Jackeline da Silva Luciano ¹, Juliana Cardoso Lacerda¹, Kemily Nunes da Silva Moya¹, Sofia Angiole Cavalcante¹, Fábio César Souza Nogueira², Gilberto Barbosa Domont², Paulo Costa Carvalho³, Priscila Ferreira de Aquino¹

Filamentous fungi that inhabit environmental niches can occasionally colonize homeothermic hosts. However, only a limited number of species possess the molecular flexibility necessary to withstand and thrive at temperatures around 37 °C. This ability to adapt to thermal stress is a key factor in the ecological transition from saprophytic lifestyles to opportunistic pathogenicity. Fusarium solani is well known for its association with human and plant infections, while Trichoderma harzianum, though not typically linked to disease, serves as a valuable comparative model due to its environmental adaptability. Investigating the fungal secretome under thermal stress conditions can provide important insights into metabolic regulation and protein secretion dynamics. So, this study aimed to analyze and compare the secretome profiles of Fusarium solani (CFAM 1313) and Trichoderma harzianum (CFAM 1308) cultivated at 28 °C and 37 °C, in order to identify differentially expressed proteins potentially related to thermal adaptation and pathogenic behavior.Fungal isolates were obtained from the Amazon Fungal Collection (CFAM/ILMD-Fiocruz) and authenticated by morphological and molecular features. Cultures were grown in malt extract liquid medium at 150 rpm for 96 h (28 °C) and 120 h (37 °C). Secretomes were filtered (0.45 µm and 0.22 µm), concentrated (Centripep 3 kDa), and precipitated with TCA-acetone. Proteins were digested with trypsin and analyzed by LC-MS/MS (Orbitrap Velos). The data were processed using PatternLab for Proteomics V software, using the Peptide Spectrum Match (PSM) approach and XIC for protein relative quantification. The differential expression was realized in software R, and functional annotation was performed using Gene Ontology, STRING, and FungiFun3. In F. solani, 62 proteins were detected at 37 °C and 56 at 28 °C, with 28 and 22 being exclusive, respectively. Upregulated proteins at 37 °C included carbonic anhydrase, 1,3-beta-glucanosyltransferase, and copper acquisition factor—linked to pH regulation, cell wall remodeling, and metal ion uptake. Downregulated proteins included aspartic peptidase and peptidase A4, suggesting metabolic reallocation. In T. harzianum, 20 secreted proteins were identified: seven exclusives to 28 °C, four to 37 °C, and nine shared. Upregulated at 28 °C were glycoside hydrolase family 55, purple acid phosphatase, Kre9/Knh1-like protein, copper acquisition factor, and WSC domain-containing protein. At 37 °C, glucan endo-1,3-beta-glucanase, glycosidase, proteinase T, and another glycoside hydrolase family 55 were observed. Shared proteins such as peptidase A1, 1,3-beta-glucanosyltransferase, and acid phosphatase showed temperature-dependent modulation. In F. solani, the presence of proteins linked to pH regulation, stress response, and nutrient uptake at 37 °C suggests early molecular adjustments for survival in host-like environments. In contrast, T. harzianum modulated its secretome without expressing virulence-associated proteins, preserving its saprophytic nature. Although limited in number, the differentially expressed proteins reveal adaptive responses to thermal stress. Carbonic anhydrase and copper acquisition factor stand out as potential markers of pathogenic potential, emphasizing the relevance of environmental fungal monitoring under climatic shifts.

Agradecimentos: The authors thanks FAPEAM (POSGRAD Program), CAPES (Funding Code 001), and PROEP/ILMD-FIOCRUZ AMAZÔNIA – LDMAIS for financial support.