Matthew P. Hirakawa

The /`obligate diploid/' Candida albicans forms mating-competent haploids

Candida albicans, the most prevalent human fungal pathogen, is considered to be an obligate diploid that carries recessive lethal mutations throughout the genome. Here we demonstrate that C. albicans has a viable haploid state that can be derived from diploid cells under in vitro and in vivo conditions, and that seems to arise through a concerted chromosome loss mechanism. Haploids undergo morphogenetic changes like those of diploids, including the yeast–hyphal transition, chlamydospore formation and a white-opaque switch that facilitates mating. Haploid opaque cells of opposite mating type mate efficiently to regenerate the diploid form, restoring heterozygosity and fitness. Homozygous diploids arise spontaneously by auto-diploidization, and both haploids and auto-diploids show a similar reduction in fitness, in vitro and in vivo, relative to heterozygous diploids, indicating that homozygous cell types are transient in mixed populations. Finally, we constructed stable haploid strains with multiple auxotrophies that will facilitate molecular and genetic analyses of this important pathogen.

Discovery of a phenotypic switch regulating sexual mating in the opportunistic fungal pathogen Candida tropicalis

Sexual reproduction can promote genetic diversity in eukaryotes, and yet many pathogenic fungi have been labeled as obligate asexual species. It is becoming increasingly clear, however, that cryptic sexual programs may exist in some species, and that efficient mating requires the necessary developmental switch to be triggered. In this study we investigate Candida tropicalis, an important human fungal pathogen that has been reported to be asexual. Significantly, we demonstrate that C. tropicalis uses a phenotypic switch to regulate a cryptic program of sexual mating. Thus, diploid a and α cells must undergo a developmental transition to the mating-competent form, and only then does efficient cell-cell conjugation take place resulting in the formation of stable a/α tetraploids. We show that both the phenotypic switch and sexual mating depend on the conserved transcriptional regulator Wor1, which is regulated by temperature in other fungal species. In contrast, C. tropicalis mating occurs efficiently at both 25 °C and 37 °C, suggesting that it could occur in the mammalian host and have direct consequences for the outcome of an infection. Transcriptional profiling further reveals that ≈400 genes are differentially expressed between the two phenotypic states, including the regulatory factor Wor1. Taken together, our results demonstrate that C. tropicalis has a unique sexual program, and that entry to this program is controlled via a Wor1-mediated, metastable switch. These observations have direct implications for the regulation and evolution of cryptic sexual programs in related fungal pathogens.

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.

Candida albicans white and opaque cells undergo distinct programs of filamentous growth.

The ability to switch between yeast and filamentous forms is central to Candida albicans biology. The yeast-hyphal transition is implicated in adherence, tissue invasion, biofilm formation, phagocyte escape, and pathogenesis. A second form of morphological plasticity in C. albicans involves epigenetic switching between white and opaque forms, and these two states exhibit marked differences in their ability to undergo filamentation. In particular, filamentous growth in white cells occurs in response to a number of environmental conditions, including serum, high temperature, neutral pH, and nutrient starvation, whereas none of these stimuli induce opaque filamentation. Significantly, however, we demonstrate that opaque cells can undergo efficient filamentation but do so in response to distinct environmental cues from those that elicit filamentous growth in white cells. Growth of opaque cells in several environments, including low phosphate medium and sorbitol medium, induced extensive filamentous growth, while white cells did not form filaments under these conditions. Furthermore, while white cell filamentation is often enhanced at elevated temperatures such as 37°C, opaque cell filamentation was optimal at 25°C and was inhibited by higher temperatures. Genetic dissection of the opaque filamentation pathway revealed overlapping regulation with the filamentous program in white cells, including key roles for the transcription factors EFG1, UME6, NRG1 and RFG1. Gene expression profiles of filamentous white and opaque cells were also compared and revealed only limited overlap between these programs, although UME6 was induced in both white and opaque cells consistent with its role as master regulator of filamentation. Taken together, these studies establish that a program of filamentation exists in opaque cells. Furthermore, this program regulates a distinct set of genes and is under different environmental controls from those operating in white cells.

MTL-independent phenotypic switching in Candida tropicalis and a dual role for Wor1 in regulating switching and filamentation.

Phenotypic switching allows for rapid transitions between alternative cell states and is important in pathogenic fungi for colonization and infection of different host niches. In Candida albicans, the white-opaque phenotypic switch plays a central role in regulating the program of sexual mating as well as interactions with the mammalian host. White-opaque switching is controlled by genes encoded at the MTL (mating-type-like) locus that ensures that only a or α cells can switch from the white state to the mating-competent opaque state, while a/α cells are refractory to switching. Here, we show that the related pathogen C. tropicalis undergoes white-opaque switching in all three cell types (a, α, and a/α), and thus switching is independent of MTL control. We also demonstrate that C. tropicalis white cells are themselves mating-competent, albeit at a lower efficiency than opaque cells. Transcriptional profiling of C. tropicalis white and opaque cells reveals significant overlap between switch-regulated genes in MTL homozygous and MTL heterozygous cells, although twice as many genes are white-opaque regulated in a/α cells as in a cells. In C. albicans, the transcription factor Wor1 is the master regulator of the white-opaque switch, and we show that Wor1 also regulates switching in C. tropicalis; deletion of WOR1 locks a, α, and a/α cells in the white state, while WOR1 overexpression induces these cells to adopt the opaque state. Furthermore, we show that WOR1 overexpression promotes both filamentous growth and biofilm formation in C. tropicalis, independent of the white-opaque switch. These results demonstrate an expanded role for C. tropicalis Wor1, including the regulation of processes necessary for infection of the mammalian host. We discuss these findings in light of the ancestral role of Wor1 as a transcriptional regulator of the transition between yeast form and filamentous growth.

Novel Structural Components of the Ventral Disc and Lateral Crest in Giardia intestinalis

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment.

NETosis in Neonates: Evidence of a Reactive Oxygen Species–Independent Pathway in Response to Fungal Challenge

Release of neutrophil extracellular traps (NETs) is a significant antimicrobial host defense mechanism in adults. In neonates, fungal sepsis is a frequent cause of morbidity and mortality and may be a consequence of inadequate neutrophil defense functions. Like neutrophils from adult donors, we found that neutrophils from neonates formed robust cellular aggregates and released NETs in response to fungal β-glucan and Candida albicans hyphae when presented with extracellular matrix. Therefore, in response to fungal stimulation, neonatal neutrophils are capable of NETosis. Neonate susceptibility to fungal infections may not be due to an inability of their neutrophils to produce NETs.

Parasexuality and ploidy change in Candida tropicalis.

ABSTRACT Candida species exhibit a variety of ploidy states and modes of sexual reproduction. Most species possess the requisite genes for sexual reproduction, recombination, and meiosis, yet only a few have been reported to undergo a complete sexual cycle including mating and sporulation. Candida albicans, the most studied Candida species and a prevalent human fungal pathogen, completes its sexual cycle via a parasexual process of concerted chromosome loss rather than a conventional meiosis. In this study, we examine ploidy changes in Candida tropicalis, a closely related species to C. albicans that was recently revealed to undergo sexual mating. C. tropicalis diploid cells mate to form tetraploid cells, and we show that these can be induced to undergo chromosome loss to regenerate diploid forms by growth on sorbose medium. The diploid products are themselves mating competent, thereby establishing a parasexual cycle in this species for the first time. Extended incubation (>120 generations) of C. tropicalis tetraploid cells under rich culture conditions also resulted in instability of the tetraploid form and a gradual reduction in ploidy back to the diploid state. The fitness levels of C. tropicalis diploid and tetraploid cells were compared, and diploid cells exhibited increased fitness relative to tetraploid cells in vitro, despite diploid and tetraploid cells having similar doubling times. Collectively, these experiments demonstrate distinct pathways by which a parasexual cycle can occur in C. tropicalis and indicate that nonmeiotic mechanisms drive ploidy changes in this prevalent human pathogen.

Parasex Generates Phenotypic Diversity de Novo and Impacts Drug Resistance and Virulence in Candida albicans

Candida albicans is a diploid fungus that is a frequent cause of mucosal and systemic infections in humans. This species exhibits an unusual parasexual cycle in which mating produces tetraploid cells that undergo a nonmeiotic program of concerted chromosome loss to return to a diploid or aneuploid state. In this work, we used a multipronged approach to examine the capacity of parasex to generate diversity in C. albicans. First, we compared the phenotypic properties of 32 genotyped progeny and observed wide-ranging differences in fitness, filamentation, biofilm formation, and virulence. Strikingly, one parasexual isolate displayed increased virulence relative to parental strains using a Galleria mellonella model of infection, establishing that parasex has the potential to enhance pathogenic traits. Next, we examined parasexual progeny derived from homothallic, same-sex mating events, and reveal that parasex can generate diversity de novo from identical parental strains. Finally, we generated pools of parasexual progeny and examined resistance of these pools to environmental stresses. Parasexual progeny were generally less fit than control strains across most test conditions, but showed an increased ability to grow in the presence of the antifungal drug fluconazole (FL). FL-resistant progeny were aneuploid isolates, often being diploid strains trisomic for both Chr3 and Chr6. Passaging of these aneuploid strains frequently led to loss of the supernumerary chromosomes and a concomitant decrease in drug resistance. These experiments establish that parasex generates extensive phenotypic diversity de novo, and that this process has important consequences for both virulence and drug resistance in C. albicans populations.

Sexual biofilm formation in Candida tropicalis opaque cells

Candida albicans and Candida tropicalis are opportunistic fungal pathogens that can transition between white and opaque phenotypic states. White and opaque cells differ both morphologically and in their responses to environmental signals. In C. albicans, opaque cells respond to sexual pheromones by undergoing conjugation, while white cells are induced by pheromones to form sexual biofilms. Here, we show that sexual biofilm formation also occurs in C. tropicalis but, unlike C. albicans, biofilms are formed exclusively by opaque cells. C. tropicalis biofilm formation was dependent on the pheromone receptors Ste2 and Ste3, confirming the role of pheromone signalling in sexual biofilm development. Structural analysis of C. tropicalis sexual biofilms revealed stratified communities consisting of a basal layer of yeast cells and an upper layer of filamentous cells, together with an extracellular matrix. Transcriptional profiling showed that genes involved in pheromone signalling and conjugation were upregulated in sexual biofilms. Furthermore, FGR23, which encodes an agglutinin‐like protein, was found to enhance both mating and sexual biofilm formation. Together, these studies reveal that C. tropicalis opaque cells form sexual biofilms with a complex architecture, and suggest a conserved role for sexual agglutinins in mediating mating, cell cohesion and biofilm formation.