Browsing by Subject "Candida albicans"

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    Characterization of the ESCRT pathway in Candida albicans.
    (2010-02) Wolf, Julie Marie
    iv Abstract The human opportunistic pathogen Candida albicans has a signal transduction pathway unique to fungi, called the Rim101 pathway. The Rim101 pathway regulates the proteolytic activation of the transcription factor, Rim101, the activation of which is required for growth at neutral-alkaline pH. Many genes regulated by Rim101 play a role in C. albicans virulence, including genes involved in filamentation, cell wall structure, adhesion, and nutrient acquisition. The Rim101 pathway consists of two complexes: a signaling complex at the plasma membrane and a processing complex inside the cell, and both of these complexes are required for Rim101 activation. Rim101 activation also requires members of a second pathway, the endosomal sorting complex required for transport (ESCRT) pathway. The ESCRT pathway is required to generate multivesicular bodies prior to vesicle fusion with the vacuole. The ESCRT pathway consists of several polyprotein complexes recruited sequentially to the endosomal membrane to generate an intraluminal vesicle. The role of the ESCRT pathway has not been well characterized in C. albicans, and study of the ESCRT pathway is complicated by the secondary effect many ESCRT mutations have on Rim101 processing. These studies sought to separate iv Abstract The human opportunistic pathogen Candida albicans has a signal transduction pathway unique to fungi, called the Rim101 pathway. The Rim101 pathway regulates the proteolytic activation of the transcription factor, Rim101, the activation of which is required for growth at neutral-alkaline pH. Many genes regulated by Rim101 play a role in C. albicans virulence, including genes involved in filamentation, cell wall structure, adhesion, and nutrient acquisition. The Rim101 pathway consists of two complexes: a signaling complex at the plasma membrane and a processing complex inside the cell, and both of these complexes are required for Rim101 activation. Rim101 activation also requires members of a second pathway, the endosomal sorting complex required for transport (ESCRT) pathway. The ESCRT pathway is required to generate multivesicular bodies prior to vesicle fusion with the vacuole. The ESCRT pathway consists of several polyprotein complexes recruited sequentially to the endosomal membrane to generate an intraluminal vesicle. The role of the ESCRT pathway has not been well characterized in C. albicans, and study of the ESCRT pathway is complicated by the secondary effect many ESCRT mutations have on Rim101 processing. These studies sought to separate iv Abstract The human opportunistic pathogen Candida albicans has a signal transduction pathway unique to fungi, called the Rim101 pathway. The Rim101 pathway regulates the proteolytic activation of the transcription factor, Rim101, the activation of which is required for growth at neutral-alkaline pH. Many genes regulated by Rim101 play a role in C. albicans virulence, including genes involved in filamentation, cell wall structure, adhesion, and nutrient acquisition. The Rim101 pathway consists of two complexes: a signaling complex at the plasma membrane and a processing complex inside the cell, and both of these complexes are required for Rim101 activation. Rim101 activation also requires members of a second pathway, the endosomal sorting complex required for transport (ESCRT) pathway. The ESCRT pathway is required to generate multivesicular bodies prior to vesicle fusion with the vacuole. The ESCRT pathway consists of several polyprotein complexes recruited sequentially to the endosomal membrane to generate an intraluminal vesicle. The role of the ESCRT pathway has not been well characterized in C. albicans, and study of the ESCRT pathway is complicated by the secondary effect many ESCRT mutations have on Rim101 processing. These studies sought to separate ESCRT function from Rim101 function, and to investigate ESCRT pathway function in C. albicans virulence. In these studies, ESCRT and Rim101 pathway separation is demonstrated (1) at distinct domains on a single protein known to be part of both pathways by using alanine scanning mutagenesis and (2) at ESCRT pathway complexes by using deletion mutagenesis. The ESCRT pathway is demonstrated here to play a wholly Rim101-independent role in C. albicans virulence.
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    A conservative mechanism of polarization drives hyphal growth in the opportunistic yeast pathogen Candida albicans
    (2014-08) Pulver, Rebecca
    In eukaryotes, different cell morphologies are generated by fine-tuning the spatiotemporal regulation of the polarized growth machinery. Studying hyphal growth in the multimorphic opportunistic yeast pathogen Candida albicans provides a unique opportunity to understand how highly polarized cell structures are generated and maintained, and has the potential to provide insight into mechanisms of pathogenesis. Hyphal cell morphology requires that polarized growth machinery be held at hyphal tips over extreme distances and through multiple cell cycles. Deletion of the bud-site selection GTPase Rsr1 in C. albicans results in defects in cell size and shape not observed in studies of its ortholog in the related yeast Saccharomyces cerevisiae. This suggests that, in addition to its role in bud site selection, Rsr1 has expanded function in C. albicans, which impacts polarized growth and the generation of the hyphal morphology. Here, I show that loss of Rsr1 results in changes to a hyphal-specific tip structure, the Spitzenkörper, a downstream developmental indicator of Cdc42 signaling, and key regulator of polarized growth. Also, my results show that Rsr1's function impacts the spatiotemporal distribution of Bem1 a marker of the active form of Cdc42. Interestingly, the changes in the distribution of Cdc42 activity are also correlated with reduced expression of the hyphal transcriptional program. In addition, I also show the differential effects of the guanine nucleotide binding states of Rsr1, through the manipulation of the Rsr1 GAP, Bud2, and GEF, Bud5. Through the action of Bud2, Rsr1-GDP acts as a global inhibitor that limits competitive, stochastically-activated clusters of Cdc42, and also as a lateral inhibitor of growth at hyphal tips that strongly influences the overall width of the hypha. In contrast, Rsr1-GTP, through Bud5 activity, is needed to efficiently nucleate single clusters of Cdc42 activity during hyphal emergence, and also contributes to the extremely narrow morphology of the hypha. Altogether, the data presented here suggest that Rsr1 cycling supports a conservative mechanism of polarization that optimizes the efficiency with which polarization occurs, which is required for the maintenance of polarized growth.
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    Data supporting "Single-cell detection of copy number changes reveals dynamic mechanisms of adaptation to antifungals in Candida albicans"
    (2024-07-10) Zhou, Xin; Hilk, Audrey; Solis, Norma V; Scott, Nancy; Beach, Annette; Soisangwan, Natthapon; Billings, Clara L; Burrack, Laura S; Filler, Scott G; Selmecki, Anna; Selmecki@umn.edu; Anna Selmecki; University of Minnesota Selmecki lab
    Genomic copy number changes are associated with antifungal drug resistance and virulence across diverse fungal pathogens. Despite the high prevalence of both gain and loss events, the rate and dynamics of these genomic changes in the presence of antifungal drugs is not known for any organism. We optimized a dual-fluorescent reporter system in the diploid pathogen Candida albicans to quantify copy number variation (CNV) and loss of heterozygosity (LOH) at the single cell level with flow cytometry. We followed the rate and dynamics of CNV and LOH at two distinct genomic locations during parallel evolution experiments in the presence and absence of antifungal drugs in vitro and in a murine model of infection. Copy number changes were rapid and dynamic during adaptation to three different concentrations of the azole drug fluconazole. The fluorescent reporters revealed competing sub-populations with distinct genotypes in the drug-evolved lineages. Extensive whole genome sequencing identified recurrent genotypes that cause increased competitive fitness in the presence of antifungal drug. Specifically, at low concentrations of drug, chromosome 3 (Chr3) LOH frequently arose and led to significantly increased competitive fitness. In contrast, at high drug concentrations, Chr3 and Chr6 aneuploidy arose together, and these concurrent aneuploidies conferred multi-azole tolerance. In the murine model, copy number changes were only detected in isolates recovered from mice treated with fluconazole, and included whole-genome duplication events resulting in polyploidy. This study provides the first quantitative evidence for the incredible speed at which diverse genotypes arise and undergo dynamic population-level fluctuations during adaptation to antifungal drugs in vitro and in vivo.  
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    Generation of phenotypic diversity in the fungal pathogen Candida Albicans
    (2010-09) Zacchi, Lucia F.
    Microbial organisms have a diverse array of mechanisms to obtain phenotypic variation. Phenotypic variation not only enhances population fitness and competitiveness for a specific niche but it is also critical for the survival of a population to unexpected environmental changes. Further, in pathogenic organisms, phenotypic variation is directly associated with virulence. Therefore, besides of the contribution to our understanding of microbial evolution, dissecting the mechanisms that lead to phenotypic variation in pathogenic organisms is very clinically relevant. Candida albicans is the most successful opportunistic fungi that infect humans. C. albicans is an obligate diploid yeast with an almost exclusive clonal form of reproduction. In the absence of meiosis to introduce variation in the population, C. albicans needs alternative mechanisms to achieve variability, such as the colony morphology phenotypic switching (CMPS). CMPS is the formation of colonies that have an altered, heritable, and low frequency reversible morphology. CMPS is associated with pathogenesis in C. albicans: variant colony morphologies have been isolated during infections in humans and show an altered expression of diverse virulence factors, including the secretion of hydrolytic enzymes and resistance to antifungal drugs. Despite the potential role of CMPS in the pathogenesis of C. albicans, little is known about the mechanisms that regulate this phenomenon. In our lab, we serendipitously identified a negative regulator of CMPS in C. albicans: the Kelch protein Mds3. Mds3 had been previously associated with other morphogenetic processes in fungi, but the biological role of Mds3 in the cell was unknown. Therefore, my goals in this dissertation were to understand the function of Mds3 in the cell and to use this knowledge to gain insights into C. albican's CMPS mechanisms and regulation. Through a combination of bioinformatic, biochemical, and genetic analyses we found that Mds3 appears to be a large Kelch/BTB cytoplasmic scaffold protein that functions as a regulator of two major signaling cascades, the TOR and Ras pathways (Chapters 2, 3, and 5). With this information, I was able to identify more CMPS regulators that belong to these pathways and environmental signals that regulate CMPS and which are all strongly associated with signaling through these pathways (Chapters 4 and 5). Analyses of morphologically switched mds3delta/delta strains indicated that the phenotypic switch is accompanied by an increase in the sensitivity to the TOR inhibitor rapamycin, which suggests an increased dependence on TOR function in the switched strains. Further, the phenotypic switch was also accompanied by increased sensitivity to genotoxic agents and sometimes also by karyotypic rearrangements and aneuploidies (Chapter 4). Increased DNA damage and genomic instability are mechanisms associated with phenotypic variation in several highly diverse organisms, and could also be mechanisms leading to the phenotypic switch in C. albicans (Chapter 4). Taken together, I propose a model for CMPS in C. albicans in which defects in the signaling through the TOR and Ras signal transduction pathways as cells become nutrient limited and stressed lead to the accumulation of genetic and epigenetic alterations that eventually cause the phenotypic switch.
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    Genome-wide studies of replication origins in Candida albicans reveal their conserved and distinct features
    (2013-04) Tsai, Hung-Ji
    Faithful DNA replication is required for genome inheritance during cell division. To ensure complete duplication of the entire genome, the initiation of replication occurs at multiple loci along the chromosomes. The determinants to regulate where and when DNA replication initiates in higher eukaryotes are little known. Not only the properties of nucleotide sequences, but also the dynamic chromatin structure are highly controlled to define replication origins. The major goal in my doctoral thesis is to understand the features of replication origins and their impact on genome organization in the pathogenic yeast Candida albicans. I combined computational genomics and experimental approaches to address the following research objectives. First, I investigated the distinct replication features of centromeres. Centromere is a specialized chromosomal locus required for chromosome segregation during cell division. Importantly, Candida centromeres are epigenetically defined regional centromeres, similar to centromeres in higher eukaryotes. I found that centromeres constitutively replicate first on each chromosome and this early replication event is linked to the epigenetic nature of centromeres. Furthermore, aligning ORC binding sites with conserved nucleosome depletion patterns throughout the genome revealed the locations of potential chromosomal origins. Strikingly, origin DNA conferred ARS (autonomously replication sequences) function on a linear plasmid vector. Thus, I performed a genome-wide ARS screen to identify the consensus sequences (ACS) for ARS function, and I identified a unique 15 bp ACS motif. This motif is required for origin activity on the plasmid, and it functions in the chromosomal context when associated with appropriately positioned nucleosomes. Thus, despite the presence of a regional, sequence-independent centromere, C. albicans requires a specific sequence motif for replication origin function. Taken together, I comprehensively mapped the origins of replication in the C. albicans genome and characterized their conserved and distinct features. Importantly, the discovery of the ACS motif allowed us to develop the first plasmid shuttle vector for use as a genetic tool in C. albicans. All work here facilitates the study of C. albicans as a promising model organism for understanding fungal pathogenesis and eukaryotic genome organization.
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    Immunity against Candida albicans skin infection
    (2016-05) Kashem, Sakeen
    Candida albicans is a dimorphic commensal fungus that colonizes the healthy human skin, mucosa and reproductive tract. C. albicans is also a predominant opportunistic fungal pathogen, leading to disease manifestations such as disseminated candidiasis and chronic mucocutaneous candidiasis (CMC). The differing host susceptibilities to the sites of C. albicans infection have revealed tissue compartmentalization with tailoring of immune responses based on site of infection. Furthermore, extensive studies of host genetics in rare cases of CMC have identified conserved genetic pathways involved in the immune recognition and response to the extracellular pathogens. In this dissertation, we focus on mouse skin as a site of C. albicans infection and define the mechanisms behind innate and adaptive resistance to C. albicans skin infection. iii