Uncovering virulence pathways facilitated by proteolysis in HIV and a HIV associated fungal pathogen, Cryptococcus neoformans

Peptidases are enzymes that cleave proteins or peptides, irreversibly modifying their substrates. Proteolytic activity is essential for many biological processes, however these enzymes are also associated with many pathological states. In particular, peptidases are frequently used by pathogenic organisms for purposes such as host invasion, immune system evasion and nutrient acquisition. Both HIV and the HIV/AIDS associated fungal pathogen, Cryptococcus neoformans, are known to employ peptidases to promote virulence. HIV expresses a protease that has a well characterized role in maturation of infectious viral particles by cleaving viral polyproteins into their individual constituents. However, in this work we present evidence that this enzyme has a dual purpose. Our results indicate that in addition to processing viral proteins, HIV protease cleaves numerous host-proteins to optimize the host environment for viral replication. We conducted a study of HIV protease host-protein interactions using an affinity purification-mass spectrometry based approach with tagged, inactive HIV protease. Over 50 candidate substrate proteins were identified through this technique. We then individually studied over 40 of these proteins, revealing that eight are cleaved by HIV protease in vitro. Through experiments assaying viral replication in combination with siRNA knock-down of host proteins, we discovered one of these host-proteins, the eukaryotic translation initiation factor 3d (eIF3d) restricts viral reverse transcription. Therefore, we hypothesize that cleavage of this protein by HIV protease may promote reverse transcription during viral infection.In this work we also investigate the secreted peptidases of C. neoformans, an opportunistic fungal pathogen responsible for over 600,000 deaths every year. This organism is known to secrete peptidases but the functions, regulation and characteristics of these enzymes have remained mysterious. We used an unbiased activity based approach to detail the specificity and regulation of extracellular peptidase activity produced by this pathogen grown under two different culture conditions. We pinpoint a principle enzyme responsible for secreted peptidase activity and find that this enzyme is important for tolerance to low pH environments as well as virulence in a mouse model of cryptococcus infection. Using knowledge of the substrate specificity of this enzyme, we screen a focused library of small molecule inhibitors, identifying four potent compounds.Our study of the functions and characteristics of peptidases from viral and fungal pathogens reveals important information regarding the host-pathogen biology of these organisms. Our results may be informative for the development of alternative therapeutic strategies for combating these intractable pathogens. Furthermore, the approaches and techniques we describe are useful for the investigation of proteolytic activity secreted by other disease-causing organisms