Jaqueline
Villanueva Govea
Piezo2 is a mechanosensitive ion channel responsible for many sensational experiences including proprioception and light touch sensitivity. Human Piezo2 has 6 alternatively spliced exons, producing various isoforms with tissue-specific expression differences. However, the effect of alternative splicing in human Piezo2 on intrinsic channel properties remains undiscovered. To address this, I first analyzed electrophysiological data collected in my lab measuring the sensitivity of human Piezo2 isoforms to membrane tension, the physical stimulus these ion channels sense. My analysis showed human Piezo1, human Piezo2, and isoforms of human Piezo2 differ in their sensitivity to membrane tension, suggesting that alternative splicing may control mechanosensing by human Piezo2. Second, I focused on the single channel conductance of human Piezo2 isoforms. Piezo2 has a high level of structural homology compared to Piezo1. The only splice variant of mouse Piezo1, mouse Piezo1.1, lacks amino acids known to form a structure that plugs the pore, thereby modifying its single channel conductance. Since an alternatively spliced exon in Piezo2, exon 33, corresponds to the same amino acids absent in mouse Piezo1.1, I asked if exon 33 in human Piezo2 affects single channel conductance. Through cell-attached patch-clamp electrophysiology, I recorded single channel opening events. I found that a construct lacking all exons (Piezo2Min) has a significantly greater single channel conductance (g = 32.1 ± 3.6 pS) than a construct containing all exons (Piezo2Max) (g = 20.4 ± 2.2 pS). These results indicate that alternative splicing in human Piezo2 modifies single channel conductance in addition to tension sensitivity. My current focus is on investigating the necessity and sufficiency of exon 33 in determining single channel conductance of human Piezo2. These findings indicate that alternative splicing in human Piezo2 is a mechanism of modulation for intrinsic channel properties and is utilized physiologically to meet tissue-specific mechanosensing and channel conductance needs. From Hypothetical to Functional: Characterizing Two Related Mannoproteins in Cryptococcus neoformans.
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Jaqueline Villanueva Govea
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Cryptococcus neoformans is the causative agent of cryptococcal meningoencephalitis, an infection that leads to brain inflammation and meninges. A defining feature of C. neoformans is its extracellular capsule, consisting of polysaccharides, proteins, and other molecules that protect the yeast from environmental stresses and bodily immune defenses. Mannoproteins are examples of glycoproteins also found in the capsule. Mannoproteins in C. neoformans can be immunogenic and stimulate the host's immune protection. Little is known about how mannoproteins contribute to the virulence and survival of C. neoformans in vivo. Our previous transcriptomic work on Cryptococcus gene expression in human cerebral spinal fluid (CSF) has identified several genes encoding mannoproteins with a high abundance of transcripts. This study focuses on two predicted glycosylphosphatidylinositol (GPI)-anchored mannoproteins, CNAG_05424 and CNAG_01272, which are likely paralogs. Strains carrying a single or double gene deletion were constructed to aid in the characterization of these genes in C. neoformans. First, plasmids were constructed to facilitate the deletion of the genes at their native site. Next, sgRNA was designed to allow us to use the CRISPR-CAS9 strategy to construct the deletion strain. We successfully deleted single and double deletion strains lacking CNAG_01272 and CNAG_05424. A CNAG_05424 deletion strains was tested in a mouse inhalation cryptococcosis model to assess its role in virulence. The fungal burden was reduced in mice infected lacking CNAG_05424 compared to the wild type strain (H99). Phenotypic characterization of these strains revealed changes in stress resistance, as observed by reduced colony growth size under stress conditions. These results provide strong evidence that supports the conclusion that CNAG_05424 contributes to fungal survival and host-pathogen interaction. We demonstrate that these previously uncharacterized mannoproteins significantly affect Cryptococcus virulence, further advancing our understanding of genes that contribute to the pathogenesis of Cryptococcus.
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Duke University / 2025
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Jaqueline Villanueva Govea