Identification of Isoform-Specific Intersectin Mutants in Human Pathogenic Fungus Cryptococcus neoformans
Publication Date
July 2019
Location
LSU Health Medical Education Building
Document Type
Abstract
Start Date
26-7-2019 9:00 AM
End Date
26-7-2019 12:00 PM
Description
Cryptococcus neoformans is an encapsulated basidiomycetous fungus that is prevalently found in the environment including the fecal matter of avians, especially of pigeons. C. neoformans can infect immunocompromised and healthy individuals leading to the development of life-threatening meningoencephalitis and lung infections. In fact, cryptococcosis is the fourth most common opportunistic infection in HIV patients. Immunosuppressed individuals, such as those who have undergone organ transplants, are also at a leveled risk of infection. As a result of limited treatment options, there are high mortality and morbidity rates amongst infected individuals. Reaching a greater understanding of the underlying pathogenesis mechanisms of C. neoformans is crucial to the development of potential treatment options. Virulence factors including melanin, a polysaccharide capsule, and extracellular urease are critical to the virulence of C. neoformans. It is understood that the secretion of these virulence factors to the cell surface is made possible by means of intracellular tracking. During hematogenous dissemination, C. neoformans displays an inclination for the host brain through its ability to infiltrate the usually impermeable blood-brain barrier to enter the central nervous system. However, uncertainty exists on the mechanisms adopted by the fungi to thrive within the host CNS after entry. Previous research has identified cryptococcal intersectin 1 (Cin1) as a novel endocytic protein significant to C. neoformans growth, virulence, and intracellular trafficking. Cin1 contains multiple domains and is expressed as two isoforms, Cin1-S (short isoform) and Cin1-L (long isoform), as the result of alternate mRNA splicing. Interestingly, Cin1¬L is highly homologous to human CNS-specific intersectin 1, ITSN1, whose expression of the long isoform ITSN1-L is CNS-specific. Given that Cin1-L shares high sequence homology with ISNT1-1 and Cin1-S was previously shown to have a significant survival advantage in the CNS through a murine model of cryptococcosis, we hypothesize that Cin1 and its isoform formation may play a role in infection, especially in CNS. To test this hypothesis, we plan to create a Cin1-L mutant for infection in comparison to Cin1-S. Cryptococcus neoformans wild type strain JEC21 was previously transformed with the CIN1-L allele linked to G418 drug resistance and 57 transformants were collected. The mutant strains were screened using colony-PCR amplification of the CIN1-L allele by DNA sequencing. The 30 initially screened transformants displayed either the wild type sequence or both the wild type and the CIN1-L allele, and none contained the CIN1-L allele alone. Screening of the remaining transformants are in progress. Once the Cin1-L mutants are obtained, they will be used to infect the mouse model. By comparing with infection of Cin1-S mutant, our study will help to examine the mechanisms of the neurotropic property and pathogenesis of C. neoformans.
Recommended Citation
Hill, Haley L., "Identification of Isoform-Specific Intersectin Mutants in Human Pathogenic Fungus
Cryptococcus neoformans" (2019). Summer Research Internship Program. 15.
https://digitalscholar.lsuhsc.edu/srip/2019/undergrad/15
Identification of Isoform-Specific Intersectin Mutants in Human Pathogenic Fungus Cryptococcus neoformans
LSU Health Medical Education Building
Cryptococcus neoformans is an encapsulated basidiomycetous fungus that is prevalently found in the environment including the fecal matter of avians, especially of pigeons. C. neoformans can infect immunocompromised and healthy individuals leading to the development of life-threatening meningoencephalitis and lung infections. In fact, cryptococcosis is the fourth most common opportunistic infection in HIV patients. Immunosuppressed individuals, such as those who have undergone organ transplants, are also at a leveled risk of infection. As a result of limited treatment options, there are high mortality and morbidity rates amongst infected individuals. Reaching a greater understanding of the underlying pathogenesis mechanisms of C. neoformans is crucial to the development of potential treatment options. Virulence factors including melanin, a polysaccharide capsule, and extracellular urease are critical to the virulence of C. neoformans. It is understood that the secretion of these virulence factors to the cell surface is made possible by means of intracellular tracking. During hematogenous dissemination, C. neoformans displays an inclination for the host brain through its ability to infiltrate the usually impermeable blood-brain barrier to enter the central nervous system. However, uncertainty exists on the mechanisms adopted by the fungi to thrive within the host CNS after entry. Previous research has identified cryptococcal intersectin 1 (Cin1) as a novel endocytic protein significant to C. neoformans growth, virulence, and intracellular trafficking. Cin1 contains multiple domains and is expressed as two isoforms, Cin1-S (short isoform) and Cin1-L (long isoform), as the result of alternate mRNA splicing. Interestingly, Cin1¬L is highly homologous to human CNS-specific intersectin 1, ITSN1, whose expression of the long isoform ITSN1-L is CNS-specific. Given that Cin1-L shares high sequence homology with ISNT1-1 and Cin1-S was previously shown to have a significant survival advantage in the CNS through a murine model of cryptococcosis, we hypothesize that Cin1 and its isoform formation may play a role in infection, especially in CNS. To test this hypothesis, we plan to create a Cin1-L mutant for infection in comparison to Cin1-S. Cryptococcus neoformans wild type strain JEC21 was previously transformed with the CIN1-L allele linked to G418 drug resistance and 57 transformants were collected. The mutant strains were screened using colony-PCR amplification of the CIN1-L allele by DNA sequencing. The 30 initially screened transformants displayed either the wild type sequence or both the wild type and the CIN1-L allele, and none contained the CIN1-L allele alone. Screening of the remaining transformants are in progress. Once the Cin1-L mutants are obtained, they will be used to infect the mouse model. By comparing with infection of Cin1-S mutant, our study will help to examine the mechanisms of the neurotropic property and pathogenesis of C. neoformans.
Comments
Mentor: Ping Wang, PhD, Department of Microbiology, Immunology, and Parasitology