Self-regulation of Candida albicans population size during GI colonization

Abstract

Interactions between colonizing commensal microorganisms and their hosts play important roles in health and disease. The opportunistic fungal pathogen Candida albicans is a common component of human intestinal flora. To gain insight into C. albicans colonization, genes expressed by fungi grown within a host were studied. The EFH1 gene, encoding a putative transcription factor, was highly expressed during growth of C. albicans in the intestinal tract. Counterintuitively, an efh1 null mutant exhibited increased colonization of the murine intestinal tract, a model of commensal colonization, whereas an EFH1 overexpressing strain exhibited reduced colonization of the intestinal tract and of the oral cavity of athymic mice, the latter situation modeling human mucosal candidiasis. When inoculated into the bloodstream of mice, both efh1 null and EFH1 overexpressing strains caused lethal infections. In contrast, other mutants are attenuated in virulence following intravenous inoculation but exhibited normal levels of intestinal colonization. Finally, although expression of several genes is dependent on transcription factor Efg1p during laboratory growth, Efg1p-independent expression of these genes was observed during growth within the murine intestinal tract. These results show that expression of EFH1 regulated the level of colonizing fungi, favoring commensalism as opposed to candidiasis. Also, different genes are required in different host niches and the pathway(s) that regulates gene expression during host colonization can differ from well-characterized pathways used during laboratory growth.

Figure 1. Expression of C. albicans Genes

Expression of genes of interest in cDNA preparations was determined by qRT-PCR as described in Materials and Methods. Results were normalized using actin expression and are expressed relative to a reference sample of laboratory-grown log phase cells. (A) Gene expression in C. albicans cells recovered from the oral cavities or esophagi of IGB piglets. Closed circles, WT C. albicans cells (SC5314) recovered from the tongue, roof of the mouth, or esophagus; bars, geometric means. (B) Gene expression in C. albicans cells recovered from the intestinal tract. Closed circles, WT C. albicans cells (SC5314) recovered from the large intestine of an IGB piglet; closed triangles, WT (SC5314 or DAY185), or mutant (efg1 cph1 double null) C. albicans cells recovered from the cecum of Swiss Webster mice; open triangles, WT C. albicans (SC5314) recovered from the ileum of Swiss Webster mice; bars, geometric means. (C) Gene expression during laboratory growth. White bars, log phase C. albicans grown in rich medium at 37 °C; black bars, post-exponential phase C. albicans grown in rich medium at 37 °C; grey bars, hyphal C. albicans grown in RPMI-serum at 37 °C; error bars, standard deviation. (D) Comparison of gene expression in WT C. albicans (DAY185) and in an efg1 deletion mutant grown in RPMI-serum at 37 °C. Cross-hatched bars, relative ECE1 expression; white bars, relative RBT1 expression; black bars, relative RBT4 expression; error bars, standard deviation.

Figure 2. C. albicans Cells Colonizing the Murine Intestinal Tract Are Predominantly in the Yeast Form

(A and B) Contents of the ileum of mice inoculated with C. albicans strain CKY368, WT, GFP-expressing, day 3 post-inoculation (A) or strain CKY374, efh1 null mutant, GFP-expressing day 13, post-inoculation (B). Fluorescence micrographs show the morphology of yeast-form cells (A) or hyphal-form cells (B). (C) Quantitation of cellular morphology.

Figure 3. Deletion of EFH1 Alters Murine Intestinal Colonization

WT, efh1 deletion mutant, or EFH1 reconstituted null mutant were orally inoculated by gavage into Swiss Webster mice. At various days post-inoculation, the amounts of C. albicans in fecal pellets and in organs of the intestinal tract were measured. (A) CFUs per gram of cecum contents from mice sacrificed on the indicated days post-inoculation. Each symbol represents a sample from a different mouse. (Composite results from several experiments.) Red circles, WT C. albicans strain DAY185 (23 mice); blue triangles, efh1 deletion mutant strain CKY366 (24 mice); black diamonds, EFH1 reconstituted strain, CKY373 (ten mice); open symbols, no colonies detected; bars, geometric means. p-Value was determined using the t test with log transformed data. ** indicates p < 0.000005. (B) CFUs per gram of fecal pellet. Mice were sampled repeatedly and each symbol represents a sample from a different mouse. Different numbers of mice are shown on different days because some mice were sacrificed earlier and some mice were sampled on different days. Red circles, WT C. albicans strain DAY185 (ten mice); blue triangles, efh1 deletion mutant strain CKY366 (12 mice); black diamonds, EFH1 reconstituted strain, CKY373 (seven mice); open symbols, no colonies detected; bars, geometric means. p-Value was determined using the t test with log transformed data. * indicates p < 0.0003. (C) Correlation between CFUs measured in several organs and in fecal pellets. Light blue diamonds, fecal pellets (labeled F); pink triangles, cecum contents (labeled C); open circles, ileum contents (labeled I); squares, stomach contents (labeled S). CFU/gm of cecum contents, stomach contents, and fecal pellets correlated well, while the CFU/gm ileum was more variable. (D) Mice were colonized with WT strain DAY185 (red circles) or efh1 null mutant CKY366 (blue triangles) or were not inoculated with C. albicans (not shown). Mice were sacrificed on day 21 post-inoculation, CFUs from cecum contents were determined by plating, and C. albicans DNA in cecum contents was quantified by qPCR as described in Materials and Methods. The correlation between CFUs in 70 mg of pelleted cecum contents (x-axis) and DNA (in arbitrary units) determined by qPCR (y-axis) is shown. Each symbol represents a different mouse.

Figure 4. Attenuated Colonization of Mice by an EFH1 Overexpressing Strain

Cells of WT strains CKY363 or DAY185, efh1 deletion mutant strain CKY366, and EFH1 overexpressing strain CKY364 were orally inoculated by gavage. At various days post-inoculation, fresh fecal pellets were recovered from inoculated mice and the amount of C. albicans per gm was measured. Red circles, CKY363 or DAY185 (WT) (seven mice); blue triangles, efh1 deletion mutant strain CKY366 (six mice); dark green diamonds, CKY364 (EFH1 overexpressing) (eight mice); bars, geometric means. Composite results of two experiments are shown. Different numbers of mice are shown on different days because some mice were sacrificed earlier and some mice were sampled on different days.

Figure 5. EFH1 Overexpression Reduces Colonization of the Oral Cavity in Immunodeficient Mice

Cells of strains CKY363 (WT) or CKY364 (EFH1 overexpressing, OE) were mixed in a 1:1 ratio with strain RMIS1 (Nou^R marked WT) and introduced into the oral cavities of nude mice by swabbing. On various days post-inoculation, the oral cavities were sampled by swabbing. Swabs were rubbed on YPD SA plates and the colonies were replica-plated to YPSnourseothricin to determine the ratio of Nou^S to Nou^R colonies. The CI is defined as the ratio of Nou^S/Nou^R colonies at time x, divided by their ratio in the inoculum. Each symbol represents a sample taken from an individual animal. Composite results of two experiments are shown. Red circles, CKY363 (WT) mixed with RMIS1 (seven mice); dark green diamonds, CKY364 (EFH1 overexpressing) mixed with RMIS1 (six mice). Day ≥5 includes samples taken at either day 5 or day 6. Samples that yielded very few colonies (<10) were not included on the graph. A statistically significant difference (p < 0.01 by t test) was observed at day ≥5.

Figure 6. rbt1 and rbt4 Mutants Colonize the Murine Intestinal Tract at WT Levels

Cells of WT strains DAY185 or F2U, rbt1 deletion mutant strain BCa 7–4, rbt4 null mutant strain BCa 11–3, ece1 null mutant CAW19–1, CKY362 (ECE1 reconstituted), and CKY376 (yhb1 yhb5 double null mutant) were orally inoculated by gavage. At various days post-inoculation, fresh fecal pellets were recovered from inoculated mice and the amount of C. albicans per gm was measured. Composite results of two experiments are shown. Different numbers of mice are shown on different days because some mice were sacrificed earlier and some mice were sampled on different days. (A) Red circles, DAY185 or F2U (WT) (six mice); green triangles, rbt1 deletion mutant strain BCa 7–4 (six mice); blue diamonds, rbt4 deletion mutant strain Bca 11–3 (six mice); bars, geometric means. (B) Red circles, DAY185 (WT) (six mice); green triangles, yhb1 yhb5 double null mutant strain CKY376 (six mice); bars, geometric means. (C) Red circles, DAY185 or F2U (WT) (six mice); green triangles, ece1 deletion mutant strain CAW19–1 (six mice); black diamonds, ECE1 reconstituted mutant strain CKY362 (four mice); bars, geometric means. Open symbols, no colonies detected. Day ≥ 14 indicates samples taken on day 14 or day 15.