Transcriptomic analysis of the influence of arachidonic acid on polymicrobial biofilms of Candida albicans and Pseudomonas aeruginosa

The interaction of clinically relevant microorganisms is the focus of various studies, e.g. the interaction between the pathogenic yeast, Candida albicans, and the bacterium, Pseudomonas aeruginosa and these interactions can alter the outcome of infection, growth dynamics of each species and antimicrobial resistance of pathogens. During infection, both C. albicans and P. aeruginosa can elicit the release arachidonic acid (AA) from host cells membranes through the action of phospholipases. This polyunsaturated fatty acid can be transformed into immune-modulating compounds, termed eicosanoids, by both host-derived and microbial-derived enzymatic reactions. In its free form, AA can affect the growth of both C. albicans and P. aeruginosa, inhibiting the morphogenesis of C. albicans as well as reducing resistance towards antifungal agents. However, the mechanism of this is unknown. Previous studies on the effect of polyunsaturated fatty acids have indicated a possible alteration in plasma membrane organisation and permeability. Our group aimed to address how AA affects C. albicans in both single species biofilms, as well as in polymicrobial biofilms with P. aeruginosa. RNAseq was performed on single and polymicrobial biofilms in the presence and absence of a sub-inhibitory (100 µM) concentration of AA. Differential expression was determined between C. albicans single species biofilms in the presence and absence of AA. Secondly, the influence of co-incubation of C. albicans with P. aeruginosa in the absence of AA was evaluated to identify novel facets of interaction not previously identified, and to establish a baseline to determine the effect of AA on C. albicans in polymicrobial biofilms. Lastly, the effect of AA on C. albicans in polymicrobial biofilms was determined through comparison with polymicrobial biofilms in the absence of AA. This study provides a comprehensive analysis of the effect of AA and both co-incubation of C. albicans with P. aeruginosa focused on the transcriptome. Four sets of samples in triplicate (n = 3) were analysed consisting of single species or polymicrobial biofilms prepared in 20 mL RPMI-1640 medium and incubated at 37ºC for 6 hours to allow biofilm formation. These include single species biofilms of C. albicans exposed to 100 µM arachidonic acid (CAA; arachidonic acid dissolved in ethanol) compared to single species biofilms of C. albicans exposed to ethanol control (CET; ethanol equivalent to amount arachidonic acid added - final concentration of 0,076% ethanol v/v). Secondly, polymicrobial biofilms consisting of C. albicans and P. aeruginosa (exposed to 0,076% ethanol; CPET) were compared to CET. Lastly, polymicrobial biofilms exposed to 100 µM AA (CPAA) were compared to CPET. Total RNA extraction was performed and subjected to sequencing on the Illumina NextSeq 500. Resultant FastQ files were subjected to a relevant bioinformatics pipeline to determine differential expression.