CUR - The Council on Undergraduate Research

Investigating the link between pH regulation and membrane trafficking in Cryptococcus neoformans.

Allyson Hardin1, Jiyoung Kang2, Kristen John1, J. Andrew Alspaugh3, and Michael S. Price2,3 1Dept. of Biology and Chemistry and 2Dept. of Molecular and Cellular Sciences, Liberty University, Lynchburg, VA, 3Department of Medicine, Duke University.

Cryptococcus neoformans initiates infection via fungal pneumonia in the lungs. This is often misdiagnosed as a bacterial pneumonia as they are much more common. C. neoformans has the unique capability to cross the blood-brain barrier, allowing it to enter the central nervous system (CNS). Once there it causes meningoencephalitis, which is often fatal due to difficulties in treatment. This is a serious condition in those that are immunocompromised as they are more susceptible to infection. When C. neoformans enters the more neutral/alkaline environment of the human host, the Rim pathway senses this change and alters gene expression in order to survive. Therefore, the Rim pathway is an attractive target for mitigating disease. A published study identified C. neoformans gene CNAG_02291 as associated with survival at alkaline pH. My goal is to delete this gene in the wild-type C. neoformans strain KN99α. This will be done by fusing the 5’ and 3’ flanking sequence for the gene (~1kb each) with the NEO resistance marker for G418 resistance by overlap PCR as described elsewhere (Davidson et al., 2002). Following gel purification of the fused knockout construct, we will introduce the construct into the wild-type C. neoformans strain KN99α. The CNAG_02291 gene should be replaced by the NEO resistance gene by homologous recombination, resulting in a gene deletion. Virulence and changes in pH-related phenotypes will be assessed in order to determine the function of the gene in the Rim pathway. 

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