The Tst Gene Differential Expression In Staphylococcus Aureus And The Role Of TSST-1 In Accelerating The Development Of Atherosclerosis

BackgroundStaphylococcus aureus(S.aureus)is one of the most common pathogenic bacteria in hospitals and communities.The severity of infection by S.aureus is related to the amount of produced toxins.Toxic shock syndrome toxin-1(TSST-1),coded by tst,is a kind of pyrogenic toxin generated by S.aureus and can lead to toxic shock syndrome(TSS)with a mortality of 50%,if not treated promptly.In part one and part two of this study,we investigated the prevalence of tst gene,detected the gene expression level,and preliminarily explored the possible mechanism of the differential expression of tst.These data may provide an important theoretical basis of surveillance of tst-positive S.aureus infection.Atherosclerosis(AS)is recognized as a disease of chronic inflammatory disorder.Considerable evidence in epidemiology,clinical pathology and animal model indicates that infections with bacteria or viruses are conducive to chronic inflammatory processes and thus to the pathogenesis of AS.TSST-1,a member of superantigen,can cause systemic inflammatory response of the body.Recent investigations in rabbits displayed that chronic TSST-1 exposure induced systemic inflammation and had an impact on the development of diabetes mellitus type Ⅱ(DM Ⅱ),As one of the common features of DM II and atherosclerosis,chronic inflammation exerts significant effects on both diseases.Thus,we hypothesized that TSST-1 could contribute to the progress of atherosclerosis by similar effects to that of DM Ⅱ.In the third part of this study,we administered TSST-1 by miniosmotic pumps to New Zealand White rabbits,and evaluated aortic atherosclerosis using histological and morphometric methods.Additionally,possible mechanisms involved in TSST-1-mediated effects were also explored.We hope our results can provide an important information for searching potential targets for therapeutic intervention in AS.In conclusion,the aims of this study are to detect the prevalence and expression of tst gene in clinical S.aureus,explore the mechenisms of differential expression of tst,and demonstrate the role of chronic TS ST-1 exposure in development of AS.We divided our research in the following three parts.Part one Prevalence and molecular characterization of the Staphylococcus aureus strains harboring tst geneObjective To investigate the prevalence of tst gene and the molecular characterization of the S.aureus strains carrying tst.Methods Nine hundred and sixteen isolates of S.aureus were collected from seven hospitals in Shanghai and Zhejiang Province and subjected to detection of tst,mecA and mecC genes by polymerase chain reaction(PCR).The genetic types were determined for the tst gene positive isolates using agr,SCCmec(for MRSA),PFGE,MLST and spa typing motheds.Results Of the 916 isolates,208 carried tst gene(22.7%),and 665 were mecA positive.All isolates were mecC negative.Out of the 665 MRSA isolates,198 hosted the tst gene(29.8%).The most common agr and SCCmec types were agr 2(97.0%)and SCCmecⅡ(94.4%),respectively.The ST5(68.8%)is the most frequent clone.As for spa type,82.3%of the tst gene positive MRSA were t002.PFGE typing showed that 29.3%and 19.2%of the MRSA were clustered into PFGE type E and type F,respectively.Among the 251 methicillin-sensitive S.aureus(MSSA)isolates,only 10 were tst gene positive(4.0%),among which the ST5,t002 and agr1/2 were predominant.Of note,all the tst positive MSS A isolates were clustered into PFGE type B(n=4)and H(n=6).Conclusions The tst gene positive MRS A isolates mainly belonged to ST5-agr2-t002-SCCmecⅡ(39.3%).The prevalence of tst gene in MRSA is significantly higher than that in MSS A,which means clinicians should pay close attention to the infections caused by these strains.Part two Detection of tst mRNA level and preliminarily exploration of tst gene differential expression mechanismObjective To investigate the expression of tst mRNA in clinical S.aureus and preliminarily explore the mechanism of tst differential expression.Methods Based on the genetic characterization of S.aureus harboring tst gene investigated in part one,30 isolates were chosen for the detection of tst mRNA expression.Subsequently,strains with high and low tst mRNA expression were picked out for the preliminarily exploration of the mechanism of tst gene differential expression.PCRs were performed to amplify tst gene promoter and coding sequence,and qRT-PCR was used to determine the expression quantity of RNAⅢ、sigB、sarA、sarT、ccpA、srrA、srrB、rot and sarT genes.Mechanism of promoter mutation causing differential expression of tst was verified by carrier construction,subsequent electrotransformation to RN4220,and detecting the tst expression by qRT-PCR.Results Out of the selected 30 S.aureus isolates harboring tst gene,the relative transcript levels of tst gene in 73.3%(22 isolates)of the strains range from 3 to 5(the amount of tst in isolate RJ24,an isolate with the lowest tst mRNA expression,was set to be 1).Strain SJ0951,whose tst gene expression was 8.4 times higher than that of strain RJ24,was the strain with the highest tst gene expression level.Two strains with high-expression and low-expression,respectively,were chosen to investigate the mechanism of tst differential expression.No significant mutation was detected in tst ORF and no differential expressions were found in sigB,sarA,srrB and rot genes among high-and low-expressed strains.However,a base T deleted at the location of-114 in tst gene of high-expressed strains was detected.The tst expression regulated by a base T deleted promoter is 2.4 times as much of that controlled by non-base deleted promoter in RN4220.Moreover,high expression of srrA mRNA in LS1956,sarT mRNA in RJ24,RNAIII in H-SJ0951 and ccpA in L-HK471 were found.Conclusions Differential expression of TSST-1 in clinical S.aureus might be related to the diverse expression of various transcriptional regulators,and the mutation of tst promoter.Part three Chronic S.aureus superantigen TSS T-1 exposure accelerates the development of atherosclerosis in rabbitsObjective To investigate that how the chronic staphylococcus aureus superantigen TSST-1 exposure influences the development of atherosclerosis in rabbits.Methods The animals were randomly divided into 4 groups of five animals each,designated as Normal Diet(ND)group,Normal Diet +TSST-1(ND+TSST-1)group,High Fat Diet(HFD)group,and High Fat Diet + TSST-1(HFD + TSST-1)group.Alzet miniosmotic pumps,containing either TSST-1 or PBS with a constant pumping rate,were implanted subcutaneously in New Zealand White rabbits in the week 6.All rabbits were euthanized at the end of the experiments.The obtained aortas were dissected and stained with Oil Red O,and the atheroma plaque area was evaluated using the image analysis software ImageJ.Aortic arches were dyed with hematoxylin and eosin(H&E).The intimal and medial thicknesses were quantitatively measured using the image analysis software Leica Qwin pro.To visualize the cellular components in the lesion area,the primary antibodies,including muscle actin monoclonal antibody(mAb,HHF35)and mAbs against macrophages(RAM11),were used for immunohistochemical staining.The levels of serum lipids,endotoxin,and tumor necrosis factor alpha(TNF-α)were also analyzed.Results Except for triglycerides,TSST-1 treatment did not change the other serum lipids quantities and body weights of the rabbits.However,the gross atherosclerotic lesion area in the aortic arch was increased by 15.3%in HFD+TSST-1 group compared to that in HFD group.In ND+TSST-1 and HFD +TSST-1 groups,the structure of the vascular media was changed with obvious migration and proliferation of vascular smooth muscle cells(SMCs).Histological and immunohistochemical analyses revealed TSST-1 exposure increased the number of SMCs,but had no significant effect on the numbers of macrophages within lesions.Moreover,the serum TNF-α level increased,whereas the endotoxin concentration remained unchanged.Conclusions Our data validated that chronic stimulation with TSST-1 induced inflammation,and facilitates,not in collaboration with endotoxin,the progression of atherosclerosis,which may be associated with migration and proliferation of vascular SMCs.