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

Yersinia enterocolitica exploits different pathways to accomplish adhesion and toxin injection into host cells.

The current paradigm suggests that Yersinia enterocolitica (Ye) adheres to host cells via the outer membrane proteins Yersinia adhesin A (YadA) or invasin (Inv) to facilitate injection of Yops by the type III secretion system. In this process Inv binds directly to β1 integrins of host cells while YadA may bind indirectly via extracellular matrix proteins to β1 integrins. Here we challenged this paradigm and investigated the requirements for Yop injection. We demonstrate that Inv- but not YadA-mediated adhesion depends on β1 integrin binding and activation, and that tight adhesion is a prerequisite for Yop injection. By means of novel transgenic cell lines, shRNA approaches and RGD peptides, we found that YadA, in contrast to Inv, may use a broad host cell receptor repertoire for host cell adhesion. In the absence of β1 integrins, YadA mediates Yop injection by interaction with αV integrins in cooperation with yet unknown cofactors expressed by epithelial cells, but not fibroblasts. Electron microscopic and flow chamber studies revealed that a defined intimate contact area between Ye and host cells resulting in adhesion forces resisting shear stress is required for Yop injection. Thus, the indirect binding of YadA to a broad extracellular matrix (ECM) binding host cell receptor repertoire of different cell types makes YadA a versatile tool to ensure Yop injection. In conclusion, given the differential expression of the outer membrane proteins Inv and YadA in the course of Ye infection and differential expression of integrins by various host cell populations, the data demonstrate that Ye is flexibly armed to accomplish Yop injection in different host cell types, a central event in its immune evasion strategy

Modulation of the fungal-host interaction by the intra-species diversity of C. albicans

The incidence of human infections caused by the opportunistic fungal pathogen Candida albicans is on the rise due to increasing numbers of immunosuppressed patients. The importance of the immune system in preventing overgrowth of the colonizing fungus and thereby limiting infection is well recognized and host protective mechanisms widely investigated. Only recently, it was recognized that the natural diversity in the fungal species could also influence the outcome of the interaction between the fungus and the host. C. albicans strain-specific differences are complex and their regulation at the genomic, genetic, and epigenetic level and by environmental factors is only partially understood. In this review, we provide an overview of the natural diversity of C. albicans and discuss how it impacts host-fungal interactions and thereby affects the balance between commensalism versus disease

A Peptide Derived from the Highly Conserved Protein GAPDH Is Involved in Tissue Protection by Different Antifungal Strategies and Epithelial Immunomodulation

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has an important role not only in glycolysis but also in nonmetabolic processes, including transcription activation and apoptosis. We report the isolation of a human GAPDH (hGAPDH) (2-32) fragment peptide from human placental tissue exhibiting antimicrobial activity. The peptide was internalized by cells of the pathogenic yeast Candida albicans and initiated a rapid apoptotic mechanism, leading to killing of the fungus. Killing was dose-dependent, with 10μgml (3.1μM) and 100μgml hGAPDH (2-32) depolarizing 45% and 90% of the fungal cells in a population, respectively. Experimental C. albicans infection induced epithelial hGAPDH (2-32) expression. Addition of the peptide significantly reduced the tissue damage as compared with untreated experimental infection. Secreted aspartic proteinase (Sap) activity of C. albicans was inhibited by the fragment at higher concentrations, with a median effective dose of 160mgl−1 (50μM) for Sap1p and 200mgl−1 (63μM) for Sap2p, whereas Sap3 was not inhibited at all. Interestingly, hGAPDH (2-32) induced significant epithelial IL-8 and GM-CSF secretion and stimulated Toll-like receptor 4 expression at low concentrations independently of the presence of C. albicans, without any toxic mucosal effects. In the future, the combination of different antifungal strategies, e.g., a conventional fungicidal with immunomodulatory effects and the inhibition of fungal virulence factors, might be a promising treatment option

CFU of LGG and <i>C</i>. <i>albicans</i> used for different experiments.

<p>CFU of LGG and <i>C</i>. <i>albicans</i> used for different experiments.</p

<i>L</i>. <i>rhamnosus</i> GG (LGG) protects against <i>C</i>. <i>albicans</i> infection (adapted from [29]).

<p>(A) <i>C</i>. <i>albicans</i>-induced release of lactate dehydrogenase (LDH). Semi-thin sections of <i>C</i>. <i>albicans</i>-infected RHOEs pretreated either with PBS (B) or LGG (C). Images are representative for three individual experiments. Scale bars equal 100 μm. Supernatants of RHOEs were further analyzed for cytokines. Content of interleukin-8 (IL8; D), granulocyte macrophage colony-stimulating factor (GM-CSF; E) and IL1α (F) was quantified. n = 3–7 *<i>p</i><0.05, **<i>p</i><0.01, ***<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

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