Biology subjects

Beyhan, S.

Publications and source records attributed to Beyhan, S..

6 recordsLinked to original sources

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

Transcriptional regulatory features associated with Coccidioides immitis phase transition

Coccidioidomycosis (Valley Fever) is an emerging endemic fungal infection with a rising incidence and an expanding geographic range. It is caused by Coccidiodes, which are thermally dimorphic fungi that grow as mycelia in soil but transition in the lung to form pathogenic spherules. The regulatory mechanisms underlying this transition are not understood. Exploiting capped small (cs)RNA-seq, which identifies actively initiated stable and unstable transcripts and thereby detects acute changes in gene regulation with remarkable sensitivity, here we report the changes in architectural organization and key sequence features underlying phase transition of this highly pathogenic fungus. Spherule transition was accompanied by large-scale transcriptional reprogramming, functional changes in transcript isoforms, and a massive increase in promoter-distal transcription of ncRNAs. Analysis of spherule-activated regulatory elements revealed a motif predicted to recruit a WOPR family transcription factor, which are known regulators of virulence in other fungi. We identify CIMG_02671 as a C. immitis WOPR homologue and show that it activates transcription in a WOPR motif-dependent manner, suggesting it is an important regulator of pathogenic phase transition. Collectively, this also highlights csRNA-seq as a powerful means to identify transcriptional mechanisms that control pathogenesis.

immunology↗

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↗

bioRxiv2019

A Distinct Contractile Injection System Found in a Majority of Adult Human Microbiomes

An imbalance of normal bacterial groups such as Bacteroidales within the human gut is correlated with diseases like obesity. A current grand challenge in the microbiome field is to identify factors produced by normal microbiome bacteria that cause these observed health and disease correlations. While identifying factors like a bacterial injection system could provide a missing explanation for why Bacteroidales correlates with host health, no such factor has been identified to date. The lack of knowledge about these factors is a significant barrier to improving therapies like fecal transplants that promote a healthy microbiome. Here we show that a previously ill-defined Contractile Injection System is carried in the gut microbiome of 99% of individuals from the United States and Europe. This type of Contractile Injection System, we name here Bacteroidales Injection System (BIS), is related to the contractile tails of bacteriophage (viruses of bacteria) and have been described to mediate interactions between bacteria and diverse eukaryotes like amoeba, insects and tubeworms. Our findings that BIS are ubiquitous within adult human microbiomes suggest that they shape host health by mediating interactions between Bacteroidales bacteria and the human host or its microbiome.

microbiology↗

bioRxiv2019

Bacterial Phage Tail-like Structure Kills Eukaryotic Cells by Injecting a Nuclease Effector

Many bacteria interact with target organisms using syringe-like structures called Contractile Injection Systems (CIS). CIS structurally resemble headless bacteriophages and share evolutionarily related proteins such as the tail tube, sheath, and baseplate complex. Recent evidence shows that CIS are specialized to puncture membranes and often deliver effectors to target cells. In many cases, CIS mediate trans-kingdom interactions between bacteria and eukaryotes, however the effectors delivered to target cells and their mode of action are often unknown. In this work, we establish an in vitro model to study a CIS called Metamorphosis Associated Contractile structures (MACs) that target eukaryotic cells. We show that MACs kill two eukaryotic cell lines, Fall Armyworm Sf9 cells and J774A.1 murine macrophage cells through the action of a newly identified MAC effector, termed Pne1. To our knowledge, Pne1 is the first CIS effector exhibiting nuclease activity against eukaryotic cells. Our results define a new mechanism of CIS-mediated bacteria-eukaryote interaction and are a first step toward understanding structures with the potential to be developed as novel delivery systems for eukaryotic hosts.

microbiology↗

bioRxiv2019

Opposing signaling pathways regulate morphology in response to temperature in the fungal pathogen Histoplasma capsulatum

Phenotypic switching between two opposing cellular states is a fundamental aspect of biology, and fungi provide facile systems to analyze the interactions between regulons that control this type of switch. A long-standing mystery in fungal pathogens of humans is how thermally dimorphic fungi switch their developmental form in response to temperature. These fungi, including the subject of this study, Histoplasma capsulatum, are temperature-responsive organisms that utilize unknown regulatory pathways to couple their cell shape and associated attributes to the temperature of their environment. H. capsulatum grows as a multicellular hypha in the soil that switches to a pathogenic yeast form in response to the temperature of a mammalian host. These states can be triggered in the laboratory simply by growing the fungus either at room temperature (where it grows as hyphae) or at 37{degrees}C (where it grows as yeast). Prior worked revealed that 15-20% of transcripts are differentially expressed in response to temperature, but it is unclear which transcripts are linked to specific phenotypic changes such as cell morphology or virulence. To elucidate temperature-responsive regulons, we previously identified four transcription factors (Ryp1-4) that are required for yeast-phase growth at 37{degrees}C; in each ryp mutant, the fungus grows constitutively as hyphae regardless of temperature and the cells fail to express genes that are normally induced in response to growth at 37{degrees}C. Here we perform the first genetic screen to identify genes required for hyphal growth of H. capsulatum at room temperature and find that disruption of the signaling mucin MSB2 results in a yeast-locked phenotype. RNAseq experiments reveal that MSB2 is not required for the majority of gene expression changes that occur when cells are shifted to room temperature. However, a small subset of temperature-responsive genes is dependent on MSB2 for its expression, thereby implicating these genes in the process of filamentation. Disruption or knockdown of an Msb2-dependent MAP kinase (HOG2) and an APSES transcription factor (STU1) prevents hyphal growth at room temperature, validating that the Msb2 regulon contains genes that control filamentation. Notably, the Msb2 regulon shows conserved hyphal-specific expression in other dimorphic fungi, suggesting that this work defines a small set of genes that are likely to be conserved regulators and effectors of filamentation in multiple fungi. In contrast, a few yeast-specific transcripts, including virulence factors that are normally expressed only at 37{degrees}C, are inappropriately expressed at room temperature in the msb2 mutant, suggesting that expression of these genes is coupled to growth in the yeast form rather than to temperature. Finally, we find that the yeast-promoting transcription factor Ryp3 associates with the MSB2 promoter and inhibits MSB2 transcript expression at 37{degrees}C, whereas Msb2 inhibits accumulation of Ryp transcripts and proteins at room temperature. These findings indicate that the Ryp and Msb2 circuits antagonize each other in a temperature-dependent manner, thereby allowing temperature to govern cell shape and gene expression in this ubiquitous fungal pathogen of humans.

microbiology↗