Project title : The role of prostaglandin-like molecules in host-pathogen interaction of Candida parapsilosis

Candida parapsilosis is one of the major human fungal pathogen, which primarily causes invasive infection among low birth weight infants. Despite its increasing prevalence very little is known about the mechanisms involved in its interaction with the human host. The eicosanoids are 20C long chain fatty acid molecules which are mainly derived from arachidonic acid. They are the oxygenated metabolites of arachidonic acid produced mostly by the enzymatic action of COX and LOX enzyme. There are different eicosanoids produced in the mammalian host belong to the different subfamilies, which include prostaglandins, thromboxane, leukotrienes, lipoxins, resolvins and HETEs (Hydroperooxy-eicosatraenoic acid). These eicosanoids are the bioactive signaling lipid molecules, which plays a major role not only in infection and inflammation but also in resolution of inflammation. Although fungi lack the homologue of Cox/Lox enzymes still they can produce prostaglandins from exogenous arachidonic acid. In recent years it has been shown by different groups that human pathogenic fungi like Candida albicans, Candida parasilosis, Cryptococcus neoformans, Paracoccidioides brasiliensis and Aspergillus fumigatus also produce prostaglandin molecules which is similar to the host. In case of C. albicans, it has been found that two genes namely OLE2 (Fatty acid desaturase homologue) and FET3 (Multicopper oxidase homologue) is involved the prostaglandinE2 (PGE2) production. However, it has been shown from our lab that, unlike the closely related pathogenic fungus C. albicans, OLE2 (fatty acid desaturase gene) is not involved in the biosynthesis of Prostaglandin in C. parapsilosis. The current work involves the analysis of the novel genes or pathways for different eicosanoids production in C.  parapsilosis including prostaglandins and how these fungal lipid mediators are involved in inflammation and host pathogen interaction during candidiasis.

To identify genes for eicosanoid biosynthesis in C. parapsilosis, we performed RNA-seq analysis after growing the cells in presence of arachidonic acid, the main precursor for eicosanoid production. The sequence analysis (in collaboration with Dr. Toni Gabaldon’s Lab, CRG, Barcelona) showed that total 68 genes were upregulated and 83 genes were downregulated. From the upregulated genes we found that 14.5% genes are involved in lipid metabolism. Six probable candidate genes were selected by GO term analyses which are involved in the lipid metabolism process. Knock out (KO) mutants for those 6 genes were generated by fusion PCR technique. The 6 mutants were then analyzed for the eicosanoid production. In the beginning mainly the cell free supernatant was analyzed after growing the cells in presence of 100uM arachidonic acid. The eicosanoid analysis was done in the LC/MS facility in collaboration with Dr. Martin Giera’s lab, LUMC, Leiden. The analysis showed that other than different prostaglandins (PGD2, PGE2 and 15-K-PGE2) C. parapsilosis also produce lipoxinA4 molecules, which is a novel finding according to our knowledge. From the 6 KO mutants 3 of them showed significant reduction in PGE2 and LipoxinA4 production compared to the wild type (WT) strain. Currently samples were prepared to analyze the internal eicosanoid from the mutant strains.

As the lipid mediators also have effect on the morphogenesis and biofilm production of C. albicans, the mutants were analyzed for their fitness after growing them in different temperature, pH, stress source and pseudo hypha inducing media. Few of the mutants showed prominent difference in some of the growth condition. Currently the mutants are also studied to check whether they have any difference in their ability to form biofilms, as C. parapsilosis also can biofilm, one of the pathogenic trait of this organism.

Finally, to study the role of the fungal lipid mediators in inflammation and host pathogen interaction during candida infection, human primary macrophage cells are being used as a model system (PBMC-Peripheral Blood Mononuclear Cell and PBMC derived macrophages). The recent results showed that the PBMC-DMs are more efficient in killing and phagocytose the eicosanoid KO mutants compared to the WT C. parapsilosis strain. In the ongoing experiments, the mutants are compared with the WT strain for their ability to generate inflammatory response by analyzing different pro and anti-inflammatory cytokines and chemokines (IL1β, TNFα, IL10 and IL8) by ELISA after infecting both PBMC and PBMC-DM. Preliminary results showed that the KO mutants produce higher amount of anti-inflammatory cytokine IL10 which correlates the lower production of pro-inflammatory cytokine TNFα production. The future plan is to analyze the mutants for their virulence using mouse model of systemic infection.