Beyond Melanin: Proteomics Reveals Virulence-Related Proteins in Paracoccidioides brasiliensis and Paracoccidioides lutzii Yeast Cells Grown in the Presence of L-Dihydroxyphenylalanine

Abstract

Species of the genus Paracoccidioides cause a systemic infection in human patients. Yeast cells of Paracoccidioides spp. produce melanin in the presence of L-dihydroxyphenylalanine and during infection, which may impact the pathogen’s survival in the host. To better understand the metabolic changes that occur in melanized Paracoccidioides spp. cells, a proteomic approach was performed to compare melanized and non-melanized Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells. Melanization was induced using L-dihydroxyphenylalanine as a precursor, and quantitative proteomics were performed using reversed-phase nano-chromatography coupled to high-resolution mass spectrometry. When comparing melanized versus non-melanized cells, 1006 and 582 differentially abundant/detected proteins were identified for P. brasiliensis and P. lutzii, respectively. Functional enrichment and comparative analysis revealed 30 important KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways in melanized P. brasiliensis and 18 in P. lutzii, while differentially abundant proteins from non-melanized cells from these species were involved in 21 and 25 enriched pathways, respectively. Melanized cells presented an abundance of additional virulence-associated proteins, such as phospholipase, proteases, superoxide dis-mutases, heat-shock proteins, adhesins, and proteins related to vesicular transport. The results suggest that L-dihydroxyphenylalanine increases the virulence of Paracoccidioides spp. through complex mechanisms involving not only melanin but other virulence factors as well.

Fungal Melanin and the Mammalian Immune System

“…Melanized P. brasiliensis is also highly resistant to NO, ROS, hypochlorite, and H 2 O 2 [ 116 ]. A recent proteomic analysis comparing melanized and non-melanized P. brasiliensis and P. lutzii revealed that melanization leads to an abundance of virulence-associated proteins, including heat-shock proteins, vesicular transport proteins, adhesins, superoxide dismutases, proteases, and phospholipases, which further underscores the complex mechanisms that occur along with melanin production to subvert the host [ 117 ]. As with cryptococcosis, melanin-binding antibodies are generated during murine as well as human infection with P. brasiliensis [ 114 ].…”

Updates in Paracoccidioides Biology and Genetic Advances in Fungus Manipulation

“…In addition to protease secretion, melanin is also produced as a putative factor that contributes to fungal protection in the host hostile environment. Melanin is a biopolymer produced in several organisms, and, in fungi is commonly associated with protection against several stress conditions [108]. Synthesis can occur by two distinct pathways: through he polysaccharide-synthetase pathway with endogenous substrates, and through phenoloxidases or laccases pathways using phenolic compounds such as L-3,4dihydroxyphenylalanine (L-DOPA) [109].…”