Matthew Z. Anderson

Genetic and phenotypic intra-species variation in Candida albicans

Candida albicans is a commensal fungus of the human gastrointestinal tract and a prevalent opportunistic pathogen. To examine diversity within this species, extensive genomic and phenotypic analyses were performed on 21 clinical C. albicans isolates. Genomic variation was evident in the form of polymorphisms, copy number variations, chromosomal inversions, subtelomeric hypervariation, loss of heterozygosity (LOH), and whole or partial chromosome aneuploidies. All 21 strains were diploid, although karyotypic changes were present in eight of the 21 isolates, with multiple strains being trisomic for Chromosome 4 or Chromosome 7. Aneuploid strains exhibited a general fitness defect relative to euploid strains when grown under replete conditions. All strains were also heterozygous, yet multiple, distinct LOH tracts were present in each isolate. Higher overall levels of genome heterozygosity correlated with faster growth rates, consistent with increased overall fitness. Genes with the highest rates of amino acid substitutions included many cell wall proteins, implicating fast evolving changes in cell adhesion and host interactions. One clinical isolate, P94015, presented several striking properties including a novel cellular phenotype, an inability to filament, drug resistance, and decreased virulence. Several of these properties were shown to be due to a homozygous nonsense mutation in the EFG1 gene. Furthermore, loss of EFG1 function resulted in increased fitness of P94015 in a commensal model of infection. Our analysis therefore reveals intra-species genetic and phenotypic differences in C. albicans and delineates a natural mutation that alters the balance between commensalism and pathogenicity.

The Three Clades of the Telomere-Associated TLO Gene Family of Candida albicans Have Different Splicing, Localization, and Expression Features

ABSTRACT Candida albicans grows within a wide range of host niches, and this adaptability enhances its success as a commensal and as a pathogen. The telomere-associated TLO gene family underwent a recent expansion from one or two copies in other CUG clade members to 14 expressed copies in C. albicans. This correlates with increased virulence and clinical prevalence relative to those of other Candida clade species. The 14 expressed TLO gene family members have a conserved Med2 domain at the N terminus, suggesting a role in general transcription. The C-terminal half is more divergent, distinguishing three clades: clade α and clade β have no introns and encode proteins that localize primarily to the nucleus; clade γ sometimes undergoes splicing, and the gene products localize within the mitochondria as well as the nuclei. Additionally, TLOα genes are generally expressed at much higher levels than are TLOγ genes. We propose that expansion of the TLO gene family and the predicted role of Tlo proteins in transcription regulation provide C. albicans with the ability to adapt rapidly to the broad range of different environmental niches within the human host.

A Pseudouridine Synthase Homologue Is Critical to Cellular Differentiation in Toxoplasma gondii

ABSTRACT Toxoplasma gondii is a haploid protozoan parasite infecting about one in seven people in the United States. Key to the worldwide prevalence of T. gondii is its ability to establish a lifelong, chronic infection by evading the immune system, and central to this is the developmental switch between the two asexual forms, tachyzoites and bradyzoites. A library of mutants defective in tachyzoite-to-bradyzoite differentiation (Tbd−) was created through insertional mutagenesis. This library contains mutants that, compared to the wild type, are between 20% and 74% as efficient at stage conversion. Two mutants, TBD5 and TBD8, with disruptions in a gene encoding a putative pseudouridine synthase, PUS1, were identified. The disruption in TBD8 is in the 5′ end of the PUS1 gene and appears to produce a null allele with a 50% defect in differentiation. This is about the same switch efficiency as obtained with an engineered pus1 deletion mutant (Δpus1). The insertion in TBD5 is within the PUS1 coding region, and this appears to result in a more extreme phenotype of only ∼10% switch efficiency. Complementation of TBD8 with the genomic PUS1 allele restored wild-type differentiation efficiency. Infection of mice with pus1 mutant strains results in increased mortality during the acute phase and higher cyst burdens during the chronic infection, demonstrating an aberrant differentiation phenotype in vivo due to PUS1 disruption. Our results suggest a surprising and important role for RNA modification in this biological process.

Telomeric ORFs (TLOs) in Candida spp. Encode Mediator Subunits That Regulate Distinct Virulence Traits

The TLO genes are a family of telomere-associated ORFs in the fungal pathogens Candida albicans and C. dubliniensis that encode a subunit of the Mediator complex with homology to Med2. The more virulent pathogen C. albicans has 15 copies of the gene whereas the less pathogenic species C. dubliniensis has only two (CdTLO1 and CdTLO2). In this study we used C. dubliniensis as a model to investigate the role of TLO genes in regulating virulence and also to determine whether TLO paralogs have evolved to regulate distinct functions. A C. dubliniensis tlo1Δ/tlo2Δ mutant is unable to form true hyphae, has longer doubling times in galactose broth, is more susceptible to oxidative stress and forms increased levels of biofilm. Transcript profiling of the tlo1Δ/tlo2Δ mutant revealed increased expression of starvation responses in rich medium and retarded expression of hypha-induced transcripts in serum. ChIP studies indicated that Tlo1 binds to many ORFs including genes that exhibit high and low expression levels under the conditions analyzed. The altered expression of these genes in the tlo1Δ/tlo2Δ null mutant indicates roles for Tlo proteins in transcriptional activation and repression. Complementation of the tlo1Δ/tlo2Δ mutant with TLO1, but not TLO2, restored wild-type filamentous growth, whereas only TLO2 fully suppressed biofilm growth. Complementation with TLO1 also had a greater effect on doubling times in galactose broth. The different abilities of TLO1 and TLO2 to restore wild-type functions was supported by transcript profiling studies that showed that only TLO1 restored expression of hypha-specific genes (UME6, SOD5) and galactose utilisation genes (GAL1 and GAL10), whereas TLO2 restored repression of starvation-induced gene transcription. Thus, Tlo/Med2 paralogs encoding Mediator subunits regulate different virulence properties in Candida spp. and their expansion may account for the increased adaptability of C. albicans relative to other Candida species.

Silencing Is Noisy: Population and Cell Level Noise in Telomere-Adjacent Genes Is Dependent on Telomere Position and Sir2

Cell-to-cell gene expression noise is thought to be an important mechanism for generating phenotypic diversity. Furthermore, telomeric regions are major sites for gene amplification, which is thought to drive genetic diversity. Here we found that individual subtelomeric TLO genes exhibit increased variation in transcript and protein levels at both the cell-to-cell level as well as at the population-level. The cell-to-cell variation, termed Telomere-Adjacent Gene Expression Noise (TAGEN) was largely intrinsic noise and was dependent upon genome position: noise was reduced when a TLO gene was expressed at an ectopic internal locus and noise was elevated when a non-telomeric gene was expressed at a telomere-adjacent locus. This position-dependent TAGEN also was dependent on Sir2p, an NAD+-dependent histone deacetylase. Finally, we found that telomere silencing and TAGEN are tightly linked and regulated in cis: selection for either silencing or activation of a TLO-adjacent URA3 gene resulted in reduced noise at the neighboring TLO but not at other TLO genes. This provides experimental support to computational predictions that the ability to shift between silent and active chromatin states has a major effect on cell-to-cell noise. Furthermore, it demonstrates that these shifts affect the degree of expression variation at each telomere individually.

Real-Time Evolution of a Subtelomeric Gene Family in Candida albicans

Subtelomeric regions of the genome are notable for high rates of sequence evolution and rapid gene turnover. Evidence of subtelomeric evolution has relied heavily on comparisons of historical evolutionary patterns to infer trends and frequencies of these events. Here, we describe evolution of the subtelomeric TLO gene family in Candida albicans during laboratory passaging for over 4000 generations. C. albicans is a commensal and opportunistic pathogen of humans and the TLO gene family encodes a subunit of the Mediator complex that regulates transcription and affects a range of virulence factors. We identified 16 distinct subtelomeric recombination events that altered the TLO repertoire. Ectopic recombination between subtelomeres on different chromosome ends occurred approximately once per 5000 generations and was often followed by loss of heterozygosity, resulting in the complete loss of one TLO gene sequence with expansion of another. In one case, recombination within TLO genes produced a novel TLO gene sequence. TLO copy number changes were biased, with some TLOs preferentially being copied to novel chromosome arms and other TLO genes being frequently lost. The majority of these nonreciprocal recombination events occurred either within the 3′ end of the TLO coding sequence or within a conserved 50-bp sequence element centromere-proximal to TLO coding sequence. Thus, subtelomeric recombination is a rapid mechanism of generating genotypic diversity through alterations in the number and sequence of related gene family members.

Human Genetic Determinants of Viral Diseases

Much progress has been made in the identification of specific human gene variants that contribute to enhanced susceptibility or resistance to viral diseases. Herein we review multiple discoveries made with genome-wide or candidate gene approaches that have revealed significant insights into virus-host interactions. Genetic factors that have been identified include genes encoding virus receptors, receptor-modifying enzymes, and a wide variety of innate and adaptive immunity-related proteins. We discuss a range of pathogenic viruses, including influenza virus, respiratory syncytial virus, human immunodeficiency virus, human T cell leukemia virus, human papilloma virus, hepatitis B and C viruses, herpes simplex virus, norovirus, rotavirus, parvovirus, and Epstein-Barr virus. Understanding the genetic underpinnings that affect infectious disease outcomes should allow tailored treatment and prevention approaches in the future.

A Multistate Toggle Switch Defines Fungal Cell Fates and Is Regulated by Synergistic Genetic Cues

Heritable epigenetic changes underlie the ability of cells to differentiate into distinct cell types. Here, we demonstrate that the fungal pathogen Candida tropicalis exhibits multipotency, undergoing stochastic and reversible switching between three cellular states. The three cell states exhibit unique cellular morphologies, growth rates, and global gene expression profiles. Genetic analysis identified six transcription factors that play key roles in regulating cell differentiation. In particular, we show that forced expression of Wor1 or Efg1 transcription factors can be used to manipulate transitions between all three cell states. A model for tristability is proposed in which Wor1 and Efg1 are self-activating but mutually antagonistic transcription factors, thereby forming a symmetrical self-activating toggle switch. We explicitly test this model and show that ectopic expression of WOR1 can induce white-to-hybrid-to-opaque switching, whereas ectopic expression of EFG1 drives switching in the opposite direction, from opaque-to-hybrid-to-white cell states. We also address the stability of induced cell states and demonstrate that stable differentiation events require ectopic gene expression in combination with chromatin-based cues. These studies therefore experimentally test a model of multistate stability and demonstrate that transcriptional circuits act synergistically with chromatin-based changes to drive cell state transitions. We also establish close mechanistic parallels between phenotypic switching in unicellular fungi and cell fate decisions during stem cell reprogramming.

A chromosome 4 trisomy contributes to increased fluconazole resistance in a clinical isolate of Candida albicans

Candida albicans is an important opportunistic fungal pathogen capable of causing both mucosal and disseminated disease. Infections are often treated with fluconazole, a front-line antifungal drug that targets the biosynthesis of ergosterol, a major component of the fungal cell membrane. Resistance to fluconazole can arise through a variety of mechanisms, including gain-of-function mutations, loss of heterozygosity events and aneuploidy. The clinical isolate P60002 was found to be highly resistant to azole-class drugs, yet lacked mutations or chromosomal rearrangements known to be associated with azole resistance. Transcription profiling suggested that increased expression of two putative drug efflux pumps, CDR11 and QDR1, might confer azole resistance. However, ectopic expression of the P60002 alleles of these genes in a drug-susceptible strain did not increase fluconazole resistance. We next examined whether the presence of three copies of chromosome 4 (Chr4) or chromosome 6 (Chr6) contributed to azole resistance in P60002. We established that Chr4 trisomy contributes significantly to fluconazole resistance, whereas Chr6 trisomy has no discernible effect on resistance. In contrast, a Chr4 trisomy did not increase fluconazole resistance when present in the standard SC5314 strain background. These results establish a link between Chr4 trisomy and elevated fluconazole resistance, and demonstrate the impact of genetic background on drug resistance phenotypes in C. albicans.

Growth and manipulation of a human T-cell leukemia virus type 2 full-length molecular clone.

In retrovirus research, the generation of an infectious molecular clone is a landmark event, opening up new avenues of research using the cloned virus. A full-length proviral plasmid clone of the human T-cell leukemia virus (HTLV) makes possible reproducible viral genetic studies. However, the growth of full-length infectious HTLV proviral plasmid clones in bacteria, their manipulation using molecular techniques, and further characterization of replication capacity and other biological properties are not trivial. This chapter describes successful methods used for the preparation and manipulation of the full-length HTLV-2 proviral plasmid clone pH6neo. The plasmid-borne full-length clone of HTLV-2 permits the study of the interactions and contributions of viral proteins in viral replication and cellular transformation in vitro and in animal models in vivo. These types of studies have provided and will continue to provide critical insight into understanding the virus-host interactions and ultimately the contribution of viral genes and elements to the pathogenesis of HTLV.