Candida albicans-Induced Epithelial Damage Mediates Translocation through Intestinal Barriers

Abstract

Life-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans, a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans, candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin.IMPORTANCECandida albicans, usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer.

Keywords: Candida albicans; candidalysin; host cell damage; host cell invasion; intestinal barrier; necrosis; translocation.

FIG 1

Infection of C2BBe1 IECs with wild-type C. albicans. (A) Invasion of C. albicans into C2BBe1 cells at 3 h, 4.5 h, and 6 h p.i. was quantified by differential fluorescence microscopy staining. The percentage of invasive hyphae relative to total C. albicans visible hyphae is shown. (B) Representative fluorescence microscopy images of differential staining to quantify C. albicans invasion. Extracellular C. albicans (pink), C. albicans (blue), and actin (green) are indicated. The white arrows indicate the entry point of the invading hypha. (C) Quantification of C2BBe1 barrier integrity as measured by TEER, and fungal translocation (number of translocated cells) after infection with C. albicans SC5314. (D) Release of LDH from C2BBe1 cells after infection with C. albicans SC5314. Data are presented as means ± standard deviations (SD) (error bars) from at least three independent experiments.

FIG 2

Gene Ontology (GO) term analysis of hypo- and hyperdamaging mutants. Gene deletions associated with significantly decreased (A) or increased (B) damage.

FIG 3

In vitro characterization of selected C. albicans mutants on C2BBe1 IECs. (A) C2BBe1 IECs were infected with selected C. albicans mutants, and cellular damage 24 h p.i. was quantified by LDH assay. The mean cellular damage induced by wild-type C. albicans was 865 ± 219 ng/ml LDH (dotted line). (B) The adhesion of selected C. albicans mutant strains to C2BBe1 IECs 1 h p.i. was quantified as described in Materials and Methods. The mean adhesion of WT C. albicans to C2BBe1 cells was 9.2% ± 5.7% (dotted line). (C) The invasion of selected C. albicans mutant strains into C2BBe1 IECs 5 h p.i. was quantified as described in Materials and Methods. The mean invasion level of WT C. albicans was 12.3% ± 5.9% (dotted line). (D) C2BBe1 IECs were infected with selected C. albicans mutants, and their mean hyphal length 5 h p.i. was determined. The mean length of WT C. albicans hyphae was 77.8 ± 12.6 µm (dotted line). All values are presented as mean ± SD relative to the WT. Values that are statistically significantly different are indicated by asterisks as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 4

Characterization of damage, translocation, and loss of TEER by selected and control mutants. (A) C. albicans gene deletion mutants were analyzed for their ability to damage C2BBe1 IECs by LDH assay. (B) Translocation of C. albicans gene deletion mutants across a differentiated C2BBe1 intestinal epithelial barrier. (C) Assessment of C2BBe1 epithelial barrier integrity in response to C. albicans gene deletion mutants at 24 h p.i. as measured by loss of TEER. Data are expressed as TEER loss as a percentage of the wild-type C. albicans-infected cells. Strains are arranged in clusters (I to III) according to bioinformatic analysis. Cluster I exhibited low damage, translocation, and loss of TEER. Cluster II contains wild-type-like mutants. Cluster III exhibited low damage and translocation but wild-type-like loss of TEER. All values are presented as median plus range relative to the WT (dotted line). Statistical significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 5

Bioinformatic analysis of C. albicans-induced epithelial damage, loss of epithelial integrity, and fungal translocation. Data obtained from WT and mutant strains of C. albicans for damage (LDH release), change in epithelial integrity (TEER), and translocation (CFU) were analyzed. (A) Pairwise correlation analysis of epithelial damage (LDH), barrier integrity (TEER), and translocation (CFU). In addition to the median value for each strain, the fit of a first-order polynomial and an exponential fit is shown together with a LOWESS line that describes a parameter-free smoothing to visualize the overall trend. The Bayesian information criterion (BIC) of each fit is given. Note that the sap1-3Δ/Δ strain is not visible, since it has extremely high translocation values; this data point was, however, taken into account in the calculation of correlations and in the curve fitting. (B) Correlation coefficients of the pairwise measurements presented. The respective P values are calculated using a two-tailed Student’s t test.

FIG 6

Possible translocation mechanisms of C. albicans through IECs. (A) Schematic representation of possible routes of C. albicans translocation. Possible routes of C. albicans translocation are shown as follows: I, apoptosis; II, paracellular; III, transcellular with damage; IV, transcellular without damage. TJ, tight junctions; AJ, adherens junctions; CaL, candidalysin. (B) C2BBe1 IECs were infected with WT C. albicans SC5314 for 5 h and 24 h and differentially stained. Living cells (Hoechst 33342) (blue), apoptotic cells (FITC-annexin V) (green), necrotic cells (ethidium homodimer III) (red), and late apoptotic/necrotic cells (red/green) are indicated by the color(s) indicated. Colored arrows point to examples of the stained cells. (C) A summary of statistical analysis is presented (mean ± SD; n = 3) that quantifies the proportion of live-apoptotic-necrotic staining observed in the images in panel B. (D) Quantification of caspase 3/7 activity in C2BBe1 IECs infected for 5 h and 24 h with C. albicans SC5314. Staurosporine was used as a positive control for the induction of apoptosis. Data are presented as means ± SD from three independent experiments. Caspase 3/7 activity is shown in relative light units (RLU). Values that are not significantly different (ns) are indicated. (E) Transcellular growth of WT C. albicans (BWP17+Clp30) and ece1Δ/Δ mutant hyphae through C2BBe1 IECs. C2BBe1 cells were infected with C. albicans and differentially stained at 6 h p.i. Extracellular C. albicans (pink), C. albicans (blue), and actin (green) are indicated. The white arrows show the point of invasion. (F) TEM images of C2BBe1 IECs infected with WT C. albicans (BWP17+Clp30) or ece1Δ/Δ mutant. The black arrows point to the host membrane. C.a., C. albicans; Cyt, cytoplasm; ES, extracellular space.

FIG 7

Interaction of C. albicans ECE1 mutant strains with IECs. (A) Adhesion to C2BBe1 IECs as a percentage of inoculated cells of C. albicans ECE1 mutant strains. (B) Invasion into C2BBe1 IECs as a percentage of total visible hyphae of ECE1 mutant strains. (C) C2BBe1 cells were infected with ECE1 mutant strains, and the mean hyphal length of infecting fungi 6 h p.i. was quantified. (D to G) Quantification of LDH (damage) (D), analysis of epithelial barrier integrity by dextran mobility assay (E), quantification of fungal translocation (F), and epithelial barrier integrity as measured by TEER in response to C. albicans ECE1 mutant strains (G). In panels D to G, the application of exogenous candidalysin (CaL) toxin (70 µM) alone to C2BBe1 IECs and in combination with infecting ECE1 mutant strains was assessed. Melittin (70 µM) was used as a positive control for damaging C2BBe1 cells. Data are presented as means ± SD relative to the WT from at least three independent experiments. Statistical significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant.