Identification of Interkingdom Interactions of Cryptococcus Neoformans in Pigeon Excreta Using Next Generation Sequencing
Cryptococcus neoformans is an encapsulated fungus with worldwide distribution and associated with pigeon excreta. C. neoformans causes pulmonary cryptococcosis in immunocompromised populations, which results in high mortality. C. neoformans main virulence factor is its polysaccharide capsule that confers the fungus with the ability to form biofilms. Interkingdom interactions can have a positive, neutral or negative effect on C. neoformans in relation to its virulence and/or pathogenicity. C. neoformans survival within polymicrobial and other challenging communities has been shown to affect the evolution of its virulence factors. For example, the size of C. neoformans polysaccharide capsule is modulated during interactions with bacteria and/or immune cells suggesting that understanding the fungus environmental interactions may help us understand C. neoformans pathogenesis. Here, we used Next Generation Sequencing (NGS) to identify the microbial populations (e.g. bacterial, fungal and protist) in pigeon excreta in order to better understand C. neoformans evolution of virulence. Pigeon excreta samples were collected in Texas and New York, USA. DNA extraction was conducted using the Zymo Quick-DNA Fecal Microbe Kit. PCR was conducted on the pigeon excreta samples using four different universal sets of primers to identify 16S, 18S and Internal Transcribed Spacer (ITS) regions of microorganism’s chromosomes. After amplification, gel electrophoresis and purification were conducted using 2% agarose gels and PureLink Quick Gel Extraction Kit. Sample preparation was continued using the Illumina 16S protocol for the MiSeq system contained steps like PCR cleanups, indexing, and normalizing bioinformatic analysis of sequencing data was conducting using Qiime 2.0. The sequencing identified C. neoformans in all the samples collected. Other pathogens identified included: Aspergillus flavus, A. niger, Candida Kruisii, C. parapsilosis, C. tropicalis, Lactobacillaceae species, Enterbacteriaceae species, Acetobacteraceae species, and Aranciocystis muskarensis. Further studies are warranted to elucidate the results of complex C. neoformans interactions with other microbes in the environment and the impact of these symbioses in the evolution of virulence and survival mechanisms developed by this fungus in the human host.
Cadena, Marlynn, "Identification of Interkingdom Interactions of Cryptococcus Neoformans in Pigeon Excreta Using Next Generation Sequencing" (2020). ETD Collection for University of Texas, El Paso. AAI28262230.