Primary Study On The Role Of Hog1p Gene In The Virulence Control Of Cryptococcus Neoformans Var. Spp.

The pathogenic fungus Cryptococcus neoformans can be found all around the world. Most of Cryptococcosis are caused by Cryptococcus neoformans var. neoformans, Cryptococcus neoformans var. gattii (Cryptococcus gatti) is subsequent but trend to increase year by year. Cryptococcus neoformans is an important opportunistic pathogen that causes disease in immunocompromised individuals. But in our country, most of the cryptococcosis occured in the immumocompetent host. The incidence of cryptococcosis happened in the patients with acquired immunmdeficiency syndrome(AIDS) had increased significantly during the past decades.Currently, the virulence factors of Cryptoccus neoformans which has been confirmed include growth under37℃, enzyme activity, melanin production and capsule. However, the details on the pathogenesis of Cryptoccus neoformans are little known. In order to invade host, survive and reproduce in the host, Cryptococcus neoformans have to deal with high osmotic pressure. In all fungus, pathogenic mechanism of MAPK in Candida albican has been fully described. Probably because MAPK pathway regulates morphologic change during infection:yeast phase converts to hypha phase. By so far, more and more evidence demonstrate that MAPK pathway of Cryptococcus neoformans is similar to Saccharomyces cerevisia’s and Candida albican’s but possesses some new features. Thus, we speculate that hog1p of MAPK maybe play an important role in pathogenesis of Cryptococosis. However, nearly all the study on role of HOG-MAPK pathway in virulence are focused on Cryptococcus neoformans. In this study, we will explore the effect of hog1p on virulence of Cryptococcus gattii by the knockout and reconstruction of hoglp gene and try to discover the difference between Cryptococcus serotype A strain and serotpye B strain.In the study, we have successfully disrupted the hoglp gene in Cryptococcus neoformans enviromental isolate BLS71(serotype A)and Cryptococcus gattii clinical isolate CZ2012(serotpye B) to construct the mutant strains. Then the hoglp locus was reintegrated with the wild-type allele by cotransformation to get the reconstituted strains. Differences in many virulence factors of three different strains, such as stress sensitivity, capsule and melanin production, urease activity, antifungal drug sensitivity were compared. In animal models, healthy female C57BL/6mouse were injected introvanously with0.2ml yeast suspension of an indicated concentration. Then24hours and72hours after injection, tissues of the mouse were taken, homogenated, suspension were recultured on Sabourand medium agar to see if common organs were infected in early stage. For comparing the virulence of different strains, fungal burden in each organ were calculated and the mouse(seven in each group) were monitored daily to analysis role of immunity status of mice and hoglp gene of strain in suivival length of mice. We found that hog1Δ strains exhibited significantly attenuated virulence, high sensitivity to antifungal drugs such as AMB and azoles, and some phenotype changes, such as decreased capsule synthsis and melanin production. Mouse infected with hog1Δ strain and mouse which were immunitycompetent had a longer life span, indicating that hog1mutant strains exhibit attenuated virulence and mouse are more easily infected by Cryptococcus neoformans in immunitycompromised status. However, hog1Δ strains is not signifinantly sensitive to hyperosmolarity stress. Therefore, we conclude that hoglp is an essential virulence composition of Cryptococcus neoformans and hog-MAPK pathway can provide potential targets for antifungal drugs.