Diagram of the interactions of <i>Candida albicans</i> with oral epithelial cells, host defense peptides (HDPs), and the oral microbiota.

<p>(A) <i>C</i>. <i>albicans</i> invasion of epithelial cells by receptor-mediated endocytosis. The <i>C</i>. <i>albicans</i> Als3 and Ssa1 invasins interact with E-cadherin and a heterodimer composed of the epidermal growth factor receptor (EGFR) and HER2, which activate the clathrin endocytosis pathway, resulting in the endocytosis of the fungus. (B) <i>C</i>. <i>albicans</i> invasion by active penetration, in which a progressively elongating hyphus pushes its way into the epithelial cell. (C) Host defense peptides (HDPs) released by the infected epithelial cell can kill <i>C</i>. <i>albicans</i>. However, <i>C</i>. <i>albicans</i> can resist HDPs by up-regulating the Flu1 efflux pump, which reduces intracellular HDPs, by secreting aspartyl proteases (SAPs), which degrade HDPs, and by shedding of the Msb2 mucin, which binds to and inactivates HDPs. (D) <i>C</i>. <i>albicans</i> can invade between oral epithelial cells by proteolytic degradation of intercellular junctional proteins. (E) <i>C</i>. <i>albicans</i> hyphae bind <i>Candida glabrata</i> and bacteria such as <i>Staphylococcus aureus</i> and <i>Streptococcus</i> spp. <i>C</i>. <i>albicans</i> can enhance the capacity of some of these organisms to invade epithelial cells, while some of these organisms can increase the virulence of <i>C</i>. <i>albicans</i>. (F) <i>C</i>. <i>albicans</i> secretes candidalysin, a toxin that causes epithelial damage.</p

Divergent targets of Candida albicans biofilm regulator Bcr1 in vitro and in vivo.

<p>Candida albicans is a causative agent of oropharyngeal candidiasis (OPC), a biofilm-like infection of the oral mucosa. Biofilm formation depends upon the C. albicans transcription factor Bcr1, and previous studies indicate that Bcr1 is required for OPC in a mouse model of infection. Here we have used a nanoString gene expression measurement platform to elucidate the role of Bcr1 in OPC-related gene expression. We chose for assays a panel of 134 genes that represent a range of morphogenetic and cell cycle functions as well as environmental and stress response pathways. We assayed gene expression in whole infected tongue samples. The results sketch a portrait of C. albicans gene expression in which numerous stress response pathways are activated during OPC. This one set of experiments identifies 64 new genes with significantly altered RNA levels during OPC, thus increasing substantially the number of known genes in this expression class. The bcr1Δ/Δ mutant had a much more limited gene expression defect during OPC infection than previously reported for in vitro growth conditions. Among major functional Bcr1 targets, we observed that ALS3 was Bcr1 dependent in vivo while HWP1 was not. We used null mutants and complemented strains to verify that Bcr1 and Hwp1 are required for OPC infection in this model. The role of Als3 is transient and mild, though significant. Our findings suggest that the versatility of C. albicans as a pathogen may reflect its ability to persist in the face of multiple stresses and underscore that transcriptional circuitry during infection may be distinct from that detailed during in vitro growth.</p

Genome Mining of a Prenylated and Immunosuppressive Polyketide from Pathogenic Fungi

Activation of the polycyclic polyketide prenyltransferase (pcPTase)-containing silent clusters in <i>Aspergillus fumigatus</i> and <i>Neosartorya fischeri</i> led to isolation of a new metabolite neosartoricin (<b>3</b>). The structure of <b>3</b> was solved by X-ray crystallography and NMR to be a prenylated anthracenone. <b>3</b> exhibits T-cell antiproliferative activity with an IC₅₀ of 3 μM, suggestive of a physiological role as an immunosuppressive agent

Nerita virginea

Activation of the polycyclic polyketide prenyltransferase (pcPTase)-containing silent clusters in <i>Aspergillus fumigatus</i> and <i>Neosartorya fischeri</i> led to isolation of a new metabolite neosartoricin (<b>3</b>). The structure of <b>3</b> was solved by X-ray crystallography and NMR to be a prenylated anthracenone. <b>3</b> exhibits T-cell antiproliferative activity with an IC₅₀ of 3 μM, suggestive of a physiological role as an immunosuppressive agent

Functional convergence of <i>gliP</i> and <i>aspf1</i> in <i>Aspergillus fumigatus</i> pathogenicity

<p>Gliotoxin contributes to the virulence of the fungus <i>Aspergillus fumigatus</i> 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 Δ<i>gliP</i> mutant. We found that the Δ<i>gliP</i> mutation led to increased expression of <i>aspf1</i>, which specifies a secreted ribotoxin. Prior studies have shown that <i>aspf1</i>, like <i>gliP</i>, 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 <i>aspf1</i>, a Δ<i>aspf1</i> single mutant and a Δ<i>aspf1</i> Δ<i>gliP</i> double mutant were constructed. Both Δ<i>aspf1</i> and Δ<i>gliP</i> single mutants had reduced lethality in non-neutropenic mice, and a Δ<i>aspf1</i> Δ<i>gliP</i> double mutant had a greater reduction in lethality than either single mutant. Analysis of mice infected with these mutants indicated that the presence of <i>gliP</i> is associated with massive apoptosis of leukocytes at the foci of infection and inhibition of chemokine production. Also, the combination of <i>gliP</i> and <i>aspf1</i> is associated with suppression of CXCL1 chemokine expression. Thus, <i>aspf1</i> contributes to <i>A. fumigatus</i> pathogenicity in non-neutropenic mice and its up-regulation in the Δ<i>gliP</i> mutant may partially compensate for the absence of gliotoxin.</p> <p><b>Abbreviations</b>:PAS: periodic acid-Schiff; PBS: phosphate buffered saline; ROS: reactive oxygen species; TUNEL: terminal deoxynucleotidyl transferase dUTP nick-end labeling</p

Activation and Alliance of Regulatory Pathways in <i>C. albicans</i> during Mammalian Infection

<div><p>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 <i>Candida albicans</i> 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 <i>ZAP1</i> or the Zap1-dependent zinc transporter gene <i>ZRT2</i> restores pathogenicity to a <i>sut1</i> 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.</p></div

Gene expression response to caspofungin treatment during infection.

<p>(A). Changes in expression levels for 248 <i>C</i>. <i>albicans</i> environmentally responsive genes are presented for caspofungin treated versus untreated cells at 24 hr postinfection (“Kidney,” <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s006" target="_blank">S6 Data</a>), in vitro in YPD at 30°C (“YPD,” from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.ref030" target="_blank">30</a>]), and in vitro in RPMI at 37°C (“RPMI,” <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s006" target="_blank">S6 Data</a>). These environmentally responsive genes are the same ones for which expression was measured during the time-course of infection depicted in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.g001" target="_blank">Fig. 1</a>. For comparison, the expression ratios of the same genes at 12 hr postinfection relative to the inoculum are shown (“12 hr/0 hr,” from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.g001" target="_blank">Fig. 1</a>). The data are presented in heat map format. Regions “1” and “2” are expanded on the right to make gene names legible. (B). Expression levels for 231 <i>C</i>. <i>albicans</i> transcription factor genes were measured for caspofungin treated versus untreated cells at 24 hr postinfection (“In vivo caspo-induced,” <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s006" target="_blank">S6 Data</a>) and in vitro in RPMI at 37°C (“In vitro caspo-induced,” <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s006" target="_blank">S6 Data</a>). Significantly up-regulated transcription factor genes are listed (≥2-fold change and <i>p</i> < 0.05). For comparison, the significantly down-regulated transcription factor genes at 12 hr postinfection are listed (“Early down-regulated,” <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s006" target="_blank">S6 Data</a>). All numerical data for this figure are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s007" target="_blank">S7 Data</a>.</p

Gene expression during murine infection.

<p>Expression levels for 114 genes are compared in three murine infection models: oropharyngeal candidiasis (48 hr postinfection; oropharyngeal candidiasis [OPC]) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.ref013" target="_blank">13</a>], kidney infection (12, 24, and 48 hr [this study]), and intra-abdominal infection (48 hr postinfection; IAC) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.ref012" target="_blank">12</a>]. Expression levels are presented as ratios to levels in the inoculum samples used in this study (stationary phase, YPD), and shown as a heat map. Expanded portions illustrate genes induced during oral infection, during all three types of infection, and during abdominal infection. All numerical data for this figure are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s007" target="_blank">S7 Data</a>.</p

Expression of <i>C</i>. <i>albicans</i> transcription factor genes during invasive infection.

<p>Changes in expression levels during mouse kidney invasion for 231 <i>C</i>. <i>albicans</i> transcription factor genes (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s004" target="_blank">S4 Data</a>) are presented in a heat map format. Mean values of biological triplicates are shown for up-regulation (yellow) and down-regulation (blue) of genes at 12, 24, and 48 hr postinfection relative to mean inoculum levels (0 hr). Portions of the heat map are expanded to illustrate representative early up-regulated genes (top), late genes (middle), and early down-regulated genes (bottom). In these portions, individual samples are presented separately to indicate reproducibility. The early and late gene classes were defined as described in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.g001" target="_blank">Fig. 1</a> legend. All numerical data for this figure are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s007" target="_blank">S7 Data</a>.</p

Effect of <i>ZAP1</i> and <i>ZRT2</i> overexpression in a <i>sut1Δ/Δ</i> mutant.

<p>(A). Expression of zinc acquisition genes <i>ZAP1</i>, <i>PRA1</i>, <i>ZRT1</i>, and <i>ZRT2</i> was measured by nanoString at 24 hr postinfection in the wild type, the <i>zap1Δ/Δ</i> mutant, the <i>sut1Δ/Δ</i> mutant, and the <i>sut1Δ/Δ</i> mutant that overexpresses <i>ZAP1</i>. The mean of triplicate determinations is shown. (B). Mouse survival was determined after inoculation with the wild-type strain, the <i>sut1Δ/Δ</i> mutant, the <i>sut1Δ/Δ+pSUT1</i> complemented strain, the wild-type strain that overexpresses <i>ZAP1</i>, and the <i>sut1Δ/Δ</i> mutant that overexpresses <i>ZAP1</i>. Mouse survival was significantly better after infection with the <i>sut1Δ/Δ</i> mutant than after infection with the wild-type strain, the <i>sut1Δ/Δ+pSUT1</i> complemented strain, or the <i>sut1Δ/Δ</i> mutant that overexpresses <i>ZAP1</i> (<i>p</i> < 0.05 by the log-rank test). (C). Mouse survival was determined after inoculation with the wild-type strain that overexpresses <i>ZRT2</i> and the <i>sut1Δ/Δ</i> mutant that overexpresses <i>ZRT2</i>. Survival data after inoculation with the wild-type strain and the <i>sut1Δ/Δ</i> mutant from panel B are also included for comparison; all infections shown in panels B and C were carried out in parallel. The specific strains used and the dose of viable cells per mouse, as determined by plating the inocula, were: CW696 (wild type) 4.4 × 10⁵; CW704 (<i>sut1</i>) 4.9 × 10⁵; CW1035 (<i>SUT1</i> complement) 3.2 × 10⁵; WX134 (WT <i>ZAP1-OE</i>) 3.35 × 10⁵; WX102 (<i>sut1 ZAP1-OE</i>) 3.65 × 10⁵; WX144 (<i>sut1 ZRT2-OE</i>) 4.85 × 10⁵; WX137 (WT <i>ZRT2-OE</i>) 3.75 × 10⁵. Complete genotypes are given in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s012" target="_blank">S1 Table</a>. All numerical data for this figure are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s007" target="_blank">S7 Data</a>.</p