Structural characterization of the galactomannan polysaccharide in the fungus H. capsulatum and its in silico study of its metabolic synthesis pathway for subsequent investigation as a virulence factor.

BRICEÑO FERNANDEZ V; CUESTAS ML; ORLOWSKI J; NUSBLAT A

Congreso; XIV Congreso de la Asociación Argentina de Bioinformática y Biología Computacional (XIV CA2B2C).; 2024

Histoplasmosis is a pulmonary disease endemic to the Americas caused by the thermallydimorphic fungus Histoplasma capsulatum. In immunosuppressed individuals, such as those with HIV/AIDS or organ transplants, it can lead to severe clinical manifestations. A key virulence factor of H. capsulatum is galactomannan (GM), a cell wall polysaccharide composed of mannose and galactofuranose. The furanose configuration of galactose, absent in mammals, is responsible for its antigenicity. Despite its discovery over 50 years ago, the biological function, metabolic pathways, and genes involved in GM metabolism have been minimally explored in H. capsulatum.In this study, we characterized GM chemically and bioinformatically in both the yeast andmycelial phases of H. capsulatum. We also identified the putative metabolic pathwayresponsible for GM synthesis and a molecular target as the first step toward generating a GMdeficient strain to assess its role in virulence. GM was purified using ultrafiltration, proteolytic digestion, and ion exchange resins, achieving ≥95% purity. Structural analysis using GPCHPLC, NMR, and gas chromatography confirmed a 1:1 mannose-to-β-D-galactofuranose ratio with a molecular weight of 29 kDa.Bioinformatic analysis identified orthologs of GM synthesis genes from Aspergillus fumigatus, a phylogenetically related fungus. Thirteen putative genes corresponding to four stages of the GM synthesis pathway were found: mannose chain synthesis (cmsa, cmsb, gmt1, and Anp), galactofuran synthesis (ugm1, glfb, gfsa, and UgeA), UDP-Galp formation pathway (Uge3, Uge4, Uge5, and UGP1), and cell wall anchoring (Dfg3). A comparative analysis across 33 fungal genomes revealed gene divergences in the GM pathway, constructing a cladogram rooted with a non-GM-producing bacterium as an outgroup. Transcriptomic studies highlighted UDP-galactopyranose mutase as essential for GM synthesis. Given its absence in mammals, we aim to generate a strain deficient in this enzyme and study its pathogenicity in vitro and in vivo.