P443 Altered expression of fungal CotH, human glucose-regulated protein 78 (GRP78), and predicted miRNAs in macrophages and model diabetic mice infected with Rhizopus oryzae

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectives Rhizopus oryzae is one of the most common causes of mucormycosis. Among the virulence factors of the Mucorales, CotH protein has recently been identified, which causes the invasion of R. oryzae into endothelial cells. In this study, we aimed to examine the reaction between GRP78 at the level of human cells and different groups of mice and CotH3 at the surface of the R. oryzae hyphae. We evaluated the relative expression of GRP78 and CotH3 genes and changes in the expression of some miRNAs that target the human GRP78 gene.MethodsIn this study, the relative changes in gene expression were studied. In three groups (1) Macrophages derived from human monocytes: monocytes from the blood of healthy donors were isolated using Ficoll and in RPMI 1640 medium containing FCS 10% and with penicillin-streptomycin after 2 weeks were differentiated into macrophages. Two groups were investigated, including control and infected with R. oryzae hyphae, for 6 and 16 hours after infection. (2) Hematogenous dissemination mucormycosis model: Seven groups of male BALb/c mice were examined in control, infected, and treated groups with Liposomal amphotericin B. (3) Human mucormycosis: in this study, two samples of patients with rhinocerebral mucormycosis with diabetes mellitus treated and untreated with liposomal amphotericin B were examined. Total RNA extraction and cDNA synthesis were performed from the studied samples. The relative expression changes of the target genes and miRNAs were evaluated using real-time PCR carried out using Sybrgreen-based detection methods.ResultsMonocyte-derived macrophages had a steady pattern in relative changes in gene expression. An increase in expression of two genes, GRP78 and CotH3, was observed in the samples, and all miRNAs targeted by the GRP78 gene included hsa - miR-16-5p; hsa -miR-335-5p and hsa -miR-93-3p showed a decreasing pattern. In the mice mucormycosis model, relative gene expression changes were observed, and mmu-miR-181b-5p showed increased expression deviation in all groups. The clinical sample of diabetic patients with untreated rhinocerebral mucormycosis also had a consistent pattern of GRP78 and CotH3 increased gene expression. The hsa-miR-16-5p and hsa-miR-335-5p have increased expression, while hsa-miR-93-3p decreased expression.ConclusionAfter validation, these micro RNAs can be used as valuable markers in mucormycosis detection and treatment processes.