Search

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.

Next generation sequencing has unlocked a wealth of genotype information for microbial populations, but phenotyping remains a bottleneck for exploiting this information, particularly for pathogens that are difficult to manipulate. Here, we establish a method for high-throughput phenotyping of mixed cultures, in which the pattern of naturally occurring single-nucleotide polymorphisms in each isolate is used as intrinsic barcodes which can be read out by sequencing. We demonstrate that our method can correctly deconvolute strain proportions in simulated mixed-strain pools. As an experimental test of our method, we perform whole genome sequencing of 66 natural isolates of the thermally dimorphic pathogenic fungus Coccidioides posadasii and infer the strain compositions for large mixed pools of these strains after competition at 37°C and room temperature. We validate the results of these selection experiments by recapitulating the temperature-specific enrichment results in smaller pools. Additionally, we demonstrate that strain fitness estimated by our method can be used as a quantitative trait for genome-wide association studies. We anticipate that our method will be broadly applicable to natural populations of microbes and allow high-throughput phenotyping to match the rate of genomic data acquisition.

The ability of the human fungal pathogen Cryptococcus neoformans to adapt to variable copper (Cu) environments within the host is key for successful dissemination and colonization. During pulmonary infection, host alveolar macrophages compartmentalize Cu into the phagosome and C. neoformans Cu-detoxifying metallothioneins, MT1 and MT2, are required for survival of the pathogen. In contrast, during brain colonization the C. neoformans Cu + importers Ctr1 and Ctr4 are required for virulence. Central for the regulation and expression of both the Cu detoxifying MT1/2 and the Cu acquisition Ctr1/4 proteins is the Cu-metalloregulatory transcription factor Cuf1, an established C. neoformans virulence factor. Due to the importance of the distinct C. neoformans Cu homeostasis mechanisms during host colonization and virulence, and to the central role of Cuf1 in regulating Cu homeostasis, we performed a combination of RNA-Seq and ChIP-Seq experiments to identify differentially transcribed genes between conditions of high and low Cu. We demonstrate that the transcriptional regulation exerted by Cuf1 is intrinsically complex and that Cuf1 also functions as a transcriptional repressor. The Cu- and Cuf1-dependent regulon in C. neoformans reveals new adaptive mechanisms for Cu homeostasis in this pathogenic fungus and identifies potential new pathogen-specific targets for therapeutic intervention in fungal infections., (© 2018 John Wiley & Sons Ltd.)

Multiple lines of evidence implicate chromatin in the regulation of premessenger RNA (pre-mRNA) splicing. However, the influence of chromatin factors on cotranscriptional splice site usage remains unclear. Here we investigated the function of the highly conserved histone variant H2A.Z in pre-mRNA splicing using the intron-rich model yeast Schizosaccharomyces pombe Using epistatic miniarray profiles (EMAPs) to survey the genetic interaction landscape of the Swr1 nucleosome remodeling complex, which deposits H2A.Z, we uncovered evidence for functional interactions with components of the spliceosome. In support of these genetic connections, splicing-specific microarrays show that H2A.Z and the Swr1 ATPase are required during temperature stress for the efficient splicing of a subset of introns. Notably, affected introns are enriched for H2A.Z occupancy and more likely to contain nonconsensus splice sites. To test the significance of the latter correlation, we mutated the splice sites in an affected intron to consensus and found that this suppressed the requirement for H2A.Z in splicing of that intron. These data suggest that H2A.Z occupancy promotes cotranscriptional splicing of suboptimal introns that may otherwise be discarded via proofreading ATPases. Consistent with this model, we show that overexpression of splicing ATPase Prp16 suppresses both the growth and splicing defects seen in the absence of H2A.Z., (© 2017 Nissen et al.; Published by Cold Spring Harbor Laboratory Press.)

The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence virulence, but in most cases the substrate specificity and regulation of these enzymes remains poorly understood. The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets. We report here an unbiased analysis of secreted peptidase activity and specificity in C. neoformans using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations. Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions. To uncover the substrate preferences of individual enzymes and interrogate their biological functions, we constructed and profiled a ten-member gene deletion collection of candidate secreted peptidases. Through this deletion approach, we characterized the substrate specificity of three peptidases within the context of the C. neoformans secretome, including an enzyme known to be important for fungal entry into the brain. We selected a previously uncharacterized peptidase, which we term Major aspartyl peptidase 1 (May1), for detailed study due to its substantial contribution to extracellular proteolytic activity. Based on the preference of May1 for proteolysis between hydrophobic amino acids, we screened a focused library of aspartyl peptidase inhibitors and identified four high-affinity antagonists. Finally, we tested may1Δ strains in a mouse model of C. neoformans infection and found that strains lacking this enzyme are significantly attenuated for virulence. Our study reveals the secreted peptidase activity and specificity of an important human fungal pathogen, identifies responsible enzymes through genetic tests of their function, and demonstrates how this information can guide the development of high affinity small molecule inhibitors., Competing Interests: The authors have declared that no competing interests exist.

Eukaryotic gene expression requires that RNA Polymerase II (RNAP II) gain access to DNA in the context of chromatin. The C-terminal domain (CTD) of RNAP II recruits chromatin modifying enzymes to promoters, allowing for transcription initiation or repression. Specific CTD phosphorylation marks facilitate recruitment of chromatin modifiers, transcriptional regulators, and RNA processing factors during the transcription cycle. However, the readable code for recruiting such factors is still not fully defined and how CTD modifications affect related families of genes or regional gene expression is not well understood. Here, we examine the effects of manipulating the Y 1 S 2 P 3 T 4 S 5 P 6 S 7 heptapeptide repeat of the CTD of RNAP II in Schizosaccharomyces pombe by substituting non-phosphorylatable alanines for Ser2 and/or Ser7 and the phosphomimetic glutamic acid for Ser7. Global gene expression analyses were conducted using splicing-sensitive microarrays and validated via RT-qPCR. The CTD mutations did not affect pre-mRNA splicing or snRNA levels. Rather, the data revealed upregulation of subtelomeric genes and alteration of the repressive histone H3 lysine 9 methylation (H3K9me) landscape. The data further indicate that H3K9me and expression status are not fully correlated, suggestive of CTD-dependent subtelomeric repression mechansims that act independently of H3K9me levels., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C. neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We characterize the Polycomb system that assembles repressive subtelomeric domains of H3K27 methylation (H3K27me) in the yeast Cryptococcus neoformans. Purification of this PRC2-like protein complex reveals orthologs of animal PRC2 components as well as a chromodomain-containing subunit, Ccc1, which recognizes H3K27me. Whereas removal of either the EZH or EED ortholog eliminates H3K27me, disruption of mark recognition by Ccc1 causes H3K27me to redistribute. Strikingly, the resulting pattern of H3K27me coincides with domains of heterochromatin marked by H3K9me. Indeed, additional removal of the C. neoformans H3K9 methyltransferase Clr4 results in loss of both H3K9me and the redistributed H3K27me marks. These findings indicate that the anchoring of a chromatin-modifying complex to its product suppresses its attraction to a different chromatin type, explaining how enzymes that act on histones, which often harbor product recognition modules, may deposit distinct chromatin domains despite sharing a highly abundant and largely identical substrate-the nucleosome., (Copyright © 2015 Elsevier Inc. All rights reserved.)