| Objective:As the major human opportunistic pathogen, Candida albicans commonly cause chronic and persistent infections, which is closely associated with biofilm formation. The possible mechanisms include:restricted penetration of drugs through the biofilm matrix; physiological state of cells in biofilms; expression of resistance genes; membrane sterol composition in biofilms; a small number of’persister’cells. Recent studies have suggested that it is the persisters that is responsible for resistance.It has been confirmed that the mechanism of bacterial persisters formation mainly includes the following two aspects:heterogeneity in growth rates of persisters. deriving from non-growing and dormant cell subsets; SOS reaction caused by environmental changes such as starvation. So, does it have effect on Candida albicans persisters formation that changing the nutritional conditions?In our work, we investigate the influence of starvation on the Candida albicans biofilm and persisters formation to confirm the environmental mechanism of bacterial persisters formation. Then, we detect expression of the related genes in persisters by a real-time Taqman RT-PCR assay for single cells to establish a convenient and feasible method for the further studies of persisters.Method:(1) Influence of starvation on the Candida albicans biofilm and persisters formation.The growth curve was made by CFU counts methods in different culture times to find the exponential and stationary phases. Candida albicans in exponential, stationary and starvation phases were developed respectively in96-well microtiter plates to form24h biofilms model. Then the biofilms before and after amphotericin B (100μg/ml) treatment were quantified the number of live cells. Statistical analysis was performed by SPSS17.0software package. The biofilm structure was observed by confocal scanning laser microscopy.(2) Expression of the related genes in persisters by a real-time Taqman RT-PCR assay for single cells.We isolated persister cells and non-persister cells by micropipette under an inverted microscope, and then detected the expression of EFG1, ERG2, MKC1, GIN4in persisters by a real-time Taqman RT-PCR assay for single cells. Statistical analysis was performed by SPSS17.0software package.Results:(1) Biofilm formation capacity of Candida albicans in exponential, stationary and starvation phases reduced successively (P<0.05). The biofilms formed by starved C.albicans were predominately composed of yeast cells, compared with cells in exponential and stationary biofilms, which displayed filamentous growth. The ratios of persisters increased successively (P<0.05).(2) The real-time Taqman RT-PCR assay for single cells which was used in this study successfully detected the expression of EFG1, ERG2MKC1, GIN4in persisters. Expression of MKC1in persister cells and non-persister cells had no significant difference (P>0.05). EFG1, ERG2and GIN4were overexpressed in persister cells significantly (P<0.05).Conclusion:(1) The formation of Candida albicans persisters can be influenced by environment events, and starvation can increase the percentage of persister cells.(2) The self-drawn micropipette can successfully isolate persister cells, and the real-time Taqman RT-PCR assay for single cells used in this study is a convenient technique with high sensitivity to detect expression of related genes in persisters.(3) Formation of Candida albicans persisters is closely related with EFG1, ERG2and GIN4, and is regulated by multiple signaling pathways. |
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