Cuticle Staphylococcus And The Identification Of Mycobacteria Biofilm Related Genes And Functional Studies

Staphylococcus epidermidis is a leading cause of hospital-acquired infections, mostly associated with the use of medical devices in seriously ill or immunocompromised patients. Currently, the clonal characteristics of S. epidermidis in the hospital environment in China are unknown; neither is it known why these sequence types are easily disseminated in the hospital setting. In this study, multilocus sequence typing (MLST) was employed for the clonal analysis of80S. epidermidis isolates collected from patients with S. epidermidis infections. MLST revealed a total of16different sequence types among these isolates. ST2, which contained exclusively ica-positive,IS256-positive and biofilm-forming isolates, represented the majority of clinical strains tested. Of the S. epidermidis strains circulating in the hospital environment in China, as many as96.25%are resistant to meticillin. Four staphylococcal chromosomal cassette mec (SCCmec) types were identified among the total80S. epidermidis isolates, none of the strains carried a SCCmec I cassette. All of the ST2isolates carried the SCCmec type Ⅲ cassette. Taken together, the combination of biofilm-forming ability and antibiotic resistance helps ST2become successfully established within nosocomial environments, and promotes the device-related infection and bacteraemia. These results are in accordance with the virulence potential of biofilm formation in S. epidermidis.(Part Ⅰ)Biofilm is of definition that bacteria embedded in secrated excellular matix attach to orgnic or unorganic surface, then proliferate and accumulate, finally constitute the mature biofilm. Bacteria in biofilm makes them escaping from the immune system of host and disseminating to form new niche. Deep investigation of molecular mechenism of biofilm formation will be helpful to preclude and manage infections caused by Staphylococcus epidermidis efficiently. In this study, a mutant library of the clinical isolate S. epidermidis1457was constructed using mariner-based transposon mutagenesis. About a thousand mutants were screened and twelve mutants inserted in eleven genes were identified as significantly defective in biofilm formation. Among those eleven genes, ica and aap were previously reported as important biofilm factors, the other nine genes have not been previously implied in biofilm formation of S. epidermidis and included genes encoding a general stress protein (SERP0541), a polysaccharide biosynthesis protein (SERP0142), the redox-sensing transcriptional repressor Rex (SERP1657), and hypothetical proteins (SERP0270, SERP2282). We focused on a mutant in which the transposon had inserted in a gene with unknown function, SERP0541, which is annotated as a gene encoding a GSP13-like general stress response protein. The gene was named ygs (encoding an unknown general stress protein). Firstly, in comparison between mutant and wild type strain S. epidermidis1457, we found that the ygs mutant showed decreased biofilm formation and decreased production of PIA in the second step. Then we cloned this gene on a shuttle plasmid and transformed the plasmid into the mutant strain, which restored the wild type phenotype. It demonstrated ygs is a novel gene important for biofilm formation in S. epidermidis. To examine whether the altered biofilm formation that we observed in the mutant strain was due to a change in the transcription of the ica operon responsible for PIA production, we analyzed transcription of the icaB and icaR gene by quantitative real-time PCR. Expression of icaB during early-exponential growth phase was9times lower in the mutant strain than in the wildtype strain, whereas expression of icaR was2-3times higher in the mutant strain than in the wildtype strain. This indicates that ygs postively regulates the expression of the icaABCD gene through icaR at the transcriptional level. Furthermore, various stresses, including heat, pH, high osmolarity and ethanol affected survival of the ygs mutant to a significantly higher degree than the wild-type strain and led to increased expression of ygs. These results are in accordance with the putative involvement of ygs in stress-response gene regulation and indicate that ygs influences biofilm development by controlling PIA-dependent biofilm accumulation. Moreover, ygs had a significant impact on the formation of biofilms and metastatic disease in two catheter-related rat infection models. Our study shows that the ygs gene controls S. epidermidis biofilm accumulation and stress resistance, representing a key regulator of both structural and physiological biofilm characteristics with a significant impact on biofilm-associated infection.(Part Ⅱ)Mycobacterium also have the ability to form biofilm. It has been reported that M. tuberculosis forms biofilms which harbour a drug-tolerant population that persist despite exposure to high levels of antibiotics. This disease remains a major health problem. Drug resistant tuberculosis, especially multidrug resistant(MDR) and extensive drug resistant(XDR) tuberculosis are the new challenges of tuberculosis control. Therefore, studying the molecular mechanism of mycobacterial biofilm became crucial important. Recently M. smegmatis and M. marinum have been used as model organisms to study M. tuberculosis pathogenesis. In order to identification and characterization novel biofilm-related genes of M. tuberculosis, a MycoMarT7mariner transposon mutagenesis library was screened. Mutants with altered biofilm formation were selected, and the insertion sites were identified using sequencing. We identified a mutant with distinct biofilm formation. The transposon inserted aceE gene. Disruption of aceE resulted in strikingly altered colony morphology, sliding motility and acid sensitity, suggesting a role for aceE gene in cell wall structure and function. The phenotypes of the aceE mutant were fully complemented by M. smegmatis aceE. The changes in colony morphology and biofilm formation suggested that the aceE mutation affected some components of the cell wall.2D-TLC analysis revealed that the amount of TAGs and MDAGs in the mutant cell wall was decreased, whereas the amount of TAGs in the complemented strain can restore to the wildtype level. This suggested TAGs play a role in colony morphology and biofilm formation of mycobacteria. We also found the aceE mutant grow slower than the wildtype in the7H9medium which containing glucose or glycerol as carbon source., but when taking acetate as carbon source, there were no difference in the wildtype and mutant. It indicated aceE takes part in glucose or glycerol intake of M. smegmatis.(Part Ⅲ)By screening the M. marinum mutagenesis library, surprisingly, three mutants with transposon insertion sites in a single gene were identified. The mutated gene is mmaA4, which is necessary for the biosynthesis of methoxy-and keto-mycolic acids in M.tb and BCG. M.tb carries three types of mycolic acids:di-cyclopropanated a-mycolic acids and oxygenated, methoxy-and keto-mycolic acids. Trehalose6,6’-dimycolate (TDM) which consists of mycolic acid and trehalose, also called cording factor, was considered to be associated with mycobacterial virculence for a long time. In this study, we found the mmaA4mutant of M.marinum can not form cording structure, exhibited altered colony morphology, and was defective in sliding motility and biofilm formation. It has been reported that mycolic acid correlates with biofilm formation. To investigate which oxygenated mycolic acid is more important for biofilm formation in M. marinum, we use a mmaA3mutant to study. The mmaA3mutant fail to produce methoxy-mycolates. Overproduction of MMAS3results in the loss of keto-mycolic acids with a significant increase in methoxy-mycolates. Our study demonstrated that the complemented strain need longer time to form biofilm than WT and the mmaA3mutant. Therefore, keto-mycolic acids is more important for biofilm formation. In order to further investigate the role of different mycolic acid in mycobacterial pathogenicity, adult zebra fish infected with these strains. We found zebra fish infected the mmaA3mutant all survived when zebra fish infected the wildtype and complemented strains were all dead, which suggesting methoxy-mycolates play an important role in virulence of mycobacteria.(Part Ⅳ)...