Mucormycosis is a life-threatening opportunistic infection caused by certain members of the fungal order Mucorales. This infection is associated with high mortality rate, which can reach nearly 100% depending on the underlying condition of the patient. Treatment of mucormycosis is challenging because these fungi are intrinsically resistant to most of the routinely used antifungal agents, such as most of the azoles. One possible mechanism of azole resistance is the drug efflux catalyzed by members of the ATP binding cassette (ABC) transporter superfamily. The pleiotropic drug resistance (PDR) transporter subfamily of ABC transporters is the most closely associated to drug resistance. The genome of Mucor circinelloides encodes eight putative PDR-type transporters. In this study, transcription of the eight pdr genes has been analyzed after azole treatment. Only the pdr1 showed increased transcript level in response to all tested azoles. Deletion of this gene caused increased susceptibility to posaconazole, ravuconazole and isavuconazole and altered growth ability of the mutant. In the pdr1 deletion mutant, transcript level of pdr2 and pdr6 significantly increased. Deletion of pdr2 and pdr6 was also done to create single and double knock out mutants for the three genes. After deletion of pdr2 and pdr6, growth ability of the mutant strains decreased, while deletion of pdr2 resulted in increased sensitivity against posaconazole, ravuconazole and isavuconazole. Our result suggests that the regulation of the eight pdr genes is interconnected and pdr1 and pdr2 participates in the resistance of the fungus to posaconazole, ravuconazole and isavuconazole.

The fungal membrane contains ergosterol instead of cholesterol, which offers a specific point of attack for the defense against pathogenic fungi. Indeed, most antifungal agents target ergosterol or its biosynthesis.

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Mucormycosis is a life-threatening systemic infection caused by certain members of the filamentous fungal order Mucorales (e.g., Rhizopus delemar, Lichtheimia corymbifera, and Mucor circinelloides). Mucormycosis is associated with a high mortality rate, which can be nearly 100% depending on the underlying condition of the patient. One of the main risk factors of mucormycosis has been the iron-chelating treatment. Iron is an essential nutrient for both the host and the pathogen. Therefore, organisms developed various strategies to acquire iron from the environment. Three strategies are available to microorganisms to uptake iron from the environment: acidification of the environment, reduction of ferric iron to the more soluble ferrous form, and secretion of soluble iron-chelating molecules. All fungi produce hydroxamate-type siderophore, except Mucorales fungi, which secrete a polycarboxylate siderophore, rhizoferrin. However, they also can utilize hydroxamate siderophores as xenosiderophores. In the Mucor genome database, one putative sit1 gene encoding a ferrioxamine B transporter, which belongs to the ARN/SIT subfamily of the major facilitator superfamily (MFS), coding was found. In Saccharomyces cerevisiae, Candia albicans, and Aspergillus fumigatus, siderophore-iron chelates are taken up through Arn3/Sit1 transporters. The relative transcript level of sit1 gene was measured after iron-starvation, and different siderophore treatments using quantitative real-time PCR. We have started to create and characterize (growth and germination ability, in vivo virulence assay, etc.) a sit1 knock-out mutant constructed using a CRISPR-Cas9 system. Sit1 gene showed significantly increased transcript levels using deferoxamine-iron, deferasirox-iron, and enterobactin-iron complexes. The colony diameter of the mutant strain was measured on Blood and Cas agar with (CasFe) or without iron (Cas-Fe). On CasFe agar, the growth ability of the mutant was significantly decreased compared to the control strain. In Galleria mellonella model, disruption of the sit1 gene resulted in decreased virulence. Relative transcript level of the putative rhizoferrin synthase gene (rfs) was measured in the sit1 knock-out mutant, where the gene showed an increased transcript level. Our result suggested that Sit1 is required for germination and virulence of Mucor lusitanicus and it may influence rhizoferrin production. This study was supported by the NKFI project K131796 and the grant ITM NKFIA TKP-2021-EGA-28. GN is grateful for the support of the Premium Postdoctoral Fellowship Program of the Hungarian Academy of Sciences (460 050).