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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., (Copyright © 2018 Allert et al.)

The opportunistic fungal pathogen Candida albicans thrives on human mucosal surfaces as a harmless commensal, but frequently causes infections under certain predisposing conditions. Translocation across the intestinal barrier into the bloodstream by intestine-colonizing C. albicans cells serves as the main source of disseminated candidiasis. However, the host and microbial mechanisms behind this process remain unclear. In this study we identified fungal and host factors specifically involved in infection of intestinal epithelial cells (IECs) using dual-RNA sequencing. Our data suggest that host-cell damage mediated by the peptide toxin candidalysin-encoding gene ECE1 facilitates fungal zinc acquisition. This in turn is crucial for the full virulence potential of C. albicans during infection. IECs in turn exhibit a filamentation- and damage-specific response to C. albicans infection, including NFκB, MAPK, and TNF signaling. NFκB activation by IECs limits candidalysin-mediated host-cell damage and mediates maintenance of the intestinal barrier and cell-cell junctions to further restrict fungal translocation. This is the first study to show that candidalysin-mediated damage is necessary for C. albicans nutrient acquisition during infection and to explain how IECs counteract damage and limit fungal translocation via NFκB-mediated maintenance of the intestinal barrier. Author summary: Candida albicans populations colonizing the intestine serve as the main source for systemic infections. Though normally commensal, under certain conditions, C. albicans can translocate across the intestine and into the bloodstream, leading to systemic candidiasis. Here we dissect the fungal and host activities involved in this process. We find that damage to host cells, which supports efficient translocation, is associated with active acquisition of host-cell zinc by C. albicans. At the same time, intestinal epithelial cells foster barrier integrity to limit fungal translocation independently of host damage. [ABSTRACT FROM AUTHOR]

The human pathogenic fungus Candida albicans is a frequent cause of mucosal infections. Although the ability to transition from the yeast to the hypha morphology is essential for virulence, hypha formation and host cell invasion per se are not sufficient for the induction of epithelial damage. Rather, the hypha‐associated peptide toxin, candidalysin, a product of the Ece1 polyprotein, is the critical damaging factor. While synthetic, exogenously added candidalysin is sufficient to damage epithelial cells, the level of damage does not reach the same level as invading C. albicans hyphae. Therefore, we hypothesized that a combination of fungal attributes is required to deliver candidalysin to the invasion pocket to enable the full damaging potential of C. albicans during infection. Utilising a panel of C. albicans mutants with known virulence defects, we demonstrate that the full damage potential of C. albicans requires the coordinated delivery of candidalysin to the invasion pocket. This process requires appropriate epithelial adhesion, hyphal extension and invasion, high levels of ECE1 transcription, proper Ece1 processing and secretion of candidalysin. To confirm candidalysin delivery, we generated camelid VHHs (nanobodies) specific for candidalysin and demonstrate localization and accumulation of the toxin only in C. albicans‐induced invasion pockets. In summary, a defined combination of virulence attributes and cellular processes is critical for delivering candidalysin to the invasion pocket to enable the full damage potential of C. albicans during mucosal infection. Take Aways: Candidalysin is a peptide toxin secreted by C. albicans causing epithelial damage.Candidalysin delivery to host cell membranes requires specific fungal attributes.Candidalysin accumulates in invasion pockets created by invasive hyphae.Camelid nanobodies enabled visualisation of candidalysin in the invasion pocket. [ABSTRACT FROM AUTHOR]

Extracellular matrix deposition during tubulointerstitial fibrosis (TIF), a central pathological process in patients with diabetic nephropathy (DN), is driven by locally activated, disease-relevant myofibroblasts. Myofibroblasts can arise from various cellular sources, e.g., tubular epithelial cells via a process named epithelial-to-mesenchymal transition (EMT). Transforming growth factor beta 1 (TGF-β1) and its downstream Smad signaling play a critical role in both TIF and EMT. Whereas Smad3 is one central mediator, the role of the other prominently expressed variant, Smad2, is not completely understood. In this study, we sought to analyze the role of renal Smad2 in the development of TIF and EMT during streptozotocin-induced DN by using a fibroblast-specific protein 1 (FSP1)-promotor-driven SMAD2 knockout mouse model with decreased tubular, endothelial, and interstitial Smad2 expression. In contrast to wild-type diabetic mice, diabetic SMAD2 knockout mice showed the following features: (1) significantly reduced DN and TIF (shown by KIM1 expression; periodic acid Schiff staining; collagen I and III, fibronectin, and connective tissue growth factor deposition); (2) significantly reduced tubular EMT-like changes (e.g., altered Snail1, E-cadherin, matrix metalloproteinase 2, and vimentin deposition); and (3) significantly decreased expression of myofibroblast markers (α-smooth muscle actin, FSP1). As one mechanism for the protection against diabetes-induced TIF and EMT, decreased Smad3 protein levels and, as a possible consequence, reduced TGF-β1 levels were observed in diabetic SMAD2 knockout mice. Our findings thus support the important role of Smad2 for pro-fibrotic TGF-β/Smad3 signaling in experimental DN. [ABSTRACT FROM AUTHOR]