A Novel Electronic Impedance-based Method For Real-time Monitoring Host-Bacterial Interactions

The interactions between pathogens and their hosts are complex and dynamic. Outcomes of such interactions reflect the properties of the pathogenic agent and the ability of the host to respond to infection. A better understanding of the mechanisms underlying host-pathogen interactions should contribute to improvement in treatment and control of infections. While characterization of host-pathogen interactions has advanced greatly over the past two decades, mainly through employment of cell cultures and animal models. However, the improved understanding of the cellular mechanisms of such interactions remains an elusive goal.Although some methods have been developed for studying the interaction between pathogens and hosts, most of these are static end-point assays and use a single time point after infection to assess the cellular response. Label free cell detection using cell-electrode impedance readout allows for continuously monitoring cellular responses to stimulations in real time. A label free detection system (xCELLigence system, Roche Applied Science and ACEA Biosciences Inc.) was developed recently based on microelectronic biosensor technology. This system has been applied for a wide range of cell-based assays. In this work, we developed an impedance-based assay for real-time monitoring host-bacterial interactions. The resulting time-dependent cell response profiles (TCRPs) reflected the whole process of host-bacterial interactions. The specificity and sensitivity of the TCRPs of host-bacterial interactions were confirmed using currently available endpoint assays. Our data indicate this TCRP-based technique can provide continuous and quantitative information for studying the cellular mechanism of interactions between bacteria or other pathogens with diverse host cells. Part 1:A Novel Phenotypic Pattern-based Assay for Dynamically Monitoring Host Cellular Responses to Salmonella InfectionsIn this part, a novel, label-free, real-time cell detection system based on electronic impedance sensor technology was adapted to dynamically monitor entire processes of intestinal epithelial cells and macrophages in the response to Salmonella infection. Changes in cell morphology and attachment were quantitatively and continuously recorded following Salmonella infection. The resulting impedance-based time-dependent cell response profiles (TCRPs) were compared with standard assays and showed good correlation and sensitivity. Attachment of bacteria to electrode and proliferation of host cells have no contribution to TCRPs. Biochemical assays further suggest that TCRPs are correlated with cytoskeleton-associated morphological dynamics, which can be largely attenuated by inhibitions of actin and microtubule polymerization. The TCRPs are also correlated with cell death of hosts after Salmonella infection. Collectively, our data indicate that cell-electrode impedance measurements not only provide a novel, real-time, label-free method for investigating bacterial infection but also help to advance our understanding of host responses in a more physiological continuous manner, which is beyond the scope of current endpoint assays.Part 2:Wide Application of Electronic Impedence Based Method for Studying Host-bacterial InteractionsMicroarrays currently have been widely used for studying cellular responses to bacterial infections. In this part, we choose four samples according to the TCRP of intestinal epitheial cells in the response to Salmonella for microarray analysis and found that genes involved in inflammatory and innate immunity were activated during infection. IL-24, a member of IL-10 family of cytokines, was overexpressed after Salmonella infection. The level of IL-24 mRNA was correlated with the MOI and significantly blocked by MAPK and PI3K inhibitors. Treatment of cells with IL-24 protein did not affect the adhesion of Salmonella but resisted for Salmonella invasion. IL-24 also promoted the expression of musin and showed a potential role for protecting host from Salmonella infection. Phagocytosis in mammalian immune cells is activated by attachment to Pathogen-associated molecular patterns (PAMPs), which leads to remodeling of actin cytoskeleton. Heat-inactived Salmonella and LPS coated latex beads were engulfed by macrophages. The entire process of phagocytosis was dynamically monitored by xCELLigence system. Antibiotics belonged to different class were added at different time points post-infection lead to the TCRP changes which reflected different mechanisms of these antibiotics. At last, the TCRPs of cellular responses to different bacteria showed the similarities and specificities among bacteria which deserved further investigation.To our knowledge, this is the first report of a label-free, cell-based method for dynamically monitoring the host-bacterial interaction. Research focusing on the host-bacterial interaction has been hampered due to the limited tools for effective analysis. The TCRP-based methodology, together with other emerging advances in cell-based assays, makes it possible to overcome these problems and gain clearer insights into the interaction between bacteria and their hosts.