SecretedCandida parapsilosislipase modulates the immune response of primary human macrophages

Abstract

Candida parapsilosis is an important opportunistic pathogen with increasing prevalence. Extracellular lipases have been shown to play an important role in the virulence of pathogenic Candida species. However, studying the role of secreted lipase in C. albicans is challenging due to the lack of a mutant strain deficient in all 10 lipase genes. In contrast, we have previously constructed a lipase mutant C. parapsilosis strain lacking both CpLIP1 and CpLIP2, and shown that it has significantly decreased virulence in various infection models, and is killed more efficiently by mouse macrophages. In the present study, we compared the response of human peripheral blood monocyte-derived macrophages to a wild type (wt) as well as a lipase-deficient (lip(-/-)) C. parapsilosis strain that has been previously established in our lab. Although macrophages phagocytosed both strains with similar efficiency, lipase mutants were killed more efficiently according to fluorescent microscopic analysis. The more efficient killing of lip(-/-) cells was confirmed by CFU-determinations. Phagocytosis of wt and lip(-/-)C. parapsilosis was also examined by flow cytometry, revealing that both strains were internelized to the similar extent by macrophages. Additionally, quantitative imaging analysis revealed that the rate of phagolysosome fusion was higher in case of lip(-/-)C. parapsilosis. Interestingly, macrophages stimulated with lip(-/-)C. parapsilosis showed at least 1.5-fold higher expression of TNFα, IL-1β, IL-6, IL-8, and PTGS-2 after 12 h compared with those infected with wt C. parapsilosis, as determined by qRT-PCR. Furthermore, the lip(-/-)C. parapsilosis strain induced significantly higher TNFα, IL-1β, IL-6, and IL-10 protein production in macrophages after 24 h compared with the wt strain. These findings confirm the role of fungal lipases as important virulence factors during C. parapsilosis infection.

Candida parapsilosis Secreted Lipase as an Important Virulence Factor

“…The role of fungal secreted lipases is pleiotrop as they modulate the immune response in various ways [33]. Both cellular and humoral host responses are affected and they also contribute to host tissue damage, allowing fungal cells to invade the host [33][34][35]. Based on a large number of recent studies, C. parapsilosis is considered an emerging Please provide corresponding author(s) photograph size should be 4" x 4" inches pathogen, and since its clinical relevance is clear, attention should be increased towards this NAC species.…”

“…Such enzymes include extracellular proteinases, phospholipases and lipases [30][31][32]. The role of fungal secreted lipases is pleiotrop as they modulate the immune response in various ways [33]. Both cellular and humoral host responses are affected and they also contribute to host tissue damage, allowing fungal cells to invade the host [33][34][35].…”

Candida parapsilosis produces prostaglandins from exogenous arachidonic acid and OLE2 is not required for their synthesis

“…After 3 h incubation at 37 C, the samples were stained with 0.01% acridine orange dye (Sigma, 235474) as described. 34 This method allows differentiation between live and dead yeast cells. When acridine orange binds to intact (ds) DNA, it emits green fluorescence, while binding to damaged (ss) DNA it shows red fluorescence, indicating dead cells.…”

“…Isolation of human peripheral blood mononuclear cells (PBMCs) and differentiation of monocyte-derived macrophages (MDM) (under approval of the institutional ethical review board of Szeged University) PBMC isolation and differentiation was carried out as described 34 with minor modifications. Following isolation, PBMCs were plated on different cell culture plates: 96-well for killing assay (5 £ 10 5 cell/well); tissue culture coverslips (13mm, SARSTEDT, 83.1840.002) for acridine orange staining (5 £ 10 5 cell/coverslip) or 12-well for cytokine measurement (10 7 cell/ well).…”

“…The killing assays were carried out as described. 34 Cytokine measurements Human macrophages were cultured on 12-well tissue culture plates. The in vitro infection of the macrophages was carried out at a 1:5 macrophage:yeast ratio.…”