WT and complemented strains,bothΔrelA andΔrelAΔspo T postponed biofilm formation stained by crystal violet in the 96-well polystyrene microtiter at low cell density,whereas,ΔrelA andΔrelAΔspo T delayed and failed biofilm disassembly at high cell density,respectively.Similarly,the EPSs content of WT was the higher than that inΔrelA andΔrelAΔspo T at low cell density,whereas,the EPSs content of WT was the lower than that inΔrelA andΔrelAΔspo T at high cell density.Moreover,obvious cell aggregation phenomena ofΔrelA andΔrelAΔspo T could be observed in medium when compared with that of the WT and complemented strains.Furthermore,ΔrelAΔspo T lost the flagella,and the diameters of the swimming and swarming rings ofΔrelAΔspo T were significantly smaller than that of the WT,ΔrelA and complemented strains.In addition,onlyΔrelAΔspo T exhibited smooth/translucent colony morphology and spotty pellicle at the air-liquid interface.Suprisingly,the LD₅₀ for WT was 7.5×10⁴ CFU/g,which was30-or 60-fold as that ofΔrelA orΔrelAΔspo T,respectively.These results indicated that both relA and spo T played an important role in biofilm formation/decomposition,EPSs production,cell aggregation and virulence,whereas,only spo T could affect the colony morphology,spotty pellicle at the air-liquid interface,flagella formation and motility ability in V.alginolyticus.（5）Studies on virulence-related genes expression level of different strains.Experimental results showed expression level of three EPSs-related genes excluding vps M inΔrelAΔspo T andΔrelA decreased significantly at low cell density compared with that in WT,but only that inΔrelAΔspo T increased significantly at high cell density.Similarly,all expression level of six motility-related genes inΔrelAΔspo T decreased significantly in comparison with that in WT andΔrelA.Interestingly,the expression level of rpo N inΔrelA was significantly higher than that in WT at low cell density,whereas,that inΔrelAΔspo T was significantly reduced compared with WT.These results indicated that both relA and spo T could affect the expression level of rpo N,EPSs-related and motility-related genes at low cell density,whereas,only spo T gene could inhibit the expression leve of EPSs-related genes at high cell density.In summary,we found that both of the relatively conserved（p）ppGpp synthase gene relA and synthase/hydrolase gene spo T existed in V.alginolyticus.Rel A mainly responds to amino acid starvation and mupirocin,whereas,Spo T mainly responds to environmental stresses including bile salts,ethanol,oxygen and temperature changes.Both relA and spo T could participate in virulence regulation,while spo T has a more comprehensive and greater impact on the virulence-associated phenotypes in V.alginolyticus.