Three-dimensional in vitro cell systems are a promising alternative to animals to study cardiac biology and disease. We have generated three-dimensional in vitro models of the human heart (“cardiac spheroids”, CSs) by co-culturing human primary or iPSC-derived cardiomyocytes, endothelial cells and fibroblasts at ratios approximating those present in vivo. The cellular organisation, extracellular matrix and microvascular network mimic human heart tissue. These spheroids have been employed to investigate the dose-limiting cardiotoxicity of the common anti-cancer drug doxorubicin. Viability/cytotoxicity assays indicate dose-dependent cytotoxic effects, which are inhibited by the nitric oxide synthase (NOS) inhibitor L-NIO, and genetic inhibition of endothelial NOS, implicating peroxynitrous acid as a key damaging agent. These data indicate that CSs mimic important features of human heart morphology, biochemistry and pharmacology in vitro, offering a promising alternative to animals and standard cell cultures with regard to mechanistic insights and prediction of toxic effects in human heart tissue.
Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by
S
. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g.
fim
) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically,
S
. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and
fim
-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria.
Although soluble inhibitors are frequently used to block cell binding to the extracellular matrix (ECM), mechanical stretching of a protein fibre alone can physically destroy a cell-binding site. Here, we show using binding assays and steered molecular dynamics that mechanical tension along fibronectin (Fn) fibres causes a structural mismatch between Fn-binding proteins from Streptococcus dysgalactiae and Staphylococcus aureus. Both adhesins target a multimodular site on Fn that is switched to low affinity by stretching the intermodular distances on Fn. Heparin reduces binding but does not eliminate mechanosensitivity. These adhesins might thus preferentially bind to sites at which ECM fibres are cleaved, such as wounds or inflamed tissues. The mechanical switch described here operates differently from the catch bond mechanism that Escherichia coli uses to adhere to surfaces under fluid flow. Demonstrating the existence of a mechanosensitive cell-binding site provides a new perspective on how the mechanobiology of ECM might regulate bacterial and cell-binding events, virulence and the course of infection.
To clear pathogens from host tissues or biomaterial surfaces, phagocytes have to break the adhesive bacteria-substrate interactions. Here we analysed the mechanobiological process that enables macrophages to lift-off and phagocytose surface-bound Escherichia coli (E. coli). In this opsonin-independent process, macrophage filopodia hold on to the E. coli fimbriae long enough to induce a local protrusion of a lamellipodium. Specific contacts between the macrophage and E. coli are formed via the glycoprotein CD48 on filopodia and the adhesin FimH on type 1 fimbriae (hook). We show that bacterial detachment from surfaces occurrs after a lamellipodium has protruded underneath the bacterium (shovel), thereby breaking the multiple bacterium-surface interactions. After lift-off, the bacterium is engulfed by a phagocytic cup. Force activated catch bonds enable the long-term survival of the filopodium-fimbrium interactions while soluble mannose inhibitors and CD48 antibodies suppress the contact formation and thereby inhibit subsequent E. coli phagocytosis.
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