Near surface swimming of Salmonella Typhimurium explains target-site selection and cooperative invasion.

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.

Radioproteomics stratifies molecular response to antifibrotic treatment in pulmonary fibrosis.

Antifibrotic therapy with nintedanib is the clinical mainstay in the treatment of progressive fibrosing interstitial lung disease (ILD). High-dimensional medical image analysis, known as radiomics, provides quantitative insights into organ-scale pathophysiology, generating digital disease fingerprints. Here, we used an integrative analysis of radiomic and proteomic profiles (radioproteomics) to assess whether changes in radiomic signatures can stratify the degree of antifibrotic response to nintedanib in (experimental) fibrosing ILD. Unsupervised clustering of delta radiomic profiles revealed two distinct imaging phenotypes in mice treated with nintedanib, contrary to conventional densitometry readouts, which showed a more uniform response. Integrative analysis of delta radiomics and proteomics demonstrated that these phenotypes reflected different treatment response states, as further evidenced on transcriptional and cellular levels. Importantly, radioproteomics signatures paralleled disease- and drug related biological pathway activity with high specificity, including extracellular matrix (ECM) remodeling, cell cycle activity, wound healing, and metabolic activity. Evaluation of the preclinical molecular response-defining features, particularly those linked to ECM remodeling, in a cohort of nintedanib-treated fibrosing ILD patients, accurately stratified patients based on their extent of lung function decline. In conclusion, delta radiomics has great potential to serve as a non-invasive and readily accessible surrogate of molecular response phenotypes in fibrosing ILD. This could pave the way for personalized treatment strategies and improved patient outcomes.

Engineering mechanosensitive multivalent receptor-ligand interactions: why the nanolinker regions of bacterial adhesins matter.

Inspired by bacterial adhesins, we present a promising strategy of how to engineer peptides to probe various mechanical strains of extracellular matrix fibers. Functional sequence alignment of bacterial adhesins reveals that the bacterial linkers connecting the multivalent binding motifs recognizing fibronectin show considerable heterogeneity in length. Their length regulates the tunable affinities for fibronectin fibrils when stretched into different mechanical strain states. This platform has potential applications in probing extracellular matrix fiber strains in tissues.

Stem Cell-Derived Cardiac Spheroids as 3D In Vitro Models of the Human Heart Microenvironment.

Our laboratory has recently developed a novel three-dimensional in vitro model of the human heart, which we call the vascularized cardiac spheroid (VCS). These better recapitulate the human heart's cellular and extracellular microenvironment compared to the existing in vitro models. To achieve this, human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes, cardiac fibroblasts, and human coronary artery endothelial cells are co-cultured in hanging drop culture in ratios similar to those found in the human heart in vivo. The resulting three-dimensional cellular organization, extracellular matrix, and microvascular network formation throughout the VCS has been shown to mimic the one present in the human heart tissue. Therefore, VCSs offer a promising platform to study cardiac physiology, disease, and pharmacology, as well as bioengineering constructs to regenerate heart tissue.

Electro-mechanical conditioning of human iPSC-derived cardiomyocytes for translational research.

RATIONALE: Impaired maturation of human iPSC-derived cardiomyocytes (hiPSC-CMs) currently limits their use in experimental research and further optimization is required to unlock their full potential. OBJECTIVE: To push hiPSC-CMs towards maturation, we recapitulated the intrinsic cardiac properties by electro-mechanical stimulation and explored how these mimetic biophysical cues interplay and influence the cell behaviour. METHODS AND RESULTS: We introduced a novel device capable of applying synchronized electrical and mechanical stimuli to hiPSC-CM monolayers cultured on a PDMS membrane and evaluated effects of conditioning on cardiomyocyte structure and function. Human iPSC-CMs retained their cardiac phenotype and displayed adaptive structural responses to electrical (E), mechanical (M) and combined electro-mechanical (EM) stimuli, including enhanced membrane N-cadherin signal, stress-fiber formation and sarcomeric length shortening, most prominent under the EM stimulation. On the functional level, EM conditioning significantly reduced the transmembrane calcium current, resulting in a shift towards triangulation of intracellular calcium transients. In contrast, E and M stimulation applied independently increased the proportion of cells with L-Type calcium currents. In addition, calcium transients measured in the M-conditioned samples advanced to a more rectangular shape. CONCLUSION: The new methodology is a simple and elegant technique to systematically investigate and manipulate cardiomyocyte remodelling for translational applications. In the present study, we adjusted critical parameters to optimize a regimen for hiPSC-CM transformation. In the future, this technology will open up new avenues for electro-mechanical stimulation by allowing temporal and spatial control of stimuli which can be easily scaled up in complexity for cardiac development and disease modelling.

Novel peptide probes to assess the tensional state of fibronectin fibers in cancer.

Transformations of extracellular matrix (ECM) accompany pathological tissue changes, yet how cell-ECM crosstalk drives these processes remains unknown as adequate tools to probe forces or mechanical strains in tissues are lacking. Here, we introduce a new nanoprobe to assess the mechanical strain of fibronectin (Fn) fibers in tissue, based on the bacterial Fn-binding peptide FnBPA5. FnBPA5 exhibits nM binding affinity to relaxed, but not stretched Fn fibers and is shown to exhibit strain-sensitive ECM binding in cell culture in a comparison with an established Fn-FRET probe. Staining of tumor tissue cryosections shows large regions of relaxed Fn fibers and injection of radiolabeled 111In-FnBPA5 in a prostate cancer mouse model reveals specific accumulation of 111In-FnBPA5 in tumor with prolonged retention compared to other organs. The herein presented approach enables to investigate how Fn fiber strain at the tissue level impacts cell signaling and pathological progression in different diseases.

Cardiac spheroids as promising in vitro models to study the human heart microenvironment.

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.

Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism.

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.

Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria.

Bacterial adhesion and biofilm growth can cause severe biomaterial-related infections and failure of medical implants. To assess the antifouling properties of engineered coatings, advanced approaches are needed for in situ monitoring of bacterial viability and growth kinetics as the bacteria colonize a surface. Here, we present an optimized protocol for optical real-time quantification of bacterial viability. To stain living bacteria, we replaced the commonly used fluorescent dye SYTO(®) 9 with endogenously expressed eGFP, as SYTO(®) 9 inhibited bacterial growth. With the addition of nontoxic concentrations of propidium iodide (PI) to the culture medium, the fraction of live and dead bacteria could be continuously monitored by fluorescence microscopy as demonstrated here using GFP expressing Escherichia coli as model organism. The viability of bacteria was thereby monitored on untreated and bioactive dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC)-coated glass substrates over several hours. Pre-adsorption of the antimicrobial surfaces with serum proteins, which mimics typical protein adsorption to biomaterial surfaces upon contact with host body fluids, completely blocked the antimicrobial activity of the DMOAC surfaces as we observed the recovery of bacterial growth. Hence, this optimized eGFP/PI viability assay provides a protocol for unperturbed in situ monitoring of bacterial viability and colonization on engineered biomaterial surfaces with single-bacteria sensitivity under physiologically relevant conditions.

Automated time-resolved analysis of bacteria-substrate interactions using functionalized microparticles and flow cytometry.

Surface biofouling poses an increasing problem in industrial and health care applications, driving research for surface coatings to prevent anti-microbial colonization and characterization of the efficacy of the same. The diversity and increasing sophistication of such coatings, which postulate different types of anti-microbial action on planktonic and surface adhering bacteria, challenge the suitability of current approaches to evaluate and compare the different approaches as well as the speed and accuracy at which screening can be made. We describe and provide proof of principle for a method to use microparticles functionalized with molecular coatings through self-assembly together with flow cytometry readout to evaluate Escherichia coli bacteria surface adhesion and killing efficiency. Advantages of the method are the automation of the method that allows recording an immense number of interactions and the possibility to simultaneously record effects on both surface adhering and planktonic bacteria. We demonstrate and discuss design criteria to obtain this information on two coatings, poly(L-lysine)-graft-C(3)H(6)N(+)(CH(3))(2)C(12)H(25) (PLL-g-QAC) and poly(L-lysine)-graft-poly(ethylene glycol)-C(3)H(6)N(+)(CH(3))(2)C(12)H(25) (PLL-g-PEG-QAC), which exemplify two different approaches to creating anti-microbial interfaces. Despite an apparent higher killing efficiency of the PLL-g-QAC during brief exposures, the rapid fouling of that surface quickly reduces its efficiency, whereas the PLL-g-PEG-QAC coating showed greater promise in reducing the growth and interfacial colonization of bacteria over longer time scales.

Stretching fibronectin fibres disrupts binding of bacterial adhesins by physically destroying an epitope.

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.