Characterization Of Serine Proteases In Pathogenesis Of Cryptococcus Neoformans

Cryptococcus neoformans, an opportunistic encapsulated yeast, has emerged as the major causative agent of fungal meningoencephalitis in immunocompromised as well as immunocompetent hosts worldwide. C. neoformans is the most common mycological agent of morbidity and mortality in AIDS patients. It has been estimated that there are over 1 million new cases of cryptococcal infection in HIV-infected patients each year, and approximately 625,000 deaths are associated with cryptococcal meningoencephalitis. Several C. neoformans’ features, such as polysaccharide capsule synthesis, melanin production, and growth at host physiological temperatures are associated with virulence. Identification of the essential complex regulatory mechanisms in control of the cryptococcal virulence composite and its networks will be helpful in identifying targets to abrogate disease caused by C. neoformans.Aspartic and serine proteases are two families of proteolytic enzymes present in many unicellular and multicellular eukaryotes, including vertebrates, virus, plants, as well as in fungi. The aspartic proteinases are a family of enzymes involved in a number of important biological processes. In animals, the enzyme renin has a hypertensive action through its role in the renin-angiotensin system. The retroviral aspartic proteinases, such as the HIV proteinase, are essential for maturation of the virus particle and inhibitors have a proven therapeutic record in the treatment of AIDS. They also play major roles in common fungal infections such as candidiasis. The aspartic proteases secreted by Candida albicans are involved in the adherence process and penetration of tissues, and in interactions with the immune system of the infected host. Serine proteases carry out a diverse array of physiological and cellular functions to blood clotting, cellular and humoral immunity, fibrinolysis, fertilization, embryonic development, protein processing and tissue remodeling. In dermatophytes, serine proteases required for adherence to the epidermis.Cryptococcal proteinases are putative virulence factors, but their role in the pathogenesis of C. neoformans is still not clear. Extracellular Serine Proteinase from C. neoformans was Purification and Characterization. Metalloprotease(Mpr1) secreted by C. neoformans play an important role in the invasion of the central nervous system(CNS). C.neoformans serine proteinase can cleave human fibronectin and may leads to BBB disruption during Cryptococcus meningitis.In our study, we compared the transcriptional profiles of C. neoformans under 3 conditions: in vivo CSF, ex vivo CSF, and YPD. Six serine proteases and five aspartic and proteases genes were choosing. To investigate the roles of the 11 genes in growth and virulence of C. neoformans, single and multiple deletions were made used Cre-Lox System which enables deleting multiple genes within a single strain. ser1△,ser2△,ser3△,ser4△,ser5△,ser6△,ser M2△,ser M3△,ser M4△,ser M5△,ser M5△+SER3,ser M5△+SER5,asp1△,asp2△,asp3△,asp4△,asp5△,asp M2△,asp M3△,asp M4△,asp M5△ mutants were constructed.Experiments include testing the mutant strains in capsule synthesis, melanin production, growth at host physiological temperatures, growth effects on nitrogen sources and virulence attributes in animal models were done. Aspartic proteases no phenotype had been observed for several gene deletion strains created so far. This suggests that additional aspartic protease genes need to be deleted to detect phenotypes and that a compensatory mechanism(s) likely exists also for this family of proteases. We focus on ser deletions.We found that Ser5 is required for the production of melanin in C.neoformans. ser M5△ is involved in high salt stress and cell-membrane stress. Ser M5△ shows less urease activity and significantly decreased protease activity. Longer incubation reveals that ser M5? has detectable protease activity. In the tail-vein injection mouse model, reduced fungal burden in brains and lung of mice infected by the ser M5△ strain was observed. The lack of a significant impact on virulence in the mouse inhalation model may be due to the observation that ser M5? can compensate the initial loss of protease activity.The studies of the serine proteases allow us to globally understand the impact of proteases and their genes on cryptococcal pathobiology, but the impacts of proteases on pathogenesis remain poorly understood in Cryptococcus. The location of Ser5 and its role in melanin production and how the interaction between proteases and host can be further studied.