Antifungal defense of probiotic Lactobacillus rhamnosus GG is mediated by blocking adhesion and nutrient depletion

Data Availability: All relevant data are available from the Gene Expression Omnibus at the following accession number: GSE97755. Funding: This work was funded by the German Research Council (DFG) Graduation College 685, Dr. Jekyll and Mr. Hyde: A systems approach to the therapy of nosocomial infections caused by Candida albicans: a commensal organism switches to a deadly pathogen/ PTJ (FKZ: 0315409BBMBF), the Dr. Manfred Plempel-foundation, the Dr. Siegried Stettendorf-Foundation, the InfectERA Program (FunComPath; BMBF FKZ 031L0001A), the Integrated Research and Treatment Center for Sepsis Control and Care (CSCC) project CanBac (BMBF, FKZ: 01EO1002), and the German Research Council (DFG) GZ:HE7565/1-1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Clotrimazole Dampens Vaginal Inflammation and Neutrophil Infiltration in Response to Candida albicans Infection

The pathology of vulvovaginal candidiasis (VVC) caused by Candida albicans is associated with a nonprotective inflammatory response and is frequently treated with clotrimazole. We investigated the mechanisms by which clotrimazole resolves VVC. Low levels of clotrimazole, which do not block fungal growth, inhibit expression of a “danger response” transcription factor, c-Fos, block production of proinflammatory cytokines, and inhibit neutrophil infiltration to the site of infection

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. IMPORTANCE Candida 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

<i>C</i>. <i>albicans</i> viability in response to LGG.

<p>(A and B adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184438#pone.0184438.ref029" target="_blank">29</a>]). (A and B) FUN1-staining of <i>C</i>. <i>albicans</i>, grown in absence (A) or presence of LGG (B). Fluorescence staining was performed after 24 h of co-culture. Images are representative of three individual experiments. Scale bars equal 10 μm. Live fungal cells are indicated by green fluorescence and a well-defined vacuole structure. Red fluorescence inside these vacuoles indicates metabolic activity (red arrows). Yellowish fluorescence indicates dead fungi (yellow arrows). LGG stains green (white arrow). (C and D) Transmission electron microscopy of <i>C</i>. <i>albicans</i>. <i>C</i>. <i>albicans</i> was grown for 24 h in absence (C) or presence (D) of LGG. Cell organelles were counted in 25 cells per condition and experiment. Nuclei are highlighted in brown, organelles in blue. Micrographs are representative for three individual experiments. Scale bars equal 0.2 μm. (E) Metabolic activity of <i>C</i>. <i>albicans</i> grown in absence or presence of LGG, Amphotericin B (Ampho B; 0.1 μg/ml) served as control. (C, D and E) n = 3 *<i>p</i><0.05.</p

Impact of LGG on <i>C</i>. <i>albicans</i> ergosterol content and glucose concentration in cell culture supernatants.

<p>(A) Extracts of <i>C</i>. <i>albicans</i> grown on TR146 monolayers pretreated with either PBS or LGG were analyzed for their ergosterol content. (B) Exemplary selected ion chromatogram of one sample, where LGG-preincubated cells were infected with <i>C</i>. <i>albicans</i>, containing cholestane (<i>m/z</i> 217), cholesterol (<i>m/z</i> 368) from TR146 cells and ergosterol (<i>m/z</i> 363) from <i>C</i>. <i>albicans</i>. (C) Monolayers of TR146 cells were preincubated with PBS or LGG 12 h prior to infection with <i>C</i>. <i>albicans</i> cells. Amount of glucose was evaluated 6 h post infection. (D) Monolayers of TR146 cells were preincubated with PBS or LGG 12 h prior to infection. Directly before infecting epithelial cells with <i>C</i>. <i>albicans</i>, glucose was added to the experiment (5 mg/ml). LDH activity was quantified 6 h post infection. n = 3 *<i>p</i><0.05, ***<i>p</i><0.001.</p

LGG mediates protection against <i>C</i>. <i>albicans</i> infection in TR146 monolayer.

<p>LDH activity in response to <i>C</i>. <i>albicans</i> infection was quantified in cell culture supernatant 6 h (A) and 2 h (B) post infection. n = 3 ***<i>p</i><0.001.</p

Preincubation of TR146 cells with LGG impairs major virulence attributes of <i>C</i>. <i>albicans</i>.

<p>(A-C) Monolayers of TR146 cells were preincubated with PBS or LGG for 12 h. In some conditions LGG was then removed by rinsing the cells with PBS (LGG off) and/or glucose was added to the medium (5 mg/ml). Then, epithelial cells were infected with <i>C</i>. <i>albicans</i> cells. (A) Adhesion of <i>C</i>. <i>albicans</i> to epithelial cells was analyzed 1 h post infection. Invasiveness (B) and hyphal growth (C) was measured 3 h post infection. (D) LDH release by TR146 cells was quantified 6 h post infection. (A-D) n = 3 *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p