Bacterial lipopolysaccharide modifies Candida albicans biofilm development at transcriptional level

Abstract

OBJECTIVE: To evaluate the effect of Gram negative bacterial lipopolysaccharides (LPS) on C. albicans hyphal development and its transcriptional regulation during biofilm formation. METHODS: The effect of LPS from P. aeruginosa ATCC 27316 and K. pneumoniae ATCC 15380 on different C. albicans strains (ATCC 90028, SC 5314, and a clinical strain) were studied using a standard biofilm assay. Candida biofilms developed on polystyrene surfaces were treated with LPS at 90 min. The effect of LPS was assessed quantitatively by XTT reduction assay (XTT) at 48h, growth curve assay and light microscopy, and qualitatively, by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM). RNA was extracted from both test and control C. albicans SC 5314 biofilms at 1h, 3h, 6h, 12h, 24h and 48h. Differential expression of hypha specific genes (HSGs); ECE1, HWP1, HYR1, RBT1, RBT4, ALS3 and ALS8 at 48h and the transcription factor EFG1 at all time points were evaluated with quantitative Real time PCR assay. Cyclic AMP levels of 48h test and control biofilms were quantified by cAMP competitive ELISA. RESULTS: XTT data showed significantly lower metabolic activities in LPS treated C. albicans SC 5314 and clinical strain biofilms (p<0.05). Growth curves exhibited constantly lower optical density in LPS treated biofilms. Light microscopy demonstrated higher proportions of budding yeasts (89%) in test biofilm compared to its control (73%). SEM and CLSM further confirmed these data. All HSGs in the test biofilm were significantly upregulated at 48h (p<0.05). EFG1 was significantly upregulated at all six time points (p<0.05). No significant difference was noted in cAMP concentration in control and test biofilms. Conclusion: Bacterial LPS acts downstream of EFG1 in cAMP/PKA signaling pathway in C. albicans and upregulates HSGs, with a paradoxical suppression of biofilm development possibly due to an increased production of budding yeasts. (Supported by CERG#HKU-7624/06M)