The Regulation Of Sulfane Sulfur In Pseudomonas Aeruginosa PAO1

Hydrogen sulfide(H2S)has been proposed as a new gaseous signaling molecule,mediating various biological functions in mammals including human.The studies on H2S signaling in eukaryotes are common,but limited in microorganisms.Several bacterial transcription factors,including CstR,FisR,and SqrR,have been identified to indirectly respond to H2S,activating sulfur-oxidizing genes.However,the regulation of H2S on other natural behaviors is needed to support that H2S is common signaling molecule in bacteria.It is difficult to precisely distinguish the functions of H2S and sulfane sulfur.Sulfane sulfur consists of elemental sulfur,persulfide(RSS-and HSS-)and polysulfide(RSn-and HSn-,n>2).Accumulating evidence shows that H2S fulfills its signaling function through sulfane sulfur.Sulfane sulfur often possesses both nucleophilic and electrophilic characteristics.It usually modifies the active cysteine thiols in proteins,altering protein activity and influencing diverse biological processes.Sulfane sulfur is a common cellular component,which can be produced from L-cysteine metabolism or from H2S oxidation.According to reported studies,cellular sulfane sulfur contents vary along with bacterial growth phases,indicating sulfane sulfur is likely to participate in signal regulations in bacterial cells.Previous research by our team shows that H2S has important physiological functions in Pseudomonas aeruginosa PAO1.A mutant with H2S-producing genes(cbs,cse,mst and cysI)being deleted displayed an apparent reduction in the production of several virulent factors and pathogenicity,but the H2S-oxidizing genes(sqr and pdo)mutant did not show apparent differences from the wild type.The specific mechanisms about how H2S regulates the pathogenicity of P.aeruginosa PAO1 and whether H2S impacts other biological process need further study.In this study,P.aeruginosa PAO1 was used as the research object to study the variation of cellular sulfane sulfur levels and how sulfane sulfur participates in regulating pathogenicity and antibiotic resistance in P.aeruginosa PAO1.The effect of sulfane sulfur on pigment production were also studied.The main contents are as follows:1.Sulfane sulfur signaling regulates LasR activity for quorum sensing in Pseudomonas aeruginosa PAO1Based on the constructed H2S-producing genes and H2S-metabolic genes mutants,the cellular sulfane sulfur level in PAO1,Pa?H2S,Pa3K and Pa7K were detected.Results showed that Pa3K contained almost the same sulfane sulfur as PAO1,but Pa?H2S and Pa7K contained clearly less sulfane sulfur than PAO1 and Pa3K did.The sulfane sulfur level was consistent with the observed phenotypes,lower level of sulfane sulfur with less production of virulence factors and pathogenicity.The RNA-seq results showed H2S has an immense influence on PAO1 gene expression,and many genes related to quorum sensing(QS)regulators and genes were down-regulated,including lasR.As the key QS regulator,PAO1 lasR null mutant could effectively disable QS.The phenotypes datas of PAO1 lasR mutant were similar to Pa?H2S,therefore,the potential role of LasR regulated by sulfane sulfur that in turn control the pathogenicity of PAO1 were speculated.To verify the above hypothesis,we constructed a mKate reporter plasmid in E coli BL21.Results indicate that LasR does not directly sense H2S,but its oxidation product sulfane sulfur such as H2Sn.Four cysteine residues were individually mutated to serine in the reporter plasmid,and results indicated that three Cys residues in DBD are critical for LasR's function.MS analysis showed sulfane sulfur directly modified Cys188,Cys201 and Cys203 of LasR,and this modification produced a persulfidation(Cys188-SSH)and trisulfidation(Cys188-SSSH)on Cys188 and a pentasulfur link between Cys201 and Cys203(Cys201-S-SSS-S-Cys203).EMSA showed that H2Sn-treated LasR did not affect its binding to target DNA.The activation of LasR by sulfane sulfur was verified via in vitro transcription-translation assays.Combined with the above datas,we speculate that the H2Sn-modified LasR is more effective in recruiting RNA polymerase to initiate transcription.We detected the expression of lasB,rhlR and lasI,activated by LasR in different growth phases and found that the transcripts corresponded well with the sulfane sulfur contents.LasR activity is regulated by both 3O-C12-HSL and cellular sulfane sulfur.The lower cellular sulfane sulfur may slow down the LasR activity even in the presence of high levels of the quorum sensing molecule.Sulfane sulfur acts a "brake"on quorum sensing autoinduction,especially in the late decline phase.Our findings provide a clear signaling pathway of H2S involving in P.aeruginosa pathogenicity via sulfane sulfur by activating LasR.The discovery may lead to new ways to prevent or treat bacterial infection.2.Sulfane sulfur regulates drug resistance via inactivating the repressor MexR in P.aeruginosa PAO1The overexpression of MexAB-OprM efflux were reported to increase the intrinsic antibiotic resistance of PAO1.We found the transcript of mexAB-oprM operon was obviously down-regulated,but no change was found in its repressor mexR based on RNA-seq analysis.We further verified the lower mexA expression in Pa?H2S with lower level of sulfane sulfur using RT-qPCR analysis.Five antibiotics were selected to study the relations between sulfane sulfur level and antibiotic resistance.Results showed that exogenous H2S could obviously increase resistances of PAO1.Since exogenous or endogenous H2S can be converted to sulfane sulfur by SQR,hence,sulfane sulfur may be involved in the regulation of MexAB-OprM expression.Both in vivo and in vitro datas indicated that sulfane sulfur rather than H2S directly inactivates MexR,derepressing the mexAB-oprM operon.Further,we determined that both Cys30 and Cys62 residues are necessary for MexR sensing of sulfane sulfur.A significant fraction of MexR formed cross-linked homodimer when treated with H2Sn,as revealed via nonreducing,denaturing SDS/polyacrylamide gel analysis and MS data revealed that H2Sn directly modified Cys30 and Cys62 of MexR,leading to the formation of disulfide bond and trisulfide bond.This modification altered the structure of MexR and decreased its target DNA binding,promoting the expression of efflux pump genes and increasing the antibiotic resistance.The E.coli MG1655 MarR was also used to verify whether it can sense sulfane sulfur.Both in vivo and in vitro experiments revealed that sulfane sulfur could be sensed by MarR Hence,sulfane sulfur is likely to be a common signal for MarR family proteins and involved in important physiological functionsThe key process that MexR repressor senses cellular sulfane sulfur in this model was proposed.The co-increase of mexR and mexA expression suggests that MexR also represses its own gene expression.At the logarithmic stage,low levels of sulfane sulfur are not sufficient to inactivate MexR.At the stationary phase,high levels of sulfane sulfur inactivate MexR and turn on the expression of efflux pump.Thus,the involvement of sulfane sulfur to regulate mexAB-oprM expression is growth phase-dependent,depending on the level of sulfane sulfur.3.Sulfane sulfur impacts the pigment production via OspR in P.aeruginosa PAO1As an oxidative stress response and pigment production regulator,its mutant leads to a significant increase in H2O2 sensitivity.By detecting the phenotype of ospR null mutant and ospR complementation strain,we discovered that overexpression of OspR results in the production of dark red pigments and an increase in drug resistance.Exogenous addition of H2S reduces the production of the dark red pigments.As a repressor,OspR could inhibit several genes transcription,such as PA2826,PA2009 and PA1897,and RT-qPCR results also verified that.According to the EMSA results,sulfane sulfur-treated OspR has a reduced affinity to its target DNA site,and Cys24 and Cys134 of OspR are mainly responsible for sensing and reacting with sulfane sulfur.Further studies on the mechanisms of sulfane sulfur participating in the regulation of OspR is needed.In conclusion,the physiological functions of sulfane sulfur in P.aeruginosa PAO1 were investigated in this study,and the signal pathways in terms of pathogenicity and antibiotic resistance were reported.Our finding enriches the understanding of the signal function of sulfane sulfur in bacteria beyond inducing sulfur-metabolizing genes expression.