| Congresso Brasileiro de Microbiologia 2023 | Resumo: 123-1 | ||||
Resumo:Cryptococcal meningitis is a serious fungal disease responsible for 181,000 deaths annually. C. neoformans is the main etiological agent of cryptococcosis in immunocompromised individuals. Diverse signaling cascades contribute to the pathogen's survival within the host environment and the successful establishment of infection. Virulence determinants allow microorganisms to adapt to the host environment, thus exacerbating the infection's severity. The calcium calcineurin signaling pathway is one of the main cascades responsible for C. neoformans virulence and survival. Calmodulin protein senses calcium influx into the cytosol and activates calcineurin phosphatase that in turn phosphorylates different targets that will act regulating transcriptional and post-transcriptional processes. Crz1 is one of the main calcineurin targets and it is involved in regulating the expression of different genes that pertain to C. neoformans survival and adaptation. Through an in silico workflow involving differential gene expression and system biology analysis we have selected a potential new component of this pathway not yet characterized. The target functional enrichment analysis and transcriptomic data suggested that its function could be related to basal cellular processes and stress responses, respectively. The target relative mRNA levels were measured by qPCR for different mutants of calcineurin pathway components (pmc1Δ and crz1Δ) in distinct conditions. Expression results have shown that the target is upregulated in cryptococcal cells exposed to host temperature. A mutant strain of the selected target was constructed, and in vitro phenotypic tests were performed to explore possible functions of the gene of interest. These tests include spot plate assay in the presence of cell wall and cell membrane stressors, osmotic stressor, antifungal drugs, calcineurin inhibitor (FK506), different pH ranges and oxidative stressor at 30°C, at 37°C and at 37oC + 5%CO2; viability and capsule induction in DMEM at 37oC + 5% CO2 during 24h, 48h and 72h; viability in in RPMI 2X containing FK506 at 30oC and 37oC; titan cell induction in induction media at 37ºC for 72h and virulence assay in an invertebrate Tenebrio molitor model. Spot plate assay results were not able to specify a mutant strain phenotype compared to wildtype. However, minimal growth differences were noticed in YPD with different alcohol range concentrations and FK506. Considering these findings, fungal viability was estimated by CFU counts after growth in RPMI 2X media with FK506 concentration ranges at 30°C and at 37oC. No statistically significant differences between mutant and wildtype strain growth were found for most of the tested ranges. Also, the viability of mutant cells appeared to be affected after growth in DMEM at 37°C + 5%CO2. Furthermore, a thicker capsule was observed for the mutant strain when compared to the wild type strain. For titan cell induction and invertebrate virulence assays, no statistically significant differences were found between strains. Considering these findings, the target gene seems to be related to different fungal stress responses. Going forward, further phenotypic tests should be performed, including assessment of expression profile in the mutant strain of genes related to various stress responses and virulence determinants production. Additionally, differences in intracellular calcium concentration and macrophage interaction should be determined between strains. Palavras-chave: Cryptococcus, CRYPTOCOCCOSIS, CALCIUM-CALCINEURIN PATHWAY, BIOINFORMATICS Agência de fomento:CAPES E FAPERGS | |||||