| S.epidermidis is the most frequently isolated member of the group of coagulase-negative staphylococci. It was regarded for a long time as relatively innocuous. In recent years it has gained substantial interest because it has become the most important cause of nosocomial infections. The most important groups of infections caused by S.epidermidis are infections on foreign bodies, such as indwalling catheters and any implanted devices. S.epidermidis infections on these devices are complicated by the formation of biofilm. The staphylococci agr is the most important locus that is responsible for the growth-phase-dependent regulation of virulence factors. Investigation of the regulatory effect of agr in S. epidermidis has not kept pace with the research done in S. aureus. Virtually little has been known about the regulatory effect of agr system on the biofilm formation in S. epidermidisPart I. Genetic polymorphism of agr locus in S. epidermidis and the association with its pathogenicityThe agr locus in staphylococci has been shown to be polymorphic, it is reported that S. aureus agr groups were associated with the pattern of S. aureus diseases. Specific PCR was used in this study to amplify the different agr groups. The results were confirmed by enzyme digestion and sequence analysis. The percentages of three agr groups in pathogenic S. epidermidis are 68.2% of agr group I, 19.3% of agr group II and 12.5% of agr group III espectively. In the isolates isolated from healthy people, there were 39% of agr group I, 51% of agr group II and 10% of agr group III respectively. The results had a signiuficant statistical difference (P<0.01). Thepercentages of agr group I of S. epidermidis isolated from catheters and blood were higher than that isolated from the other clinical specimens. Genetic polymorphism of the agr locus in S. epidermidis was linked to its pathogenicity. Agr group I was associated with pathogenicity while most isolates isolated from healthy people were agr group II.Part II. Construction of S.epidermidis agr group I deletion mutant via homologous recombinationIn order to construct S. epidermidis agr group I deletion mutant and getting the agr group I negative mutant and wild-type strain with the same gene background except agr gene. Plasmid pBT2-Aagr was constructed for homologous recombination of the agr system of S.epidermidis by the insertion of erythromycin resistance gene and two PCR-amplified agr-flanking regions into plasmid pBT2. The homologous recombination vector was firstly transformed to S.aureus RN4220 by electroporation and then transformed to S. epidermidis. S. epidermidis with recombination vector was incubated at 40 °C. S. epidermidis agr deletion mutant was selected. Restriction endonucleases results indicated that the homologous recombination vector was correct. S. epidermidis accepted the recombination vector. The agr group I deletion mutant was proved by antibiotic-resistance > southern blot and direct sequencing of the chromosomal DNA at the borders of the PCR-derived regions. S. epidermidis agr group I deletion mutant was successful constructed, most of sequences of S. epidermidis agr group I were replaced by erythromycin resistance gene. It could be of value in investigating the relationship between agr group I gene and the important functional genes related to biofilm-froming and the virulence related proteins.Part III. Effect of agr group I on the biofilm formation and pathogenicity in S. epidermidisTo characterize the effect of agr group I on the biofilm formation in S. epidermidis, semi-quantitative biofilm assay was used to detect the biofilm formation in agr groupI mutant and wild type. The result showed that biofilm formation in agr group I mutant was significantly higher than that in wild type. This morphological conversion was also confirmed by scanning electron microscopy. To demonstrate the influence of agr group I gene on the pathogenicity of S. epidermidis, agr group I mutant and wild type were studied in a rat central venous catheter (CVC) - associated infection model. The number of bacteria recovered from the liver, kidney and heart of infected animals was remarkably higher for the agr group I mutant compared to wild type. In summary, the biofilm formation and pathogenicity in agr group I mutant were increased compared to wild type.Part IV. Proteomic analysis of the impact of agr group I on the biofilm formation in S. epidermidisIt was recently reported that agr regulated biofilm formation in S.epidermidis by regulation of a?/￡(autolysin) and 8-toxin expression. There are many other important factors influencing the biofilm formation in S. epidermidis besides atlE, such as fbe (fibrinogen-binding protein). The regulatory effect of agr system on them is unknown. A proteomic analysis of S.epidermidis agr group I mutant and S.epidermidis wild type was carried out. Differential levels of gene expression in S.epidermidis agr group I mutant and S.epidermidis wild type were subsequently analyzed by quantitative real-time reverse transcription-PCR assay. The results showed that many proteins (14/19) were up-regulated in agr group I mutant. They could be classified into four categories according to their function: (1) proteins for sugar metabolism (2) proteins for cell wall biosynthesis (3) proteins for specific protein biosynthesis (4) proteins with unknown function. The mRNA and protein of ClpP were up-regulated significantly, which is essential for biofilm formation. At the same time, the mRNA and protein of D-alanine-D-alanine ligase also showed increased, which is important for cell wall formation. However, the level of fbe gene expression was equivalent to wild-type strain. Our data indicated that agr system regulated biofilm formation in S.epidermidis not only by regulation of atlE and 8-toxin expression, but also by regulation of ClpP protein expression. This was also the first report that agr could regulate cell wall formation in S.epidermidis by regulation of D-alanine-D-alanine... |
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