Papel dos fatores de transcrição NirA e AreA na assimilação de nitrogênio em Paracoccidioides lutzii

Abstract

The Paracoccidioides complex consists of thermodimorphic fungi, the etiological agents of Paracoccidioidomycosis (PCM), a systemic and endemic mycosis in Latin America. The acquisition of nutrients is one of the factors contributing to its virulence in the host, with nitrogen being one of the key nutrients essential for the survival of the fungus and the production of molecules such as proteins and nucleic acids. In this regard, the pathogen employs various mechanisms for nitrogen acquisition, as the host restricts nutrients through nutritional immunity. One of these mechanisms is Nitrogen Catabolic Repression (NCR), which ensures secondary nitrogen sources are available when preferred sources are not. NCR is mediated by the GATA transcription factor AreA, which is crucial for the successful acquisition of non-preferred nitrogen sources. For the assimilation of nitrate and nitrite, the NirA transcription factor is required, which physically interacts with AreA in Aspergillus nidulans, and is an important regulator of nitrogen assimilation. In the Paracoccidioides complex, this mechanism has not yet been fully elucidated. Thus, we aimed to evaluate the influence of NirA and AreA on the NCR mechanism in P. lutzii. To achieve this, a silenced strain for the AreA gene (AS-AreA) of P. lutzii was developed using antisense RNA technology mediated by Agrobacterium tumefaciens (ATMT). Growth curve analysis by optical density (OD) of the wild-type (WT) and silenced mutant (AS-AreA) strains of P. lutzii in selective medium supplemented with Sodium Nitrate (10mM) as a non-preferred nitrogen source, and Ammonium Sulfate (10mM) as a preferred source, showed that the AreA transcription factor may be related to nitrate assimilation in the Paracoccidioides genus. Additionally, through propidium iodide staining of WT and AS-AreA cells, it was observed that the cells maintained good viability even after 48 hours in medium without a preferred nitrogen source, indicating the presence of alternative mechanisms for nitrogen uptake and maintenance of cellular activities. The analysis of NirA expression by qPCR under the studied conditions revealed overexpression in the AS-AreA strain in nitrate. However, even with overexpression, growth analysis reinforces the importance of AreA for cell growth. Therefore, the preliminary results suggest the importance of the AreA-NirA interaction for nitrate assimilation in the NCR mechanism, but further analyses are needed to corroborate these findings.