| Staphylococcus aureus(S.aureus)is an important pathogen that can cause a variety of human infectious diseases,including skin/soft tissue infections,sepsis,and infective endocarditis.One major problem of S.aureus infection is that this pathogen could quickly develop resistance to all kinds of used antibiotics,which adds difficulty to clinical treatment.Glycopeptide antibiotics such as vancomycin are considered as the last line of drug in clinical treatment of S.aureus infections.However,with increasing clinical use of vancomycin,S.aureus clinical isolates with reduced susceptibility to vancomycin emerged.Particularly,the emergence and prevalence of vancomycin-intermediate S.aureus(VISA)has become a global health concern.Cell wall thickening is a common feature of VISA strains,while the molecular mechanisms underlying VISA formation are complicated and remain unclear.Currently,most studies on the mechanisms of VISA formation focus on the mutations in certain genes,and researches from other perspectives are limited.In-depth elucidation of mechanism of VISA formation is an important foundation to develop new treatment strategies against VISA infections.Post translational modification(PTM)is a dynamic and reversible chemical process that plays an important role in various bacterial physiological activities.Protein succinylation(Ksucc)is a newly discovered PTM,which can dramatically affect protein functions by insertion of the succinyl group to the ε-NH2 of lysine(K)residue through enzymologically or non-enzymologically ways.Increasing evidence suggests that lysine succinylation affects a variety of biological processes,including tricarboxylic acid cycle,glycolysis,and protein metabolism.However,the relationship between protein succinylation and antibiotic resistance is unclear.In this study,we aim to illustrate the regulatory role of succinylation on bacterial vancomycin resistance.We firstly identified SaCobB as a desuccinylase in a VISA strain XN108,of which the vancomycin MIC value is12 μg/m L,and confirmed that SaCobB could significantly influence bacterial resistance to vancomycin.Next,by performing label-free-based quantitative analysis of lysine succinylome and constructing mur A gene mutant of XN108,the MurA protein was found to play key role in SaCobB-promoted vancomycin resistance.Furthermore,site-specific mutation and enzyme activity determination were performed.All these results together revealed that SaCobB negatively regulates MurA succinylation to promote vancomycin resistance in VISA.The main research contents and results are as following:1.Effect of SaCobB on vancomycin resistance in S.aureus.In this section,the Western blot analysis was performed to measure the succinylation profiles of seven S.aureus strains with different vancomycin resistance phenotypes,and variable succinylation levels presented among the strains were detected.Then,a protein,which was highly conserved with Cob B of E.coli,named NAD-dependent protein deacetylase of the SIR2 family was found in the VISA strain XN108 through homology analysis,and it was named SaCobB.The XN108-ΔSacob B mutant strain was constructed,and Western blot revealed a higher succinylation level of XN108-ΔSacob B compared with that of XN108-WT.After that,several main biological functions of XN108 and XN108-ΔSacob B were compared,and few changes in the growth rate,hemolytic activity,and bacterial colonization in vivo between these two strains were observed.However,the vancomycin MIC of XN108-ΔSacob B decreased from 12 μg/m L to 8 μg/m L.The population analysis further confirmed the increased vancomycin susceptibility of XN108-ΔSacob B when compared with XN108 wild-type(WT)strain.Transmission electron microscopy(TEM)revealed that the cell wall of XN108-ΔSacob B strain was remarkably thinner than XN108-WT.All the results above were reproduced in the standard VISA strain Mu50.These results indicated that SaCobB could promote vancomycin resistance in VISA strains.2.Label-free-based quantitative analysis of lysine succinylomes in XN108-ΔSacob B and XN108-WT.The lysine succinylomes of XN108-ΔSacob B and XN108-WT were analyzed by using label-free-based quantitative liquid chromatography-mass spectrometry(LC-MS/MS)method.A total of 4,321 succinylation sites on 979 proteins were identified.Among them,3260 succinylation sites on 799 proteins were identified in XN108-WT,and 4011 succinylation sites on 942 proteins were identified in XN108-ΔSacob B.The numbers of succinylation sites and proteins which were quantified and normalized in both two strains were 2,936 and 741 respectively.By comparing the succinylomes between XN108-ΔSacob B and XN108-WT strains,98 sites on 83 proteins were hypersuccinylated,and only 3 sites on 2 proteins were hyposuccinylated after Sacob B deletion..Notably,most of the enzymes involved in peptidoglycan synthesis pathway MurA–Mur G were found to be succinylated.These results suggested that SaCobB may promote vancomycin resistance by affecting the succinylation of enzymes in charge of peptidoglycan synthesis.3.Study on the mechanism of SaCobB-promoted vancomycin resistance in VISA through negatively regulating MurA succinylation.RNA-seq and Western blot analysis were performed,and the results showed negligible transcriptional and translational differences in the genes of cell wall associated enzymes MurA–Mur G between XN108-WT and XN108-ΔSacob B.However,a significant difference in succinylation modification of MurA–Mur G was observed by modification site analysis.Then,the key enzyme MurA which exhibited the most succinylation sites was employed to investigate the mechanism by which SaCobB promotes vancomycin resistance in VISA.Deletion of mur A gene not only caused significant decrease in MIC values(from 12 μg/m L to 6 μg/m L),but also lead to thinner cell wall of XN108-Δmur A in comparison to its wild-type strain,indicating that MurA may mediate the regulation of SaCobB on vancomycin resistance in VISA.The three-dimensional structure modeling of MurA indicated that two succinylated lysine residues(K69 and K191)locate at the enzymatic center of this enzyme.The K to E mutation was introduced into these two sites in XN108 genome to mimic succinylation,and the mutants XN108-MurA(K69E)and XN108-MurA(K191E)exhibited decreased vancomycin MIC value when compared with wild-type strain.Similar results were observed in another VISA strain K65,a reverted mutant of XN108.Importantly,the vancomycin MIC of K65-MurA(K191E)decreased to 1μg/m L,presenting a vancomycin sensitive phenotype.At last,the recombinant MurA,MurA(K69E),and MurA(K191E)proteins were prepared,and the enzymatic activity assay revealed a decreased enzymatic activity of these mutant proteins compared with that in MurA in vitro.These results suggested that succinylation at K69 and K191 sites would significantly impair the function of MurA protein.Therefore,SaCobB could enhance the MurA enzyme activity by catalyzing desuccinylation at these two key residues to promote cell wall synthesis,and thereby upregulate vancomycin resistance of VISA.In conclusion,our results demonstrated that SaCobB serves as a desuccinylation enzyme in VISA strain XN108.SaCobB can affect the enzyme activity of MurA protein by regulating its succinylation level,and thus enhances cell wall synthesis and promotes vancomycin resistance in VISA.Our data provided important information regarding the effect of protein succinylation on vancomycin resistance of VISA,opening a new window for elucidating the complex mechanism of VISA formation.The results may provide new ideas and target candidates to develop therapeutic options for control of VISA infections. |
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