| The severe watery diarrheal disease caused by Vibrio cholerae still occurring in parts of the world and is a continuing threat to global public health.In recent Haiti-Nepal clinical isolates,flagellar genes mutation have been uncovered.These indicate that spontaneous mutations in flagellar genes leading to nonmotility-related mutants may be a new direction of directed evolution of pathogens in the host,but the mechanism of producing spontaneous nonmotility-related mutants in V.cholerae is still unclear.In this study,we found V.cholerae from adult mouse intestine produced high-frequency spontaneous nonmotility-related mutants that enhanced long-term host adaptation.The hotspot mutation genes of spontaneous nonmotility-related mutants are flagellar regulatory genes flrA,flrC and rpoN by sequencing.It is hypothesized that the DNA damage causing reactive oxygen species（ROS）and reactive nitrogen species（RNS）in the adult mouse intestine and the DNA repair systems of V.cholerae may be associated with the generation of spontaneous nonmotile mutants.The results indicated that only deletion of dps,a ROS-resistant gene,significantly increased the rate of nonmotile mutants.Dps is a cyclin,deletion of dps does not affect the motility and mutation frequency of V.cholerae.The dps D65A and dps F46E point mutations display decreased resistance to ROS,and exhibited an increased rate of nonmotile mutants in adult mouse intestine compared to the wild-type C6706.Removal of ROS from adult mouse intestine with N-Acetyl-L-cysteine（NAC）resulted in a decreased rate of nonmotile mutants.It indicates that Dps ROS detoxification function deficiency is one of the reasons for the generation of nonmotile mutants.This study showed thatΔdps and C6706 produce the same mutation hotspot genes,which are flagellar transcriptional regulator genes flrA,flrC and rpoN.Point mutations of flrA,flrC and rpoN showed enhanced ability to colonize adult mouse,which suggested that V.cholerae enhances adult mouse colonization by producing mutations in the flagellar region genes.Further studies found that theΔflrA,ΔflrC,andΔrpoN had a colonization advantage in adult mice,which did not correlate with the growth advantage and motility.Resistance to ROS and formation of biofilm are not responsible for enhanced colonization ofΔflrA andΔflrC.Enhanced resistance to ROS in adult mouse intestinal tract may be one of the mechanism for the increased colonization ofΔrpoN in adult mice.Proteomic and adult mouse competition assays found that methionine metabolism pathway may be related to increased colonization ofΔflrA,ΔflrC andΔrpoN in adult mice.Dps response to oxidative stress has a careful balance,increased Dps expression can confer resistance to ROS,but overexpression can also sequester iron,inactivate iron ion-dependent enzymes and inhibit bacterial growth.When the expression of Dps was decreased,V.cholerae may enhance adaptation in the host by producing nonmotility-related mutants,which may represent a novel adaptation strategy for directed evolution of pathogens in the host.First reported spontaneous mutation in flagellar transcriptional regulatory genes flrA,flrC and rpoN have a colonization advantage in adult mice,providing new insights to further understand the pathogenesis of V.cholerae,especially how it interacts with the host to develop a colonization advantage. |
