Integrated Activity and Genetic Profiling of Secreted Peptidases in Cryptococcus neoformans Reveals an Aspartyl Peptidase Required for Low pH Survival and Virulence

Abstract

The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence virulence, but in most cases the substrate specificity and regulation of these enzymes remains poorly understood. The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets. We report here an unbiased analysis of secreted peptidase activity and specificity in C. neoformans using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations. Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions. To uncover the substrate preferences of individual enzymes and interrogate their biological functions, we constructed and profiled a ten-member gene deletion collection of candidate secreted peptidases. Through this deletion approach, we characterized the substrate specificity of three peptidases within the context of the C. neoformans secretome, including an enzyme known to be important for fungal entry into the brain. We selected a previously uncharacterized peptidase, which we term Major aspartyl peptidase 1 (May1), for detailed study due to its substantial contribution to extracellular proteolytic activity. Based on the preference of May1 for proteolysis between hydrophobic amino acids, we screened a focused library of aspartyl peptidase inhibitors and identified four high-affinity antagonists. Finally, we tested may1Δ strains in a mouse model of C. neoformans infection and found that strains lacking this enzyme are significantly attenuated for virulence. Our study reveals the secreted peptidase activity and specificity of an important human fungal pathogen, identifies responsible enzymes through genetic tests of their function, and demonstrates how this information can guide the development of high affinity small molecule inhibitors.

Aspartic peptidase of Phialophora verrucosa as target of HIV peptidase inhibitors: blockage of its enzymatic activity and interference with fungal growth and macrophage interaction

“…Corroborating this statement, F. pedrosoi conidial cells are able to secrete two distinct classes of extracellular peptidases: aspartic peptidase when grown in chemically defined medium and metallopeptidase when cultivated in Kauffman complex medium 11 . These results corroborate that the medium composition could modulate the synthesis and secretion of proteolytic enzymes as previously observed for other pathogenic fungi including C. neoformans, C. albicans, Scedosporium apiospermum (formerly Pseudallescheria boydii) and Aspergillus fumigatus [37][38][39][40] . Previous studies reported that peptidase activities can change in response to environmental conditions, which are beneficial for microbial cells adaptation, including inside the host 36,37 .…”

“…In this current work, we tested several culture media (data not shown) and the aspartic peptidase activity was detected in concentrated supernatant of P. verrucosa conidia from YNB medium. This same minimal medium was also used to induce aspartic peptidase activity of C. neoformans required for its survival and virulence in acidic environments 37 . It is well known that the composition of the nutrient medium can modulate the synthesis of different bioactive molecules.…”

Role of Amino Acid Metabolism in the Virulence of Human Pathogenic Fungi

“…Further, C. neoformans produces several secreted proteases involved in the utilization of immunoglobulin and complement proteins as the sole nitrogen and carbon source in vitro [74]. The aspartyl proteinase CnMay1 and the carboxypeptidase CnCxd1 have a broad specificity and are considered to supply C. neoformans with free amino acids liberated from extracellular protein sources and a strain lacking CnMAY1 was attenuated in a competition infection assay [75]. The functionality of oligopeptide transporters in C. neoformans virulence has not been investigated, although the fungal genome encodes for six predicted OPTs [76].…”

The structural unit of melanin in the cell wall of the fungal pathogenCryptococcus neoformans

“…Indeed, alterations in the cell-wall chitin/chitosan induced a "leaky melanin" phenotype (31,32,34) or an enhanced deposition of the pigment (14). The recovery of melanin granules in the supernatant of the culture media is explained by normal cell-wall remodeling required for cellular budding (Figures 5A,B; 9A,B), which involves local modifications of the melanin scaffold by secreted peptidases (96), chitinases (97), and glucanases (62) that lead to melanin granules detachment from the cell wall. The model does not address the source of strongly associated lipids with melanin granules, but rather provides new pieces of data that should encourage experiments to continue dissecting the phenomenon of fungal cell wall melanization.…”