Effect Of Quorum-sensing Molecules On Early Stage Of Candida Albicans Biofilms Formation

Candida albicans is a frequent opportunistic pathogen in human oral. Biofilms are the most common growth mode of Candida albicans in nature and they have important significance in clinical infections.In recent years,many studies found that quorum-sensing plays essential roles in the formation and stabilization of C.albicans biofilms.It has been shown that tyrosol and farnesol are two characteristic quorum-sensing molecules(QSM)produced by Candida albicans.Many studies have focused on farnesol but the role of tyrosol is still not well known.In this study,we investigated the effect of the two QSMs on transcription of EFG1 and HTA1 of cells in early stage of Candida albicans biofilms by RT-PCR.And we observed the morphology of C.albicans cells in biofilms with inverted microscope.Also,MTT assay were tested to determine whether QSMs can affect cell proliferation in C.albicans biofilms.By all these studies,we hope to get some initial information about the effect of QSMs on early stage of C.albicans biofilm formation. Partâ… Effect of QSMs on morphology of Calbicans cells in early biofilms[Materials and Methods]C.abicans clinic isolates were obtained from oral rinse of thirty volunteers.Standardized cells of C.albicans clinic isolates and type strain were prepared in RPMI 1640 medium to a density of 1.0×10⁶ cells/ml.C.albicans biofilm models were established on the surface of 50 mL tissue culture flasks under conditions of 5%CO₂ and 37℃.Four groups were set up according to their different QSM that were used.They were:(1)100μM tyrosol(tyrosol-treated group);(2)50μM farnesol(farnesol-treated group);(3)50μM farnesol+500μM tyrosol (both-treated group);(4)no QSMs(control group).Early C.albicans biofilms that grew for 90 minutes and mature C.albicans biofilms that grew for 48 hours were observed with inverted microscope.[Results]Cells in early biofilms of tyrosol-treated group and control group were mainly filamental cells.And cells in early bilfilms of farnesol-treated group and both-treated cells were spores.Mature biofilms after 48 hours consisted of a dense network of yeasts,hyphae,pseudohyphae,and extracellular polymeric material.[Conclusion]No matter there was tyrosol or not,cells in early C.albicans biofilms were mainly filamental cells.And farnesol can obviously inhibit the formation of hyphae.When the two QSMs were both added,even tyrosol at a very high concentration can not change the inhibitory effect of farnesol to the formation of hyphae.Partâ…¡Regulation of QSMs on expression of HTA1 and EFG1 of C.albicans cells in early biofilms[Materials and Methods]Preserved C.abicans clinic isolates and type strain were reanimated.Standardized C.albicans cells were prepared in RPMI 1640 medium to a density of 1.0×10⁶ cells/ml.C.albicans biofilm models were established on the surface of 50 mL tissue culture flasks under conditions of 5%CO₂ and 37℃.Four groups were set up according to their different QSM that were used.They were:(1)100μM tyrosol (tyrosol-treated group);(2)50μM farnesol(farnesol-treated group);(3)50μM farnesol+500μM tyrosol(both-treated group);(4)no QSMs(control group).Early C.albicans biofilms that grew for 90 minutes were collected with a sterile scraper and C.albicans RNA were isolated by Trizol kit. Then RT-PCR test were carried out to investigate the difference of expression of HTA1 and EFG1 between these groups.[Results]The results of RT-PCR showed that when tyrosol exist,the expression of HTA1 was up-regulated(P＜0.05),but EFG1 was not changed(P＞0.05).And farnesol can inhibit the transcription of both genes (P＜0.05).In both-treared group,the expression of HTA1 and EFG1 were also inhibited.[Conclusion]Tyrosol can promote the expression of HTA1 of C.albicans cells in early biofilms,but it can not promote the expression of EFG1. Farnesol can obviously inhibit the expression of HTA1 and EFG1 of C.albicans cells in early biofilms.When tyrosol and farnesol were added at the same time,tyrosol was not able to change the inhibitory effect of farnesol and the latter was predominant.Partâ…¢Effect of QSMs on cell proliferation in C.albicans early biofilms[Materials and Methods]Preserved C.abicans clinic isolates and type strain were reanimated.Standardized C.albicans cells were prepared in RPMI 1640 medium to a density of 1.0×10⁶ cells/ml.C.albicans biofilm models were established on the surface of presterilized,flat-bottom, 96-well microtiter plates under conditions of 5%CO₂ and 37℃.Four groups were set up according to their different QSM that were used.They were:(1)100μM tyrosol(tyrosol-treated group);(2)50μM farnesol (farnesol-treated group);(3)50μM farnesol+500μM tyrosol (both-treated group);(4)no QSMs(control group).Following C.albicans early biofilms formation on microtiter plates,MTT solution were added to medium in each wells.The plates were then incubated at 37℃for 4 h after which the medium were decanted and DMSO were added to each wells.Then the optical density at 490 nm were measured with a microtiter plate reader to explore the effect of QSMs on cell proliferation in C.albicans biofilms.[Results]There was no difference between cell numbers of C.albicans early biofilms of tyrosol-treated group and control group(P＞0.05).In farnesol-treated group and both-treated group,proliferation of C.albicans cells were obviously inhibited(P＜0.05).[Conclusion]Tyrosol can not promote the proliferation of C.albicans cells in early biofilms.And farnesol can inhibit C.albicans proliferation. Tyrosol of high concentration was not able to absorb the inhibitory effect of farnesol on the proliferation of C.albicans cells in early biofilms.