Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Until now human pathogenic fungi were not known to possess such toxins. Here we identify the first fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signaling pathway and activates epithelial immunity. Toxin-mediated membrane permeabilization is enhanced by a positively charged C-terminus and triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name ‘Candidalysin’ for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

SummaryThe basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3, 4, 5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6, 7, 8, 9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components.

Activation of the nuclear factor erythroid 2–related factor 2 (Nrf2) pathway is critical for vascular endothelial redox homeostasis in regions of high, unidirectional shear stress (USS), however the underlying mechanosensitive mediators are not fully understood. The endothelial glycocalyx is disrupted in arterial areas exposed to disturbed blood flow that also exhibit enhanced oxidative stress leading to atherogenesis. We investigated the contribution of glycocalyx sialic acids (SIA) to Nrf2 signaling in human endothelial cells (EC) exposed to atheroprotective USS or atherogenic low oscillatory shear stress (OSS). Cells exposed to USS exhibited a thicker glycocalyx and enhanced turnover of SIA which was reduced in cells cultured under OSS. Physiological USS, but not disturbed OSS, enhanced Nrf2-mediated expression of antioxidant enzymes, which was attenuated following SIA cleavage with exogenous neuraminidase. SIA removal disrupted kinase signaling involved in the nuclear accumulation of Nrf2 elicited by USS and promoted mitochondrial reactive oxygen species accumulation. Notably, knockdown of the endogenous sialidase NEU1 potentiated Nrf2 target gene expression, directly implicating SIA in regulation of Nrf2 signaling by USS. In the absence of SIA, deficits in Nrf2 responses to physiological flow were also associated with a pro-inflammatory EC phenotype. This study demonstrates that the glycocalyx modulates endothelial redox state in response to shear stress and provides the first evidence of an atheroprotective synergism between SIA and Nrf2 antioxidant signaling. The endothelial glycocalyx therefore represents a potential therapeutic target against EC dysfunction in cardiovascular disease and redox dyshomeostasis in ageing.

IntroductionMutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamins A and C, lead to a number of premature ageing syndromes, including Hutchinson Gilford Progeria syndrome (HGPS) and Emery Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). Some causal mutations disrupt lamin A processing, resulting in the accumulation of the lamin A precursor, prelamin A, however it is not clear to what extent accumulated prelamin A contributes to DCM. Here we identify DCM patients with cardiomyocyte accumulation of prelamin A and explore the impact of prelamin A accumulation in vivo .MethodsWe generated a novel line of targeted transgenic mice that accumulate prelamin A specifically in cardiomyocytes (PLA Tg mice), by expressing an uncleavable form of prelamin A, driven by Cre expression from the myosin light chain 2 ventricular (MLC2v) promoter.ResultsImmunofluorescence staining of human DCM biopsies showed the presence of nuclear prelamin A in cardiomyocytes. PLA Tg mice were born without any obvious phenotype but manifested retarded growth by 3 weeks of age and succumbed to heart failure at ˜5 weeks. At 4 weeks, MRI showed a marked dilatation of the cardiac chambers and a decline in cardiac function in vivo . Ejection fraction (EF) was substantially depressed in PLA Tg micecompared with wildtype indicating DCM and heart failure. Cardiac histology showed marked cardiomyocyte disarray and profound fibrosis. Biochemical and microscopic analyses indicated disruption of the linkers of nucleoskeleton to cytoskeleton complex and perinuclear intermediate filament network and was supported by electron micrographs showing nuclear morphology defects. Myocardial infiltration of CD45 positive cells coincided with the expression of γ-H2AX, phosphorylated ATM and increased NF-κB signalling, which suggested an inflammatory response initiated by DNA damage. This may be related to the senescence associated secretory phenotype (SASP) as senescence associated β-galactosidase was also expressed in PLA Tg myocardium.ConclusionWe have demonstrated prelamin A accumulation in the nuclei of human DCM biopsies and show that overexpression of prelamin A in a transgenic mouse model leads to an early decline in cardiac function and premature myocardial senescence.