Gliotoxin contributes to the virulence of the fungus Aspergillus fumigatus in non-neutropenic mice that are immunosuppressed with corticosteroids. To investigate how the absence of gliotoxin affects both the fungus and the host, we used a nanoString nCounter to analyze their transcriptional responses during pulmonary infection of a non-neutropenic host with a gliotoxin-deficient ΔgliP mutant. We found that the ΔgliP mutation led to increased expression of aspf1, which specifies a secreted ribotoxin. Prior studies have shown that aspf1, like gliP, is not required for virulence in a neutropenic infection model, but its role in a non-neutropenic infection model has not been fully investigated. To investigate the functional significance of this up-regulation of aspf1, a Δaspf1 single mutant and a Δaspf1 ΔgliP double mutant were constructed. Both Δaspf1 and ΔgliP single mutants had reduced lethality in non-neutropenic mice, and a Δaspf1 ΔgliP double mutant had a greater reduction in lethality than either single mutant. Analysis of mice infected with these mutants indicated that the presence of gliP is associated with massive apoptosis of leukocytes at the foci of infection and inhibition of chemokine production. Also, the combination of gliP and aspf1 is associated with suppression of CXCL1 chemokine expression. Thus, aspf1 contributes to A. fumigatus pathogenicity in non-neutropenic mice and its up-regulation in the ΔgliP mutant may partially compensate for the absence of gliotoxin.

Abbreviations

PAS: periodic acid-Schiff; PBS: phosphate buffered saline; ROS: reactive oxygen species; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Candida albicans is a gut commensal and opportunistic pathogen. The transition between yeast and invasive hyphae is central to virulence but has unknown functions during commensal growth. In a mouse model of colonization, yeast and hyphae co-occur throughout the gastrointestinal tract. However, competitive infections of C. albicans homozygous gene disruption mutants revealed an unanticipated, inhibitory role for the yeast-to-hypha morphogenesis program on commensalism. We show that the transcription factor Ume6, a master regulator of filamentation, inhibits gut colonization, not by effects on cell shape, but by activating the expression of a hypha-specific pro-inflammatory secreted protease, Sap6, and a hyphal cell surface adhesin, Hyr1. Like a ume6 mutant, strains lacking SAP6 exhibit enhanced colonization fitness, whereas SAP6-overexpression strains are attenuated in the gut. These results reveal a tradeoff between fungal programs supporting commensalism and virulence in which selection against hypha-specific markers limits the disease-causing potential of this ubiquitous commensal-pathogen.

Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality.

Gene expression dynamics have provided foundational insight into almost all biological processes. Here, we analyze expression of environmentally responsive genes and transcription factor genes to infer signals and pathways that drive pathogen gene regulation during invasive Candida albicans infection of a mammalian host. Environmentally responsive gene expression shows that there are early and late phases of infection. The early phase includes induction of zinc and iron limitation genes, genes that respond to transcription factor Rim101, and genes characteristic of invasive hyphal cells. The late phase includes responses related to phagocytosis by macrophages. Transcription factor gene expression also reflects early and late phases. Transcription factor genes that are required for virulence or proliferation in vivo are enriched among highly expressed transcription factor genes. Mutants defective in six transcription factor genes, three previously studied in detail (Rim101, Efg1, Zap1) and three less extensively studied (Rob1, Rpn4, Sut1), are profiled during infection. Most of these mutants have distinct gene expression profiles during infection as compared to in vitro growth. Infection profiles suggest that Sut1 acts in the same pathway as Zap1, and we verify that functional relationship with the finding that overexpression of either ZAP1 or the Zap1-dependent zinc transporter gene ZRT2 restores pathogenicity to a sut1 mutant. Perturbation with the cell wall inhibitor caspofungin also has distinct gene expression impact in vivo and in vitro. Unexpectedly, caspofungin induces many of the same genes that are repressed early during infection, a phenomenon that we suggest may contribute to drug efficacy. The pathogen response circuitry is tailored uniquely during infection, with many relevant regulatory relationships that are not evident during growth in vitro. Our findings support the principle that virulence is a property that is manifested only in the distinct environment in which host-pathogen interaction occurs.

Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality.

To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which is required for the organism to achieve maximal fungal burden in the lung. The other was sltA, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the ΔsltA mutant is due in part to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators.