Gene regulatory network directing <i>C. albicans</i> proliferation in a mammalian host.

<p>(A) Gene network composed of the transcription regulators <i>RTG1/3</i>, <i>HMS1</i>, <i>ZCF21</i>, and <i>TYE7</i> (orange circles) and their target genes (black circles) as determined by full-genome chromatin immunoprecipitation. Thin lines indicate binding of the specified regulator to a target gene. About a quarter of the genes in the network (those in the middle) are targets shared by two or more regulators. (B) Relationships among the “master” regulators at the core of the network. Arrows represent protein-DNA interactions. Notice that the circuit displays multiple autoregulatory, feed-forward, and feedback loops. Adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003780#ppat.1003780-Prez1" target="_blank">[8]</a>.</p

Phagocytosis of white and opaque <i>C. albicans</i> by <i>D. melanogaster</i> S2 and <i>M. musculus</i> RAW cells.

<p>White (A,C) or opaque (B,D) <i>C. albicans</i> cells were co-incubated with S2 cells for 3.5 hours (A,B) or RAW cells for 1 hour (C,D), lightly fixed with formaldehyde, stained with rabbit anti-Candida and Cy3-labeled anti-rabbit antibodies. Cells were then stained with a DAPI solution (blue) to localize S2 or RAW cells. <i>C. albicans</i> cells that were not phagocytosed appear orange in these figures.</p

S2 cells preferentially phagocytose white <i>C. albicans</i> from a mixed white-opaque population.

<p>(A) <i>D. melanogaster</i> S2 cells were co-incubated with equal numbers of white and opaque <i>C. albicans</i> for 1, 2, and 3.5 hours. The number of S2 cells phagocytosing one or more <i>C. albicans</i> cells was determined. (B) For the same S2 cells, the number of <i>C. albicans</i> cells phagocytosed was quantified and the total number of <i>C. albicans</i> cells phagocytosed divided by the number of S2 cells scored, referred to as the phagocytic index, was plotted. 100 S2 cells were counted for each data set; values reflect the average of six data sets. For the 3.5 hour time point, statistical significance of differences from the a/a whites was determined using a t-test and differences with p<.001 are marked with an asterisk.</p

A gene regulatory network comprising <i>C. albicans</i> genes upregulated in the host.

<p>(A) Gene regulatory network depicting the established 808 target genes (orange circles) connected to their respective TRs (hubs) by dashed lines which indicate a direct interaction as determined by ChIP-chip. Dark grey circles correspond to the 153 genes upregulated in the GI tract. (B) A significant proportion of the target genes identified by ChIP-chip (<i>n</i> = 808) corresponds to genes upregulated when <i>C. albicans</i> grows in the GI tract (<i>n</i> = 408) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001510#pbio.1001510-Rosenbach1" target="_blank">[19]</a>. The hypergeometric distribution was used to evaluate the significance of the overlap and its <i>p</i>-value is indicated.</p

<em>Candida albicans</em> Commensalism and Pathogenicity Are Intertwined Traits Directed by a Tightly Knit Transcriptional Regulatory Circuit

<div><p>Systemic, life-threatening infections in humans are often caused by bacterial or fungal species that normally inhabit a different locale in our body, particularly mucosal surfaces. A hallmark of these opportunistic pathogens, therefore, is their ability to thrive in disparate niches within the host. In this work, we investigate the transcriptional circuitry and gene repertoire that enable the human opportunistic fungal pathogen <i>Candida albicans</i> to proliferate in two different niches. By screening a library of transcription regulator deletion strains in mouse models of intestinal colonization and systemic infection, we identified eight transcription regulators that play roles in at least one of these models. Using genome-wide chromatin immunoprecipitation, we uncovered a network comprising ∼800 target genes and a tightly knit transcriptional regulatory circuit at its core. The network is enriched with genes upregulated in <i>C. albicans</i> cells growing in the host. Our findings indicate that many aspects of commensalism and pathogenicity are intertwined and that the ability of this microorganism to colonize multiple niches relies on a large, integrated circuit.</p> </div

Gastrointestinal tract colonization and systemic infection screens.

<p>(A) About 45% of <i>C. albicans</i> TR deletion strains (<i>n</i> = 165) display no significant growth or colony morphology phenotype under any of 55 different laboratory growth conditions <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001510#pbio.1001510-Homann1" target="_blank">[16]</a>. We used this subset of TRs (<i>n</i> = 77) to carry out genetic screens in mouse models that recapitulate niches where <i>C. albicans</i> thrives. (B) Schematic of the GI tract colonization and bloodstream infection screens. In the GI tract colonization model we used two different approaches to recover DNA from the fecal pellets and intestinal contents: (1) DNA was prepared directly from the samples, or (2) the samples were first plated and the DNA was purified from yeasts scraped off the plates. Similar results were obtained with samples that were processed directly or that were plated.</p

Genome-wide identification of DNA regions bound by TRs.

<p>(A) Summary of experimental conditions and results of ChIP. ChIP-chip was carried out for each of the identified regulators under the conditions described. The number of intergenic regions bound by each regulator is indicated. The DNA motifs shown were derived de novo from our ChIP-chip data and are significantly enriched in the bound regions. (B) The <i>C. albicans</i> Lys14 protein binds in vitro to the DNA sequence identified by ChIP-chip. The DNA-binding domain of the <i>C. albicans</i> Lys14 protein (amino acids 1–236) was N-terminally fused to 6His and to the maltose binding protein, expressed in <i>Escherichia coli</i> and purified with Ni-NTA columns. ³²P-labeled 24-nt DNA fragments (∼0.4 nM) containing the predicted wild-type or mutant Lys14 binding site were incubated with increasing concentrations of purified Lys14 protein (0, 0.039, 0.156, 0.625, 2.5, 10, and 40 nM) for 30 min at room temperature in standard EMSA buffer and resolved in 6% polyacrylamide gels run with 0.5× TGE. The DNA fragment tested corresponds to the <i>OCH1</i> promoter. The point mutations that we inserted to disrupt the putative binding site are indicated in red.</p

Target genes required for proliferation in the host.

<p>GI tract colonization (top) and systemic infection (bottom) analyses of selected target genes upregulated in the GI tract and controlled by both <i>HMS1</i> and <i>RTG1/3</i>. Blue dots represent samples below the qPCR detection level. Mutants with statistically significant GI tract colonization impairment are in green; statistically significant systemic infection defect is indicated in red.</p

Identification of regulators that govern <i>C. albicans</i> proliferation in the murine gastrointestinal tract.

<p>(A) Log₂ (recovered/input) values for <i>C. albicans</i> TR mutants at different time points after oral inoculation by gavage in three mice. The order in which the mutants are displayed reflects hierarchical clustering. Color intensity indicates reduction (blue) or accumulation (yellow). An independent isolate of each of the top eight mutants in the panel was tested in an iteration of the screen. <i>orf19.4972</i> and <i>orf19.2730</i> did not reproduce the effect. (B) The percentage of mice with detectable levels of various <i>C. albicans</i> mutants at different time points after gavage are plotted and their <i>p</i>-values (logrank test) are indicated.</p

Identification of regulators required for <i>C. albicans</i> systemic infection.

<p>(A) Results of the systemic infection screen. Bars represent median log₂ (recovered/input) values. (B–D) Virulence analysis of selected mutant deletion strains in monotypic infections. Ten BALB/c mice were infected with wild-type <i>C. albicans</i> or one of the mutant strains by tail vein injection. We used 5.2×10⁵ cells of each strain per infection. Mice were monitored daily and sacrificed when moribund. The logrank test was used for statistical analysis: <i>p</i><0.0001 for <i>zcf21</i>; <i>p</i> = 0.0008 for <i>rtg1</i>; <i>p</i> = 0.0203 for <i>hms1</i>; <i>p</i> = 0.0166 for <i>lys14</i>; and <i>p</i> = 0.0001 for <i>rtg3</i>. (E) Summary of the TRs displaying phenotypes in the mouse models that we evaluated.</p