| Staphylococcus epidermidis (S. epidermidis) is an opportunistic pathogen which colonizes on the human skin surface and mucous membrane that rarely causes pyogenic infections in healthy individuals. But the S. epidermidis can induce infections by forming biofilms on surfaces of medical devices with the synthesis of polysaccharide intercellular adhesin (PIA) and so on. Because the cells of S. epidermidis can hide in the biofilm, the lymphocytes of host cannot attach to the medical devices, which limiting the function of host immune system. For the past few years, S. epidermidis has emerged as one of the major pathogens in nosocomial infections because of the using of medical devices.Biofilm formation is composed of two steps:initial attachment and intercellular adhesin. In the first step, many factors are found to be involved in the biofilm formation,such as AtlE and Bap(Biofilm-associated protein). In the second step, PIA which is encoded by ica operon plays a very important role by mediating cell-to-cell interconnection during biofilm formation.There are many complex regulation passageways in the bacteria biofilms which is a very complex living environment. The regulation passageways are still poorly understood in the S. epidermidis, especially in the S. epidermidis clinical strains. Two-component signal transduction systems (TCSs) contribute a lot for bacteria to sense diverse environmental cues and to adjust molecule metabolism. In this thesis we studied on the phenotypes, regulation genes involved in biofilm formation and the proteins expression of the S.E icaA+/BF-clinical strains. At the same time, we also studied on the synthesis of extracellular polymeric substances (EPSs), regulation genes and proteins involved into the biofilm formation in S.E 1457AarlS which was constructed in the early study so that the molecule mechanism of biofilem formation can be interpreted.Ⅰ. The study of transcription level of biofilm regulation genes in S.E icaA+/ BF- clinical strainsBiofilm formation is composed of two steps:single bacterial cell attachment and intercellular aggregation. As far as we know, many genes are found to be involved in the biofilm formation, such as atlE and sdrF which play a role in the attachment and aap, icaADBC as well as bap which involved in cells aggregation. accumulation-associated protein (Aap) produces a marked effect on the biofilm formation by dimerization. PIA is the major component in S. epidermidis, which is encoded by ica operon and mediates cell-to-cell interconnection during biofilm formation. Usually, the existence of icaADBC is relevant to the biofilm formation. However, in our early studies, we found that 15 S. epidermidis clinical strains had ica operon but without biofilm formation(SE icaA+/BF- clinical strains). This phenomenon suggested that there may be some other genes regulate the transcription of icaADBC and affect the biofilm formation. To investigate this question, the phenotypes(the growth curve and initial attachment), regulation genes(sarA, rsbU, srrA, arlRS and luxS) involved in biofilm formation and the proteins expression of the S.E icaA+/BF-clinical strains were detected. The biofilm formation and the existence of icaADBC were validated by a semi-quantitative microtiter plate assay and RT-qPCR respectively. We found that the icaA existed but no biofilm formation. Bacterial growth curve was determined by measuring the value of OD570. Compared to the RP62A, the growth curve of SE icaA+/BF- clinical strains were similar to that of RP62A. However, the transcription levels of icaA gene were low in the SE icaA+/ BF- clinical strains by using RT-qPCR. PIA synthesis of 15 icaA+/BF-strains were detected by WGA-HRP conjugate(wheat germ agglutinin coupled to horseradish peroxidase). With this assay, no PIA production could be detected in the icaA+/BF-strains. We carried out initial adherent assays of SE icaA+/BF-clinical strains and found that the initial attachment ability remained in clinical strains, but the aggregation between the cells was very weak. According to the report, the biofilm can be induced by trypsin in a biofilm-positive phenotype S. epidermidis strain. So the effect of trypsin on ica+/BF- isolates was detected. Among of 15 clinical strains, trypsin treatment induced cell cluster formation during primary attachment in 4 isolates in which an obvious increase in biofilm formation was observed for treating with trypsin in comparison with trypsin-free controls. Then the transcription level of aap gene was detected by real-time quantitative PCR at mid-exponential-growth phase. As the result shown, the transcription level of aap gene was higher in four strains and lower in other eight strains than that of RP62A. Next we examined the expressions of Aap in icaA+/BF- strains by Western blotting using monoclonal antibodies against Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrptl.5) and found that the expressions of Aap was very low in ica+/BF- strains.Next the transcription level of biofilm regulation genes in S.E icaA+/BF- clinical strains were detected by real-time quantitative PCR at 4 hrs and 10 hrs. Results showed that at 4 hrs the transcription levels of sarA in 4isolates were lower than RP62A. And no obviouse difference was detected in the other 11 isolates. Both of the rsbU and arlRS were low transcription in all of 15 icaA+/BF-strains. The transcription levels of srrA were lower in 10 isolates but in other 5 isolates, no obviouse difference was detected. The transcription levels of luxS in 6 isolates were lower than RP62A. At 10 hrs, the transcription levels of sarA in 2 isolates were lower than RP62A. In the other 2 isolates, the sarA transcriptions were higher than RP62A. And no obviouse differences were detected in the rest isolates. rsbU still kept low transcriptions in 14 icaA+/BF-strains. The transcription levels of arlS in 11 ica+/BF-isolates and arlR in 12 isolates were lower.The transcription levels of srrA in 10 isolates were higher than RP62A and in other 4 isolates it was lower. Only for one isolate, the transcription level of luxS was higher than RP62A, but in other 2 isolates the transcription levels of srrA were lower, no obviouse difference on the transcription level of luxS were detected in the others. The results suggested that the transcription level of arlRS may be the main reason for resulting of low transcription level in ica operon (Tab.1).Ⅱ. The effect of arlRS on synthesis of extracellular polymeric substances (EPSs) and expression of relevant protein in S. epidermidisTwo-component signal transduction systems (TCSs) contribute a lot to sense diverse environmental cues and and to adjust molecule metabolism for bacteria. In S. aureus, ArlRS is a global two-component virulence regulatory system which can interact with other regulators to modulate the expression of virulence factors.. But in S. epidermidis, ArlRS are still poorly understood. In the early study of our lab, S.E 1457AarlS was constructed and found out no biofilm was formed. In the results of part one, we found that the transcription levels of arlRS were low in all of 15 icaA+/BF- strains at 4 hrs. At 10 hrs, the transcription levels of arlRS in most of isolates were still lower. In our early studies, we found that the mutation in the arlS gene resulted in no biofilm formed in S. epidermidis. At the same time the ability of autolysis of bacterial was enhanced. Besides, ArlR can bind to the icaA promoter, which suggested that the arlRS can not only affect the biofilm formation but also regulate the transcription of ica operonTo further investigate the function of arlRS in S. epidermidis, the synthesis of extracellular polymeric substances (EPSs) biofilm regulation genes and proteins as well as the genes involved into the biofilm formation in S.E 1457△arlS were detected. The extracellular DNA (eDNA) was quantified by real-time PCR. We found that there were no obviouse difference in eDNA expression in 1457△arlS and 1457 wild type. Then PIA synthesis were detected by WGA-HRP conjugate(wheat germ agglutinin coupled to horseradish peroxidase). Compared to the 1457 wild type, no PIA production could be detected in 1457△arlS. With the detection of transcription level of ica operon, we found that icaA was low transcription. Next, global protein expression changes between 1457△arlS and 1457 wild type were detected by 2-DE. As the data shown,58 protein expression were up-regulated, and 63 protein expression were down-regulated, among which Aap expression was decreased. This result was confirmed by Western blot. The transcription level of aap was also determined by RT-qPCR and the result shown that aap was transcripted at a low level in 1457△arlS. And then the transcription levels of biofilm regulation genes in S.E 1457△arlS were detected by real-time quantitative PCR at 4 hrs. Results showed that the transcription level of icaA, rsbU and sarA were significant lower than 1457 wild type at 4 hrs, while the arlR and luxS transcription remained the same level compared to 1457 wild type. |
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