two-infection-assays-to-study-non-lethal-virulence-phenotypes-c

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in <em>C. Albicans</em> using <em>C. Elegans</em>

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic ...

Emory University

Variation in baseline ploidy is seen throughout the tree of life, yet the factors that determine why one ploidy level is maintained over another remain poorly understood. Experimental evolution studies using asexual fungal microbes with manipulated ploidy levels intriguingly reveals a propensity to return to the historical baseline ploidy, a phenomenon that we term 'ploidy drive'. We evolved haploid, diploid, and polyploid strains of the human fungal pathogen Candida albicans under three different nutrient limitation environments to test whether these conditions, hypothesized to select for low ploidy levels, could counteract ploidy drive. Strains generally maintained or acquired smaller genome sizes size (measured as total nuclear DNA through flow cytometry) in minimal medium and under phosphorus depletion compared to in a complete medium, while mostly maintained or acquired increased genome sizes under nitrogen depletion. Improvements in fitness often ran counter to changes in genome size; in a number of scenarios lines that maintained their original genome size often increased in fitness more than lines that converged towards diploidy. Combined, this work demonstrates a role for both the environment and genotype in determination of the rate of ploidy drive, and highlights questions that remain about the force(s) that cause genome size variation. This article is protected by copyright. All rights reserved.

Ploidy tug-of-war: evolutionary and genetic environments influence the rate of ploidy drive in a human fungal pathogen.

The yeast  is an opportunistic pathogen of humans, meaning that despite commensal interactions with its host, it can transition to a harmful pathogen. While  is the predominant species isolated in the human gastrointestinal mycobiome and is implicated in fungal infection, infections due to non-albicans  species are rapidly rising. Studying the factors that contribute to virulence is often challenging and frequently depends on many contexts, including host immune status and pathogen genetic background. Here, we utilize the nematode  as a perspicuous and efficient model host system to study fungal infections of  pathogens. We find that, in addition to reducing lifetime host survival, exposure to  results in delayed reproduction, which significantly reduced lineage growth over multiple generations. Furthermore, we assessed fungal pathogen virulence in  hosts compromised for innate immune function and detected increased early mortality, reduced brood sizes, and delayed reproduction relative to infected healthy hosts. Importantly, by assessing virulence in both healthy and immunocompromised host backgrounds, we reveal the pathogen potential in non-albicans  species. Taken together, we present a novel lineage growth assay to measure reduction in host fitness associated with fungal infection and demonstrate significant interactions between pathogen and host immune function that contribute to virulence. Opportunistic pathogens are commensals capable of causing disease and are serious threats to human health. It is critical to understand the mechanisms and host contexts under which opportunistic pathogens become virulent. In this work, we present a novel assay to quickly and quantitatively measure pathogen virulence in healthy and immunocompromised nematode hosts. We found that  species, one of the most prominent fungal opportunistic pathogens of humans, decrease host fitness by reducing survival and impacting host reproduction. Most importantly, by measuring virulence in hosts that have intact or compromised immune function, we can reveal the pathogenic potential of opportunistic fungal pathogens.

A Novel Virulence Phenotype Rapidly Assesses  Fungal Pathogenesis in Healthy and Immunocompromised  Hosts.

Organismal ploidy and environmental stress impact the rates and types of mutational events. The opportunistic fungal pathogen , serves as a clinically relevant model for studying the interaction between eukaryotic ploidy and drug-induced mutagenesis. In this study, we compared the rates and types of genome perturbations in diploid and tetraploid  following exposure to two different classes of antifungal drugs; azoles and echinocandins. We measured mutations at three different scales: point mutation, loss-of-heterozygosity (LOH), and total DNA content for cells exposed to fluconazole and caspofungin. We found that caspofungin induced higher mutation rates than fluconazole, although this is likely an indirect consequence of stress-associated cell wall perturbations, rather than an inherent genotoxicity. Surprisingly, we found that antifungal drugs disproportionately elevated genome and ploidy instability in tetraploid  compared to diploids. Taken together, our results suggest that the magnitude of stress-induced mutagenesis results from an interaction between ploidy and antifungal drugs. These findings have both clinical and evolutionary implications for how fungal pathogens generate mutations in response to antifungal drug stress and how these mutations may facilitate the emergence of drug resistance.

The Magnitude of  Stress-Induced Genome Instability Results from an Interaction Between Ploidy and Antifungal Drugs.

 is an opportunistic fungal pathogen of humans that is typically diploid yet has a highly labile genome tolerant of large-scale perturbations including chromosomal aneuploidy and loss-of-heterozygosity events. The ability to rapidly generate genetic variation is crucial for  to adapt to changing or stressful environments, like those encountered in the host. Genetic variation occurs via stress-induced mutagenesis or can be generated through its parasexual cycle, in which tetraploids arise via diploid mating or stress-induced mitotic defects and undergo nonmeiotic ploidy reduction. However, it remains largely unknown how genetic background contributes to  genome instability  or in the host environment. Here, we tested how genetic background, ploidy, and the host environment impacts  genome stability. We found that host association induced both loss-of-heterozygosity events and genome size changes, regardless of genetic background or ploidy. However, the magnitude and types of genome changes varied across  strain background and ploidy state. We then assessed if host-induced genomic changes resulted in fitness consequences on growth rate and nonlethal virulence phenotypes and found that many host-derived isolates significantly changed relative to their parental strain. Interestingly, diploid host-associated  predominantly decreased host reproductive fitness, whereas tetraploid host-associated  increased host reproductive fitness. Together, these results are important for understanding how host-induced genomic changes in  alter its relationship with the host. is an opportunistic fungal pathogen of humans. The ability to generate genetic variation is essential for adaptation and is a strategy that  and other fungal pathogens use to change their genome size. Stressful environments, including the host, induce  genome instability. Here, we investigated how  genetic background and ploidy state impact genome instability, both  and in a host environment. We show that the host environment induces genome instability, but the magnitude depends on  genetic background. Furthermore, we show that tetraploid  is highly unstable in host environments and rapidly reduces in genome size. These reductions in genome size often resulted in reduced virulence. In contrast, diploid  displayed modest host-induced genome size changes, yet these frequently resulted in increased virulence. Such studies are essential for understanding how opportunistic pathogens respond and potentially adapt to the host environment.

Host-Induced Genome Instability Rapidly Generates Phenotypic Variation across Candida albicans Strains and Ploidy States.

Studying fungal virulence is often challenging and frequently depends on many contexts, including host immune status and pathogen genetic background. However, the role of ploidy has often been overlooked when studying virulence in eukaryotic pathogens. Since fungal pathogens, including the human opportunistic pathogen , can display extensive ploidy variation, assessing how ploidy impacts virulence has important clinical relevance. As an opportunistic pathogen,  causes nonlethal, superficial infections in healthy individuals, but life-threatening bloodstream infections in individuals with compromised immune function. Here, we determined how both ploidy and genetic background of  impacts virulence phenotypes in healthy and immunocompromised nematode hosts by characterizing virulence phenotypes in four near-isogenic diploid and tetraploid pairs of strains, which included both laboratory and clinical genetic backgrounds. We found that  infections decreased host survival and negatively impacted host reproduction, and we leveraged these two measures to survey both lethal and nonlethal virulence phenotypes across the multiple  strains. In this study, we found that regardless of pathogen ploidy or genetic background, immunocompromised hosts were susceptible to fungal infection compared to healthy hosts. Furthermore, for each host context, we found a significant interaction between  genetic background and ploidy on virulence phenotypes, but no global differences between diploid and tetraploid pathogens were observed.

Article	Year
Ploidy tug-of-war: evolutionary and genetic environments influence the rate of ploidy drive in a human fungal pathogen. Evolution; international journal of organic evolution\| PubMed ID: 28195309	2017
A Novel Virulence Phenotype Rapidly Assesses Fungal Pathogenesis in Healthy and Immunocompromised Hosts. mSphere\| PubMed ID: 30971447	2019
The Magnitude of Stress-Induced Genome Instability Results from an Interaction Between Ploidy and Antifungal Drugs. G3 (Bethesda, Md.)\| PubMed ID: 31585926	2019
Host-Induced Genome Instability Rapidly Generates Phenotypic Variation across Candida albicans Strains and Ploidy States. mSphere\| PubMed ID: 32493724	2020
Virulence phenotypes result from interactions between pathogen ploidy and genetic background. Ecology and evolution\| PubMed ID: 32953064	2020

Meleah A. Hickman

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