Transcriptomics analysis of mixed biofilms

The critical traits contributing to the individual pathogenic potential of P. aeruginosa and C. albicans are the production of virulence (disease causing) factors (VF), the formation of biofilms and antibiotic resistance. Expression of these traits is under stringent regulation and involves the complex intertwining of transcription factors (TF), regulatory RNAs and sigma factors. We are particularly interested in cell density-dependent QS molecules determining, or somewhat related to, the expression of virulence and resistance genes. In both species, QS systems play a critical role in allowing cells to modulate gene expression in response to environmental conditions.

P. aeruginosa employs three interdependent mechanisms of QS, i.e. Las, Rhl and Pseudomonas quinolone systems. QS mechanisms control the production of enzymes and toxins such as LasA, LasB and ToxA, redox-active compounds such as phenazines, and the formation of biofilms. Also, some efflux pumps, such as MexGHI- OpmD, play a role in pumping out quoromones to the cell exterior while others, such as MexAB-OprM, are involved in antimicrobial resistance. In turn, C. albicans is known to express genes encoding adhesins, such as Als3p, and hydrolytic enzymes, such as SAP1 and PLB2, during infection. The formation of hyphae and phenotypic switching are also involved in the virulence of the fungus. C. albicans QS typically involves the production of the molecules farnesol, farnesoic acid, tyrosol, tryptophol, and phenylethyl alcohol (Mayer et al., 2013).

QS also mediates polymicrobial interactions, affecting species diversity and function (Méar et al., 2013). In vitro experiments show that P. aeruginosa may kill C. albicans either by producing toxins, such as pyocyanin, or by direct contact on its biofilm-dependent filamentous form (Trejo-Hernández et al., 2014). In turn, C. albicans may adapt its morphology in the presence of P. aeruginosa QS molecules, and may inhibit P. aeruginosa virulence through the secretion of farnesol, one of its QS molecules (Ader et al., 2011). Also, Candida spp. TBC and P. aeruginosa VAP could be a consequence of prior antibiotic treatment and acquired resistance (Hamet et al., 2012). So, the investigation of gene expression of species within biofilms and upon antimicrobial treatment is considered vital to understand inter-species communication and how it affects single-species gene expression patterns and contribute to infection.

We are conducting in vitro simulation of P. aeruginosaVAP infection and C. albicans TBC to profile the transcriptome of the pathogens in mixed biofilms and under common antimicrobial stress, namely under the effect of tobramycin and amphotericin B. Once potentially relevant genes have been identified in this way, their expression will be investigated in further detail with quantitative real-time PCR in order to determine expression kinetics in a range of conditions.


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