Genetic Modification of Closely Related Candida Species

Species from the genus Candida are among the most important human fungal pathogens. Several of them are frequent commensals of the human microbiota but are also able to cause a variety of opportunistic infections, especially when the human host becomes immunocompromised. By far, most of the research to understand the molecular underpinnings of the pathogenesis of these species has focused on Candida albicans, the most virulent member of the genus. However, epidemiological data indicates that related Candida species are also clinically important. Here, we describe the generation of a set of strains and plasmids to genetically modify C. dubliniensis and C. tropicalis, the two pathogenic species most closely related to C. albicans. C. dubliniensis is an ideal model to understand C. albicans pathogenesis since it is the closest species to C. albicans but considerably less virulent. On the other hand, C. tropicalis is ranked among the four most common causes of infections by Candida species. Given that C. dubliniensis and C. tropicalis are obligate diploids with no known conventional sexual cycle, we generated strains that are auxotrophic for at least two amino acids which allows the tandem deletion of both alleles of a gene by complementing the two auxotrophies. The strains were generated in two different genetic backgrounds for each species — one for which the genomic sequence is available and a second clinically important one. In addition, we have adapted plasmids developed to delete genes and epitope/fluorophore tag proteins in C. albicans so that they can be employed in C. tropicalis. The tools generated here allow for efficient genetic modification of C. dubliniensis and C. tropicalis, and thus facilitate the study of the molecular basis of pathogenesis in these medically relevant fungi

<em>MTL</em>–Independent Phenotypic Switching in <em>Candida tropicalis</em> and a Dual Role for Wor1 in Regulating Switching and Filamentation

<div><p>Phenotypic switching allows for rapid transitions between alternative cell states and is important in pathogenic fungi for colonization and infection of different host niches. In <i>Candida albicans</i>, the white-opaque phenotypic switch plays a central role in regulating the program of sexual mating as well as interactions with the mammalian host. White-opaque switching is controlled by genes encoded at the <i>MTL</i> (mating-type-like) locus that ensures that only <b>a</b> or α cells can switch from the white state to the mating-competent opaque state, while <b>a</b>/α cells are refractory to switching. Here, we show that the related pathogen <i>C. tropicalis</i> undergoes white-opaque switching in all three cell types (<b>a</b>, α, and <b>a</b>/α), and thus switching is independent of <i>MTL</i> control. We also demonstrate that <i>C. tropicalis</i> white cells are themselves mating-competent, albeit at a lower efficiency than opaque cells. Transcriptional profiling of <i>C. tropicalis</i> white and opaque cells reveals significant overlap between switch-regulated genes in <i>MTL</i> homozygous and <i>MTL</i> heterozygous cells, although twice as many genes are white-opaque regulated in <b>a</b>/α cells as in <b>a</b> cells. In <i>C. albicans</i>, the transcription factor Wor1 is the master regulator of the white-opaque switch, and we show that Wor1 also regulates switching in <i>C. tropicalis</i>; deletion of <i>WOR1</i> locks <b>a</b>, α, and <b>a</b>/α cells in the white state, while <i>WOR1</i> overexpression induces these cells to adopt the opaque state. Furthermore, we show that <i>WOR1</i> overexpression promotes both filamentous growth and biofilm formation in <i>C. tropicalis</i>, independent of the white-opaque switch. These results demonstrate an expanded role for <i>C. tropicalis</i> Wor1, including the regulation of processes necessary for infection of the mammalian host. We discuss these findings in light of the ancestral role of Wor1 as a transcriptional regulator of the transition between yeast form and filamentous growth.</p> </div

Products of <i>C. tropicalis</i> white×white and opaque×opaque mating maintain parental phenotypes.

<p>(A) Colony morphology of mating products. Left, product of mating <i>C. tropicalis</i> white <b>a</b> (CAY3376) with white α (CAY3391) cells. Right, product of mating opaque <b>a</b> (CAY3378) with opaque α (CAY3392) cells. Colonies were grown on SCD medium lacking histidine and arginine for 3 days at room temperature to select for mating products. (B) Cell morphology of cells taken from the corresponding colonies. Scale bars = 5 µm. (C) PCR to verify the presence of <i>MTL</i><b>a</b>1 and <i>MTL</i>α2 genes in white×white (WxW) mating products, opaque×opaque (OxO) mating products, white×opaque (WxO) mating products, and in the parental <b>a</b> and α strains. (D) Left, pictures of zygotes analyzed from white×white mating experiments and, right, pictures of zygotes analyzed from opaque×opaque mating experiments. Scale bars = 5 µm.</p

Biofilm formation is induced by <i>WOR1</i> overexpression in <i>C. tropicalis</i>.

<p>(A) Quantification of cells adhering to a 12-well polystyrene dish following a 48-hour incubation in Lee's medium. Representative images of wells appear below the corresponding strains. Data from three experimental replicates, error bars indicate SD. * = p<0.001 when compared to <i>Δwor1</i>, white, and opaque strains. (B, C) Quantification of biofilm production by colorometric assays. (B) Beta-glucan content was determined by crystal violet staining. (C) Cellular viability was measured by formazan formation upon XTT reduction. Each biofilm experiment includes three or more replicates, error bars indicate S.D. * = p<0.001 when compared to <i>Δwor1</i>, white, and opaque strains. † = p<0.001 when compared to <i>Δwor1</i>, white, and <i>pTDH3-WOR1</i> strains. (D) Morphology of adherent cells taken from (A). Percentage of filamentous cells indicated below image. Scale bars = 20 µm. Statistical significance was determined using Mann-Whitney pair-wise tests. ** = significantly different from opaque, white, and <i>αwor1</i> strains, p<0.001. * = significantly different from white and <i>αwor1</i> strains, p<0.001. (E) Genes induced in <i>MTL</i><b>a</b>/<b>a </b><i>WOR1</i> overexpressing cells under biofilm conditions. Heat map shows relative expression changes between <i>pTDH3-WOR1</i> (CAY3853) and opaque (CAY3378) strains in the adherence to plastic biofilm assay. Data set was filtered for genes with a fold-change greater than 4 and clustered by Average Linkage Clustering. Data shows average of two independent biological replicates. Arrowheads highlight <i>C. albicans</i> homologs that are biofilm regulated.</p

<i>C. tropicalis</i> a/α cells undergo a white-opaque phenotypic switch related to that in a and α cells.

<p>(A) White (W) colonies sectoring to opaque (O) in <b>a</b> (CAY1503), α (CAY1505), and <b>a</b>/α (CAY1511) strains and the corresponding cell morphologies observed from these colonies. Cells were grown in SCD at 37°C for 5 days and plated to Lee's + N-acetylglucosamine at room temperature for 10 days. Scale bars = 5 µm. (B) Opaque-white gene expression profiles of <b>a</b>/α, <b>a</b>, and α cells. cDNA prepared from white and opaque states of CAY1511 (<b>a</b>/α), CAY1504 (<b>a</b>), and CAY1505 (α) in independent experiments was hybridized against a universal reference. Opaque cell gene expression was divided by that in white cells and filtered for genes with a fold-change greater than 4 and clustered by Average Linkage Clustering. Genes with elevated expression in white cells are shown in blue and genes upregulated in opaque cells are shown in yellow. (C) Examples of genes whose expression was white-opaque regulated in <b>a</b> cells (left), <b>a</b>/α cells (middle), or in both <b>a</b> and <b>a</b>/α cells (right).</p

The white-opaque switch regulates <i>C. tropicalis</i> mating in a, α, and a/α cells.

<p>Mating frequency of (A) <b>a</b> x α (CAY1503 x CAY1505), (B) <b>a</b> x <b>a</b>/α (CAY1503 x CAY1511), and (C) α x <b>a</b>/α (CAY1505 x CAY1513) white and opaque cells. Mating experiments were performed on Spider medium for 3 days at room temperature and plated to selective media to quantify mating frequency (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003369#s4" target="_blank">Materials and Methods</a>). Error bars indicate SEM for 3 independent experiments. * = significantly different from white×white crosses, p<0.01.</p

<i>WOR1</i> is the master regulator of the white-opaque switch in <i>C. tropicalis</i>.

<p>(A) Cell morphologies of <i>Δwor1</i>, white, opaque, and <i>pTDH3-WOR1</i> (<i>WOR1</i>-overexpressing) strains. Cells were grown in Spider medium at room temperature to 0.8–1.0 OD₆₀₀. Scale bars = 5 µm. (B) Gene expression in <b>a</b>/α white cells (CAY1511), <i>Δwor1</i> cells (CAY4043), opaque cells (CAY4048), and <i>pTDH3-WOR1</i> cells (CAY4045), relative to white cells (CAY1511). Expression profiles for each state were divided by white expression values and filtered for those genes with an expression change greater than 4-fold in 4 or more experiments. (C) <i>WOR1</i> expression in <i>Δwor1</i>, white, opaque, and <i>pTDH3-WOR1</i> strains derived from <b>a</b>, α, and <b>a</b>/α cells. Expression levels measured by qRT-PCR. Error bars indicate SEM for replicate experiments from 3 different biological replicates.</p

<i>C. tropicalis WOR1</i> expression regulates filamentous growth.

<p>(A) Wild-type white, wild-type opaque, <i>αwor1</i> mutant, and <i>WOR1</i>-overexpression (<i>pTDH3-WOR1</i>) strains were grown on Spider medium at 37°C for 7 days and photographed. (B) Cells from corresponding patches in (A). The percentage of filamentous cells is shown below each image. Scale bar = 10 µm. ** = significantly different from opaque, white, and <i>Δwor1</i> strains, p<0.001. * = significantly different from white and <i>Δwor1</i>, p<0.001. ^# = significantly different from <i>Δwor1</i>, p<0.05.</p

A single N6-methyladenosine site regulates lncRNA HOTAIR function in breast cancer cells.

N6-methyladenosine (m6A) modification of RNA regulates normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we reveal that m6A regulates the breast cancer-associated human lncRNA HOTAIR. Mapping m6A in breast cancer cell lines, we identify multiple m6A sites on HOTAIR, with 1 single consistently methylated site (A783) that is critical for HOTAIR-driven proliferation and invasion of triple-negative breast cancer (TNBC) cells. Methylated A783 interacts with the m6A "reader" YTHDC1, promoting chromatin association of HOTAIR, proliferation and invasion of TNBC cells, and gene repression. A783U mutant HOTAIR induces a unique antitumor gene expression profile and displays loss-of-function and antimorph behaviors by impairing and, in some cases, causing opposite gene expression changes induced by wild-type (WT) HOTAIR. Our work demonstrates how modification of 1 base in an lncRNA can elicit a distinct gene regulation mechanism and drive cancer-associated phenotypes