Biology subjects

Voorhies, M.

Publications and source records attributed to Voorhies, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Temperature-driven isoform switching reprograms developmental gene expression in a human fungal pathogen

Post-transcriptional regulation is key to development, and yet little is known about how RNA isoform choice contributes to developmental choices in fungi. Here we assemble the first isoform-level transcriptome of Histoplasma, a ubiquitous human fungal pathogen that grows as an infectious environmental form (hyphae) or a pathogenic host form (yeast) in response to temperature. We find extensive morphology-associated longer leader and trailer isoforms, rapid temperature-driven transcription start site remodeling, and inclusion of regulatory elements in longer leaders. We observe isoform-specific patterns of ribosome and polysome association, indicating that isoform switching regulates the proteome. Our results suggest a model in which isoform diversity and rapid isoform switching are central to post-transcriptional regulation during thermal dimorphism, enabling precise and timely translation of factors needed to establish and maintain hyphal and yeast forms. These studies illuminate how eukaryotic systems utilize post- transcriptional regulation to determine developmental states in response to external stimuli.

microbiology↗

bioRxiv2026

Optimization of conidial production in the thermally dimorphic fungal pathogen Histoplasma

Sporulation is an integral process in the lifecycle of many fungal pathogens, including Histoplasma, a primary human pathogen that causes respiratory infections. Histoplasma conidia, or asexual spores, are the primary infectious particle but very little is known about them, in part due to the need for Biosafety Level 3 containment and inconsistency in generating viable conidia under laboratory conditions. Here, we identify media that consistently promote Histoplasma conidiation, yielding both micro- and macroconidia, and conditions that promote high levels of germination. We show that conidiation media and duration affect the proportion of macroconidia produced, and we demonstrate that Histoplasma strains vary in their response to these conidiation parameters. Finally, imaging studies of chitin, exposed chitin, and cell wall mannoproteins show that while micro- and macroconidia have similar cell wall compositions, strain type and conidiation media variation result in qualitative differences in staining. These optimized methods for Histoplasma conidial preparations will enable more detailed investigations into this understudied aspect of the biology of an important human fungal pathogen.

microbiology↗

bioRxiv2025

Macrophage phagocytosis of Coccidioides promotes its differentiation into the parasitic form

Coccidioides is an endemic fungus that is increasing in prevalence and can cause life threatening disease in otherwise immunocompetent people. In the environment the spores (arthroconidia) develop into hyphae, yet when they are inhaled by a mammalian host, they develop into a unique form called the spherule. The transition to spherule can be triggered in vitro with elevated temperatures and high CO2 levels, but the signals and host cells that might trigger Coccidioides spherulation in vivo are not known. We used live imaging to investigate how macrophages affect the fate of Coccidioides arthroconidia. Under tissue culture conditions, arthroconidia quickly developed into hyphae. The addition of macrophages promoted spherule development and delayed hyphal formation, indicating that innate immune cells can influence Coccidioides development into the pathogenic form. Exposure of arthroconidia to macrophage supernatants was not sufficient to stimulate spherulation, which was dependent on phagocytosis of arthroconidia by macrophages. Transcriptomics analysis of Coccidioides co-cultured with macrophages revealed a signature concordant with spherules grown in vitro and allowed the identification of a core set of spherule-specific transcripts. In addition, we identified Coccidioides transcripts with significantly higher abundance in the presence of macrophages compared to in vitro spherules, suggesting these factors may be needed to survive and thrive in the presence of innate immune cells. This work lays a foundation for uncovering host-pathogen signaling as well as Coccidioides factors that are critical for pathogenesis.

microbiology↗

bioRxiv2025

The Rpd3 histone deacetylase is critical for temperature-mediated morphogenesis and virulence in the human fungal pathogen Histoplasma

Adaptive responses to environmental stimuli are integral to the survival and virulence of microbial pathogens. The thermally dimorphic human fungal pathogen Histoplasma senses temperature to transition between a mold form in soil and a pathogenic yeast in mammalian hosts. The contributions of chromatin-modifying enzymes to the ability of Histoplasma to appropriately respond to temperature have never been explored. Through chemical inhibition and genetics, we determined that the class I histone deacetylase (HDAC) RPD3 is required for normal Histoplasma yeast morphology at 37 {degrees}C. Rpd3 regulated the expression of key morphology-specific genes, including critical virulence factors and transcription factors (TFs), was required for normal DNA-binding activity of yeast-promoting TFs, and influenced histone acetylation levels at the loci of putative pro-filamentation TFs. Furthermore, Rpd3 was required for virulence in a macrophage model of infection. Taken together, Rpd3 is a critical regulatory component that both activates the pathogenesis program and represses the filamentation program to enable thermal dimorphism in Histoplasma. This work uncovers the crucial role that chromatin regulation plays in temperature response of this ubiquitous pathogen.

microbiology↗

bioRxiv2025

An unusual genetic switch controls Mycobacterium avium pathogenesis, antibiotic resistance and colony morphology

Mycobacterium avium subspecies hominissuis (Mah) is an emerging environmental pathogen highly adapted to a wide range of niches, from treated water systems to mammalian tissues. On solid media, Mah forms two distinct colony morphologies, smooth transparent (SmT) and smooth opaque (SmO). These colony morphologies are representative of broader differential phenotypic states in which SmT cells are virulent and have high resistance to antibiotics while SmO cells are avirulent, antibiotic-sensitive and grow faster than SmT cells in culture. Importantly, Mah interconverts between these two morphotypes but the mechanism of SmT-SmO switching is unknown. Here we show that SmT-SmO switching is governed by a reversible transposition event that regulates expression of a periplasmic lipoprotein, Erp (extracellular repetitive protein). We found that transposition of IS1245, an endogenous insertion sequence, into the erp gene correlated with the SmT-SmO transition, and its precise removal coincided with the switch back to SmT. Genetic analyses showed that erp is required for maintenance of the SmT state and sufficient to drive the switch from SmO to SmT. We also identified a mutation in a periplasmic protease, MarP, that locks Mah in the SmO state and blocks erp-mediated switching to SmT. Our results indicate that Erp and MarP function in a signal transduction pathway that regulates a broad transcriptional response to periplasmic stress. Moreover, identification of components that control Mah colony morphology switching has revealed a potential new strategy for combating the inherent antibiotic resistance of Mycobacterium avium infections.

microbiology↗

bioRxiv2025

Rapid gain and loss of a chromosome drives key morphology and virulence phenotypes in Histoplasma, a fungal pathogen of humans

Heritable phenotypic switches are fundamental to the ability of cells to respond to specific conditions. Such switches are key to the success of environmental pathogens, which encounter disparate conditions as they transition between the environment and host. We determine that the copy number of chromosome seven in the thermally dimorphic fungus Histoplasma dramatically affects the rate of transition. Though Histoplasma is haploid, a second copy of this chromosome is present in natural isolates of multiple Histoplasma species and is gained and lost at a high rate. Cells carrying two copies of this chromosome exhibit aspects of the environmental transcriptome even under host-like conditions and have a competitive advantage in the transition to the environmental form. Conversely, these cells are considerably less virulent than euploid cells and have a competitive disadvantage in the mouse model of infection. Chromosome seven contains a previously unstudied transcription factor that, when overexpressed in euploid Histoplasma, is sufficient to promote some of the key phenotypes of aneuploidy. We hypothesize that rapid gain and loss of this chromosome benefits Histoplasma by increasing phenotypic variation, thus helping populations of cells survive abrupt transitions between environment and host.

genetics↗

bioRxiv2025

A remarkable expansion of secreted cysteine knot proteins reveals novel virulence factors in the human fungal pathogen Histoplasma

Identifying fungal secreted factors that influence host infection remains a key challenge in microbial pathogenesis. While secreted effectors, particularly small cysteine-rich proteins, are well characterized in plant fungal pathogens, their counterparts in mammalian pathogens remain elusive. We applied criteria from plant fungal effectors to the mammalian fungal pathogen Histoplasma, yielding a set of putative effectors highly enriched for knottins, proteins that adopt a distinctive cysteine knot fold. Using a novel algorithm, we further identified 25 putative knottins in the Histoplasma genome, revealing a significant expansion of knottin genes. Knottin domains are found in diverse molecules but play an unknown role in virulence. Functional studies of individual Histoplasma knottins demonstrated their critical roles in intracellular survival and host cell lysis during macrophage infection as well as virulence in vivo. These findings highlight the importance of knottins in fungal pathogenesis and suggest their broader relevance for discovering conserved mechanisms of host manipulation.

microbiology↗

bioRxiv2025

Genome Dynamics and Chromosome Structural Variations in Histoplasma ohiense, a fungal pathogen of humans

Histoplasma is a clinically important but understudied genus of thermally dimorphic human fungal pathogens. Histoplasma species normally transition between a multicellular sporulating hyphal form in the soil and a unicellular pathogenic yeast form in a mammalian host. Little is known about genome plasticity of Histoplasma, which we address in this study with the ultimate goal of increasing our understanding of its pathogenicity. This study addresses the Histoplasma genome and its plasticity to further our understanding Histoplasmas ability to cause severe disease. Here we present the first telomere-to-telomere genome assemblies for Histoplasma ohiense. To analyze genome alterations between isolates, we develop an analysis tool to identify genome discontinuities relative to a reference genome. We utilize this analysis to interrogate the genome of laboratory strains and natural Histoplasma isolates and discover that the previously published reference genome does not completely match the chromosome structure of the majority of isolates, instead harboring reciprocal chromosome translocations. We identify the telomere-to-telomere Histoplasma ohiense reference genome that is most representative of clinical isolates. Additionally, to determine the rate of Histoplasma genomic changes, we sequence 46 passaged isolates and calculate the mutation rate to be 2.6 x10-10 SNP/base/doubling--the first such measurement to our knowledge within the order Onygenales, which encompasses several critical fungal pathogens. Finally, we sequence populations of cells to assess genome stability over the course of a month under both yeast and hyphal growth conditions. Interestingly, we observe that transposon signal is not static over time and instead increases during growth in both the yeast and hyphal forms as well as through morphologic transitions, suggesting an increase in transposon copy number. Taken together, this work highlights the plasticity of the Histoplasma genome and presents a comprehensive genome assembly that is representative of Histoplasma ohiense natural isolates.

genomics↗

bioRxiv2024

Transcriptomic atlas of the morphologic development of the fungal pathogen Coccidioides reveals key phase-enriched transcripts

Coccidioides spp. are highly understudied but significant dimorphic fungal pathogens that can infect both immunocompetent and immunocompromised people. In the environment, they grow as multicellular filaments (hyphae) that produce vegetative spores called arthroconidia. Upon inhalation by mammals, arthroconidia undergo a process called spherulation. They enlarge and undergo numerous nuclear divisions to form a spherical structure, and then internally segment until the spherule is filled with multiple cells called endospores. Mature spherules rupture and release endospores, each of which can form another spherule, in a process thought to facilitate dissemination. Spherulation is unique to Coccidioides and its molecular determinants remain largely unknown. Here, we report the first high-density transcriptomic analyses of Coccidioides development, defining morphology-dependent transcripts and those whose expression is regulated by Ryp1, a major regulator required for spherulation and virulence. Of approximately 9000 predicted transcripts, we discovered 273 transcripts with consistent spherule-associated expression, 82 of which are RYP1-dependent, a set likely to be critical for Coccidioides virulence. ChIP-Seq revealed 2 distinct regulons of Ryp1, one shared between hyphae and spherules and the other unique to spherules. Spherulation regulation was elaborate, with the majority of 227 predicted transcription factors in Coccidioides displaying spherule-enriched expression. We identified provocative targets, including 20 transcripts whose expression is endospore-enriched and 14 putative secreted effectors whose expression is spherule-enriched, of which 6 are secreted proteases. To highlight the utility of these data, we selected a cluster of RYP1-dependent, arthroconidia-associated transcripts and found that they play a role in arthroconidia cell wall biology, demonstrating the power of this resource in illuminating Coccidioides biology and virulence.

microbiology↗

bioRxiv2024

Optimizing in vitro spherulation cues in the fungal pathogen Coccidioides

Coccidioides spp. are part of a group of thermally dimorphic fungal pathogens, which grow as filamentous cells (hyphae) in the soil and transform to a different morphology upon inhalation into the host. The Coccidioides host form, the spherule, is unique and highly under characterized due to both technical and biocontainment challenges. Each spherule arises from an environmental spore (arthroconidium), matures, and develops hundreds of internal endospores, which are released from the spherule upon rupture. Each endospore can then go on to form another spherule in a cycle called spherulation. One of the foremost technical challenges has been reliably growing spherules in culture without the formation of contaminating hyphae, and consistently inducing endospore release from spherules. Here, we present optimization of in vitro spherule growth and endospore release, by closely controlling starting cell density in the culture, using freshly-harvested arthroconidia, and decreasing the concentration of multiple salts in spherulation media. We developed a minimal media to test spherule growth on various carbon and nitrogen sources. We defined a critical role for the dispersant Tamol in both early spherule formation and prevention of the accumulation of a visible film around spherules. Finally, we examined how the conditions under which arthroconidia are generated influence their transcriptome and subsequent development into spherules, demonstrating that this is an important variable to control when designing spherulation experiments. Together, our data reveal multiple strategies to optimize in vitro spherulation growth, enabling characterization of this virulence-relevant morphology.

microbiology↗

bioRxiv2023

Recyclable CRISPR/Cas9 mediated gene disruption and deletions in Histoplasma

Targeted gene disruption is challenging in the dimorphic fungal pathogen Histoplasma due to the low frequency of homologous recombination. Transformed DNA is either integrated ectopically into the genome or maintained extra chromosomally by de novo addition of telomeric sequences. Based on a system developed in Blastomyces, we adapted a CRISPR/Cas9 system to facilitate targeted gene disruption in Histoplasma with high efficiency. We express a codon-optimized version of Cas9 as well as guide RNAs from a single ectopic vector carrying a selectable marker. Once the desired mutation is verified, one can screen for isolates that have lost the Cas9 vector by simply removing the selective pressure. Multiple mutations can then be generated in the same strain by retransforming the Cas9 vector carrying different guides. We used this system to disrupt a number of target genes including RYP2 and SRE1 where loss-of-function mutations could be monitored visually by colony morphology or color, respectively. Interestingly, expression of two guide RNAs targeting the 5 and 3 ends of a gene allowed isolation of deletion mutants where the sequence between the guide RNAs was removed from the genome. Whole-genome sequencing showed that the frequency of off-target mutations associated with the Cas9 nuclease was negligible. Finally, we increased the frequency of gene disruption by using an endogenous Histoplasma regulatory sequence to drive guide RNA expression. These tools transform our ability to generate targeted mutations in Histoplasma. ImportanceHistoplasma is a primary fungal pathogen with the ability to infect otherwise healthy mammalian hosts, causing systemic and sometimes life-threatening disease. Thus far, molecular genetic manipulation of this organism has utilized RNA interference, random insertional mutagenesis, and a homologous recombination protocol that is highly variable and often inefficient. Targeted gene manipulations have been challenging due to poor rates of homologous recombination events in Histoplasma. Interrogation of the virulence strategies of this organism would be highly accelerated by a means of efficiently generating targeted mutations. We have developed a recyclable CRISPR/Cas9 system that can be used to introduce gene disruptions in Histoplasma with high efficiency, thereby allowing disruption of multiple genes.

microbiology↗

bioRxiv2023

Chemical stimuli override a temperature-dependent morphological program by reprogramming the transcriptome of a fungal pathogen.

The human fungal pathogen Histoplasma changes its morphology in response to temperature. At 37{degrees}C it grows as a budding yeast whereas at room temperature it transitions to hyphal growth. Prior work has demonstrated that 15-20% of transcripts are temperature-regulated, and that transcription factors Ryp1-4 are necessary to establish yeast growth. However, little is known about transcriptional regulators of the hyphal program. To identify TFs that regulate filamentation, we utilize chemical inducers of hyphal growth. We show that addition of cAMP analogs or an inhibitor of cAMP breakdown overrides yeast morphology, yielding inappropriate hyphal growth at 37{degrees}C. Additionally, butyrate supplementation triggers hyphal growth at 37{degrees}C. Transcriptional profiling of cultures filamenting in response to cAMP or butyrate reveals that a limited set of genes respond to cAMP while butyrate dysregulates a larger set. Comparison of these profiles to previous temperature- or morphology-regulated gene sets identifies a small set of morphology-specific transcripts. This set contains 9 TFs of which we characterized three, STU1, FBC1, and PAC2, whose orthologs regulate development in other fungi. We found that each of these TFs is individually dispensable for room-temperature (RT) induced filamentation but each is required for other aspects of RT development. FBC1 and PAC2, but not STU1, are necessary for filamentation in response to cAMP at 37{degrees}C. Ectopic expression of each of these TFs is sufficient to induce filamentation at 37{degrees}C. Finally, PAC2 induction of filamentation at 37{degrees}C is dependent on STU1, suggesting these TFs form a regulatory circuit that, when activated at RT, promotes the hyphal program. ImportanceFungal illnesses pose a significant disease burden. However, the regulatory circuits that govern the development and virulence of fungi remain largely unknown. This study utilizes chemicals that can override the normal growth morphology of the human pathogen Histoplasma. Using transcriptomic approaches, we identify novel regulators of hyphal morphology and refine our understanding of the transcriptional circuits governing morphology in Histoplasma.

microbiology↗

bioRxiv2022

A Legionella toxin mimics tRNA and glycosylates the translation machinery to trigger a ribotoxic stress response.

Pathogens often secrete proteins or nucleic acids that mimic the structure and/or function of molecules expressed in their hosts. Molecular mimicry empowers pathogens to subvert critical host processes and establish infection. We report that the intracellular bacterium Legionella pneumophila secretes the toxin SidI (substrate of icm/dot transporter I), which possesses a transfer RNA (tRNA)-like shape and functions as a mannosyl transferase. The 3.1 [A] cryo-EM structure of SidI reveals an N-terminal domain that exhibits a characteristic inverted L-shape and charge distribution that is present in two other known protein mimics of tRNAs, the bacterial elongation factor EF-G and the mammalian release factor eRF1. In addition, we show that SidIs C-terminal domain adopts a glycosyl transferase B fold similar to a mannosyl transferase. This molecular coupling of the proteins fold and enzymatic function allows SidI to bind and glycosylate components of the host translation apparatus, including the ribosome, resulting in a robust block of protein synthesis that is comparable in potency to ricin, one of the most powerful toxins known. Additionally, we find that translational pausing activated by SidI elicits a stress response signature reminiscent of the ribotoxic stress response that is activated by elongation inhibitors that induce ribosome collisions. SidI-mediated effects on the ribosome activate the stress kinases ZAK and p38, which in turn drive an accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus, and orchestrates the transcription of stress-inducible genes that promote cell death. Thus, using Legionella and its effectors as tools, we have unravelled the role of a ribosome-to-nuclear signalling pathway that regulates cell fate.

cell biology↗

bioRxiv2022

Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. To interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc. We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement fragment C3a. Although it is known that the complement system reacts with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage susceptibility to fungi has not been elucidated. We demonstrated that whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc, the ubiquitous fungal pathogen Candida albicans, and the causative agent of Valley Fever Coccidioides posadasii. We showed that C3aR localizes to the early phagosome during H. capsulatum infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc-macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi.

microbiology↗

bioRxiv2021

The ESX-1 secretion system senses bacterial contact and prepares mycobacteria for environmental adaptation

The ESX-1 system (6-kDa early secretory antigenic target (ESAT-6) secretion system-1) is essential for Mycobacterium tuberculosis pathogenesis and conjugal transfer in Mycobacterium smegmatis, yet little is known about how its function is regulated. Live-cell fluorescence microscopy showed natively expressed ESX-1 was organized into distinct foci predominantly observed at cell-cell contacts. These foci formed when two cells touched and required a fully assembled ESX-1 system in both bacteria, suggesting the generation of an ESX-1 megacomplex across multiple membranes. The emergence of ESX-1 foci and ESX-1 secretion was environmentally dependent: foci formed in low nitrogen environments in which secretion was suppressed, yet with increasing concentrations of nitrogen, ESX-1 systems diffused along the plasma membrane and secretion was activated. Genome-wide transcriptional profiling revealed ESX-1 dependent induction of genes required for the SOS response and error prone DNA replication in high nitrogen. Based on these findings, we propose a new model of ESX-1 function where ESX-1 localization and secretion are responsive to nitrogen levels and form an integral node in the mycobacterial response to neighboring cells and environmental adaptation.

microbiology↗

bioRxiv2021

Cbp1, a rapidly evolving fungal virulence factor, forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel "binocular" fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a rapidly evolving fungal virulence factor that localizes to the cytosol to trigger host cell lysis. Author SummaryThe members of the Ajellomycetaceae fungal family are human pathogens that are responsible for a rising number of mycoses around the world. Calcium binding protein 1 (Cbp1) is a rapidly evolving virulence factor that is present in the genomes of the Ajellomycetaceae species that lead primarily intracellular lifestyles, including Histoplasma, Paracoccidioides, and Emergomyces but not Blastomyces, which remains largely extracellular during infection. Both Paracoccidioides and Histoplasma Cbp1 homologs are able to cause lysis of macrophages whereas Emergomyces homologs cannot. This result is consistent with Emergomyces africanus natural infection of macrophages, during which the yeast cells can replicate but cannot actively lyse the host cell. Despite divergence of the primary sequence of Histoplasma and Paracoccidioides Cbp1 homologs, their protein structures are remarkably similar and reveal a novel fold. During infection, Cbp1 enters the cytosol of the host macrophage, making it the first known virulence factor from an intracellular human fungal pathogen that localizes to the cytosol of the host cell. We also show that Cbp1 forms a complex with another cytosolic virulence factor, Yps-3. Taken together, these studies significantly advance our understanding of Histoplasma virulence.

microbiology↗

bioRxiv2021

The WOPR family protein Ryp1 is a key regulator of gene expression, development, and virulence in the thermally dimorphic fungal pathogen Coccidioides posadasii

Coccidioides spp. are mammalian fungal pathogens endemic to the southwestern US and other desert regions of Mexico, central and South America, with the bulk of US infections occurring in California and Arizona. In the soil, Coccidioides grows in a hyphal form that differentiates into 3-5 micron asexual spores (arthroconidia). When arthroconidia are inhaled by mammals they undergo a unique developmental transition from polar hyphal growth to isotropic expansion with multiple rounds of nuclear division, prior to segmentation, forming large spherules filled with endospores. Very little is understood about the molecular basis of spherule formation. Here we characterize the role of the conserved transcription factor Ryp1 in Coccidioides development. We show that Coccidioides {Delta}ryp1 mutants have altered colony morphology under hypha-promoting conditions and are unable to form mature spherules under spherule-promoting conditions. We analyze the transcriptional profile of wild-type and {Delta}ryp1 mutant cells under hypha- and spherule-promoting conditions, thereby defining a set of hypha- or spherule-enriched transcripts ("morphology-regulated" genes) that are dependent on Ryp1 for their expression. Forty percent of morphology-regulated expression is Ryp1-dependent, indicating that Ryp1 plays a dual role in both hyphal and spherule development. Ryp1-dependent transcripts include key virulence factors such as SOWgp, which encodes the spherule outer wall glycoprotein. Concordant with its role in spherule development, we find that the {Delta}ryp1 mutant is completely avirulent in the mouse model of coccidioidomycosis, indicating that Ryp1-dependent pathways are essential for the ability of Coccidioides to cause disease. Vaccination of C57BL/6 mice with live {Delta}ryp1 spores does not provide any protection from lethal C. posadasii intranasal infection, consistent with our findings that the {Delta}ryp1 mutant fails to make mature spherules and likely does not express key antigens required for effective vaccination. Taken together, this work identifies the first transcription factor that drives mature spherulation and virulence in Coccidioides. Author SummaryCoccidioides species, C. immitis and C. posadasii, are dimorphic fungal pathogens that commonly infect humans in North, Central, and South America, causing the respiratory fungal disease known as Valley Fever. Coccidioides grows as hyphae in the soil and differentiates into unique structures called spherules in the mammalian host. Spherules expand and internally divide to form endospores, which are released to facilitate dissemination of the pathogen within the host. The mechanisms underlying spherule differentiation remain largely unknown. In this study, we generated knockout mutants ({Delta}ryp1) of the conserved transcription factor Ryp1 in C. posadasii and characterized its role in spherule formation and virulence. We found that Ryp1 is required for the formation of mature spherules and colonization of mouse lungs. Transcriptional profiling of the {Delta}ryp1 mutant and the wild-type strain shows that Ryp1 regulates the expression of a subset of the transcripts that are either upregulated in wild-type spherules or in wild-type hyphae. These findings suggest that Ryp1 has a dual role in regulating morphology and virulence under host conditions as well as regulating genes involved in hyphal growth in the environment.

microbiology↗

bioRxiv2021

Chromosome-level genome assembly of a human fungal pathogen reveals synteny among geographically distinct species

Histoplasma capsulatum, a dimorphic fungal pathogen, is the most common cause of fungal respiratory infections in immunocompetent hosts. Histoplasma is endemic in the Ohio and Mississippi River Valleys in the United States and also distributed worldwide. Previous studies revealed at least eight clades, each specific to a geographic location: North American classes 1 and 2 (NAm 1 and NAm 2), Latin American groups A and B (LAm A and LAm B), Eurasian, Netherlands, Australian and African, and an additional distinct lineage (H81) comprised of Panamanian isolates. Previously assembled Histoplasma genomes are highly fragmented, with the highly repetitive G217B (NAm 2) strain, which has been used for most whole genome-scale transcriptome studies, assembled into over 250 contigs. In this study, we set out to fully assemble the repeat regions and characterize the large-scale genome architecture of Histoplasma species. We re-sequenced five Histoplasma strains (WU24 (NAm 1), G217B (NAm 2), H88 (African), G186AR (Panama), and G184AR (Panama)) using Oxford Nanopore Technologies long-read sequencing technology. Here we report chromosomal-level assemblies for all five strains, which exhibit extensive synteny among the geographically distant Histoplasma isolates. The new assemblies revealed that RYP2, a major regulator of morphology and virulence, is duplicated in G186AR. In addition, we mapped previously generated transcriptome datasets onto the newly assembled chromosomes. Our analyses revealed that the expression of transposons and transposon-embedded genes are upregulated in yeast phase compared to mycelial phase in G217B and H88 strains. This study provides an important resource for fungal researchers and further highlights the importance of chromosomal-level assemblies in analyzing high-throughput datasets. ImportanceHistoplasma species are dimorphic fungi causing significant morbidity and mortality worldwide. These fungi grow as mold in the soil and as budding yeast within the human host. Histoplasma can be isolated from soil in diverse regions, including North America, South America, Africa and Europe. Phylogenetically distinct species of Histoplasma have been isolated and sequenced. However, for the commonly used strains, genome assemblies have been fragmented, leading to underutilization of genome-scale data. This study provides chromosome-level assemblies of the commonly used Histoplasma strains using long-read sequencing technology. Comparative analysis of these genomes shows largely conserved gene order within the chromosomes. Mapping existing transcriptome data on these new assemblies reveals clustering of transcriptionally co-regulated genes. Results of this study highlight the importance of obtaining chromosome-level assemblies in understanding the biology of human fungal pathogens.

microbiology↗