The Effect Of Alkyl Hydroperoxidase AhpC Regulated By OxyR2 On Antioxidative Stress Of Vibrio Cholerae

Cholera is an epidemic disease of intestinal infection that can cause severe watery diarrhea,dehydration,and even death.It spreads rapidly and has a high mortality rate.It is still a life-threatening factor in developing countries.Vibrio cholerae is the causative agent of cholera.It is a Gram-negative,single-flagellate,arc-shaped bacterium with the motility,whose ecological niche is in saline coastal water and estuary.The bacteria infects the intestine through contaminated water or food.After overcoming the resistance of the stomach acid and the host immune,it can colonize the surface of the small intestine epithelial cells through the mucus layer,subsequently being excreted by diarrhea and re-entering the aquatic environment.Vibrio cholerae encounters various environmental stresses,like oxidative stress caused by reactive oxygen species during this cycle.Vibrio cholerae usually protects itself from these oxidative stress-induced injuries by regulating a series of antioxidant proteins expressed in its body.These proteins include catalase KatG,peroxidase PrxA,and DNA-binding protein Dps that have H2O2 scavenging activity.Among them,the prxA gene positively regulated by the transcription regulator OxyR plays an important role in the antioxidative stress of V.cholerae.In order to better understand how V.cholerae protects itself against oxidative stress,We have found the genes encoding hypothetical peroxidase AhpC in the genome of V.cholerae by bioinformatics and another OxyR homologue in its vicinity.It has 49%sequence homology with the original OxyR in V.cholerae,and the OxyR homologue also contains two conserved cysteine residues C206 and C215.Therefore,we renamed the original oxyR gene to oxyR1,and named the newly discovered oxyR as oxyR2.This was detected by constructing the transcriptional fusion reporter plasmid.It was found that oxyR2 positively regulates the expression of ahpC in a H2O2-independent manner,oxyRl does not affect the expression of ahpC,and C199 in OxyRl and C206 in OxyR2 are crucial for their respective regulatory functions.After oxyR2 or ahpC was knocked out in-frame,we found that the tolerance of V.cholerae to H2O2 increase through growth curve determination under H2O2 treatment and detection of bactericidal assays.In order to study the difference in gene expression of V.cholerae under oxidative stress,and the roles oxyR2 and ahpC play in their antioxidant processes,we performed transcriptome analysis using RNA sequencing against V.cholerae wild type,?oxyRl and?oxyR2 in the presence/absence of H2O2.The transcriptional profile was analyzed and found that there is an overlap between genes that are regulated by oxyR2 and genes that are regulated by oxyR1,such as ROS resistance-related genes vc0139(dps),vc2637(prxA)etc.The expression of these oxyR1-activated genes is inhibited by oxyR2.Afterwards,we further found the OxyR2-AhpC may modulate oxyRl activity and thereby inhibiting the expression of prxA by constructing the PprxA-luxCDABE reporter vector.To investigate the role of oxyR2 and ahpC in antioxidant processes,we used AKI medium supplemented with different concentration of H2O2.The anaerobic culture of V cholerae wild-type,?oxyR2 and AahpC revealed that ?oxyR2 and ?ahpC show growth defect at moderate concentration of H2O2,indicating that OxyR2-activated AhpC may help clear the low concentration of H2O2,and speculated that when H2O2 concentration exceeds a certain threshold or lack of oxyR/ahpC,oxyR1-regulated prxA expression is activated to clear higher level of H2O2.To further study the effects of the absence of oxyR2 or ahpC on the colonization and viability of V.cholerae,we performed competitive colonization experiments in suckling mice and adult mice,and survival rates detection in simulated artificial seawater as an in vitro aquatic environment.The results showed that the absence of oxyR2 or ahpC do not affect their colonization in suckling mice and adult mice,but significantly reduce their survival in artificial seawater environment.This shows that OxyR2-activated AhpC is beneficial to alleviate the low level of oxidative stress in the environment.In summary,our results indicate that oxyR2 and ahpC play an important role in relieving oxidative stress and environmental survival of V.cholerae.