The Investigation Of The Underlying Mechanisms Of Small Molecule Compounds Synergize Amphotericin B Against Candida Albicans Biofilms

ObjectiveThe most widespread fungal pathogen in humans and one of the most frequent causes of hospital-acquired infections is Candida albicans.C.albicans is responsible for common clinical conditions such as oral thrush and vaginitis;it can also lead to systemic fungal infections with high mortality rates in immunocompromised patients.C.albicans adheres to the surface of an object and forms a biofilm structure.Biofilm formation is an important step in pathogenesis,because after biofilms form on intravascular devices or catheters,they normally do not respond to conventional treatment and can give rise to life-threatening systemic infections.For these reasons,biofilms shield C.albicans from attack by the immune system,as well as block antifungal compounds from reaching cells.These attributes enable biofilms to function as infectious reservoirs,which release new propagules both during and after treatment with antifungal therapeutics.Most antibiotics kill bacteria by inhibiting a cellular target that is required for active cell growth(for example,ampicillin kills bacteria by inhibiting enzymes that synthesize cellular wall material).An alternative approach is to use antibacterial compounds that inhibit cellular targets not only in growing cells,but also in resting cells.Thus,therapeutic interventions using small-molecule inhibitors of biofilm formation would give clinicians a valuable tool for re-establishing pharmacological control over recalcitrant C.albicans infections,and the creation of an appropriate chemical screen is a key step to identifying promising bioactive compounds that address this need.Although a handful of biofilm and morphological transition inhibitors have emerged in recent years[1-3].many of these compounds suffer from mediocre potency or poor physiochemical characteristics.Therefore.the focus of research has shifted to examine the combination of antifungals with non-antifungals.Our intention was to search the small molecule compounds which have synergistic effect with amphotericin B and use sequencing tests to look for the specific target genes for the synergistic effects and try to provide new ideas for clinical therapy.MethodsCandida albicans strain 3153A biofilms growing in individual well of flat-bottom 384-well microtitre plate.We have tested 51520 small molecule compounds from Chinese National Compound Library using high-throughput screening(HTS)with antifungal susceptibility tests(ASTs).For the final two compounds,LIVE/DEAD FungaLight yeast viability kit(Invitrogen,USA)employed in the live/dead cells analysis.The analysis of cytotoxicity was performed using a cell counting kit-8.The Illumina-Solexa sequencing technology was used to detect the exact effect biological target of compounds on amphotericin B against Calbicans in gene expression of the two compounds,we also used real time qPCR to verity the sequencing results.In vivo,we used mice to verified that after using compounds and amphotericin B together against C.albicans biofilms.the inhibit rate was increased compare to the AmB group.We used the Illumina-Solexa sequencing technology to identify the gene expression differences between added with compounds groups and non-compounds group.By trying to figure out the potential expression changes in genes,we intend to find the exact genes responsible for the small molecule-amphotericin formulation.ResultsFirstly.10 compounds were identified and through the further research we finally identified two small molecule compounds which enhanced the biofilms inhibition of amphotericin B more than 40%.Focused on the two compounds,we revealed that both in vitro and in vivo that the inhibit rate was increased compare to use amphotericin B alone.There were four specific genes identified in the comparison of group AmB and AmB3 group,those four genes are responsible for the synthetic of the hypothetical protein via different pathways such as FAS2,EIF5B,GTF2EI,TFA1 and tfe pathways.As to the AmB vs.AmB 10 group,five genes were verified and they are responsible for conserved hypothetical protein.hypothetical protein and likely tryptophan synthetase alpha chain through DYNClH and TRP pathways.ConclusionThese small molecules may specifically target the C.albicans biofilms and make currently available antifungals more effective.Notably,from the sequencing results,we identified protein coding genes that may be the potential targets of the compounds’effects.For all those information we collected,we can reveal the hypothesis that by affecting those pathways though gene expression modulate,the small molecule compounds improved the antifungal effects of AmB.This will introduce a new set of problems on what is the precise biological process and how these genes affect the biofilms.which will be our focus of our future researches.