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

Expression of <i>C</i>. <i>albicans</i> environmentally responsive genes during invasive infection.

<p>Changes in expression levels during mouse kidney invasion for 248 <i>C</i>. <i>albicans</i> genes (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s001" target="_blank">S1 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). Color saturation represents the extent of the expression change, with full saturation at 10-fold up- or down-regulation. (All heat maps in this article follow the same color scale.) 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 illustrate reproducibility. We define early expression changes as significant differences between the inoculum and 12 hr sample. We define late expression changes as significant differences between the 12 and 48 hr samples. Significance refers to changes of ≥2-fold and a <i>p</i>-value < 0.05. The data for each sample were normalized to RNA levels from control gene <i>TDH3</i> before mean values were calculated. Our assignment criteria allow some dynamically regulated genes to fall into both the early and late expression classes. 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

Comparison of <i>C</i>. <i>albicans</i> proliferation-defective transcription factor mutants.

<p>(A). Heat map representation of gene expression ratios for 148 environmentally responsive genes at 24 hr postinfection (<i>rim101Δ/Δ</i>, <i>rob1Δ/Δ</i>, <i>rpn4Δ/Δ</i>, <i>sut1Δ/Δ</i>, or <i>zap1Δ/Δ</i>, each relative to the wild type) or 48 hr postinfection (<i>efg1Δ/Δ</i>, relative to the wild type). Complete data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s005" target="_blank">S5 Data</a>. (B). Yield of <i>C</i>. <i>albicans</i> RNA relative to total kidney RNA for each mutant and complemented strain at 24 hr postinfection. Asterisks mark significant differences (<i>p</i>-value < 0.05) between mutants and respective complemented strains. (C). Histopathology images of kidney sections. All samples were at 24 hr postinfection. Arrows indicate fungal cells. The scale bar corresponds to 50 microns. For panel A, the specific strains used and the dose of viable cells per mouse, as determined by plating the inocula, were: CW 696 (wild type) 8.4 × 10⁵; CW730 (<i>rob1</i>) 10.2 × 10⁵; DAY25 (<i>rim101</i>) 11.1 × 10⁵; CW1018 (<i>efg1</i>) 9 × 10⁵; CW792 (<i>rpn4</i>) 7.9 × 10⁵; CW756 (<i>zap1</i>) 8.8 × 10⁵; CW704 (<i>sut1</i>) 8.6 × 10⁵. 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

Mouse immune response gene expression during <i>Candida</i> infection.

<p>(A). RNA levels for 46 mouse immune response genes were assayed at 12, 24, and 48 hr postinfection in biological triplicates and were compared to uninfected kidney controls (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s003" target="_blank">S3 Data</a>). Mouse genes that represent early and late expression classes are presented. We define early genes as those with significantly higher expression in 12 hr postinfection samples compared to uninfected samples. We define late genes as those with no significant difference in expression between uninfected and 12 hr postinfection samples, but with significantly higher expression in 48 hr postinfection samples compared to 24 hr postinfection samples. An asterisk indicates significant differences (<i>p</i>-value < 0.05) between successive time-course samples (uninfected samples versus 12 hr, 24 hr versus 12 hr, 48 hr versus 24 hr). (B). Mice were infected with the indicated transcription factor mutants and respective complemented strains; RNA levels for 46 mouse immune response genes were determined by nanoString at 24h postinfection (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s003" target="_blank">S3 Data</a>). Induction of four early genes (the same genes shown in panel A for the wild-type strain) are shown here. All four genes were induced significantly more strongly by complemented strains than the respective mutants (<i>p</i>-value < 0.05), as indicated by the bracket and asterisk. <i>P</i>-values for expression of each gene are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s003" target="_blank">S3 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

Rim101-dependent gene regulation during invasive infection.

<p>RNA levels for 148 <i>C</i>. <i>albicans</i> environmental response genes were determined by nanoString at 24 hr postinfection for the <i>rim101</i> mutant and complemented strains (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s005" target="_blank">S5 Data</a>). (A). Expression ratios are plotted for each gene in <i>rim101Δ/Δ</i> versus wild type (X axis) and <i>rim101Δ/Δ</i> versus complemented strain (Y axis). Three genes (red data points) have significantly different expression ratios in the two comparisons. (B). Expression ratios are presented for all genes significantly down-regulated in the <i>rim101Δ/Δ</i> strain relative to the complemented strain during kidney infection (blue bars; ≥2-fold change and <i>p</i>-value < 0.05). The expression ratios for the same genes in the same strains during intra-abdominal candidiasis (IAC) infection (red bars; reported in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.ref012" target="_blank">12</a>]) or during in vitro growth in Spider medium (green bars) are displayed. Complete data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s005" target="_blank">S5 Data</a>. (C). Expression ratios are presented for all genes significantly up-regulated in the <i>rim101Δ/Δ</i> strain relative to the complemented strain during kidney infection (blue bars; ≥2-fold change and <i>p</i>-value < 0.05), during abdominal infection (red bars; reported in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.ref012" target="_blank">12</a>]) or during in vitro growth in Spider medium (green bars) are displayed. Complete data are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s005" target="_blank">S5 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

Transcription factor gene expression and function in vivo.

<p>¹ The top 30 transcription factor genes in each indicated category are based on expression data at 48 hr postinfection (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002076#pbio.1002076.s004" target="_blank">S4 Data</a>). "Highly expressed" or "Weakly expressed" refers to the rank order of nanoString counts at 48 hr postinfection. "Up-regulated" or "Down-regulated" refers to the fold change in nanoString counts at 48 hr postinfection compared to inoculum samples.</p><p>² Virulence or proliferation of a null mutant in the respective gene was compiled from information at the Candida Genome Database; n.a. = data not available. Data for <i>RPN4</i> and <i>SUT1</i> are from this study. Function was summarized from association with Gene Ontology (GO) terms "filamentous growth" (f) or "response to stress" (s).</p><p>Transcription factor gene expression and function in vivo.</p

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