Transregional Collaborative Research Center SFB-TR 84 - “Innate Immunity of the Lung: Mechanisms of Pathogen Attack and Host Defence in Pneumonia“


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Pathophysiological roles of endoplasmic reticulum stress, autophagy and microvillus inclusion during infection with Streptococcus pneumoniae (Chakraborty)


Cellular innate immunity is generally seen as the cumulative response to conserved molecular patterns on bacterial pathogens (PAMPs) detected by host cell pattern recognition receptors (PRRs). However, the presence of microbes leads to environmental changes that activate cellular responses suggesting that the microbes are sensed not only by PRRs. We postulate that the monitoring of excessive levels of cellular stress in infected cells may be just as important functionally for the direct detection and control of pathogens. We and others previously showed that the effects of virulence factors such as bacterial toxins, which are not directly recognized by PRRs, induce stress and repair pathways, such as endoplasmic reticulum (ER) stress and autophagy. Both processes are involved in reestablishment of cellular homeostasis. In more detail, autophagy enables cells to eliminate damaged organelles and misfolded protein aggregates. And during ER stress, the unfolded protein response (UPR) is activated to prevent overloading of the ER with dysfunctional proteins. Indeed in the case of the facultative intracellular pathogen Listeria monocytogenes, we showed that induction of ER stress led to a significant reduction of intracellular bacterial load. These and other data suggest that stress represents an innate mechanism.

In the first funding period we found that the induction of UPR and in particular the inhibition of protein translation and activation of autophagy marker is part of the cellular response to infection in lung epithelial H441 club cells with Streptococcus pneumoniae. Both cellular responses were also observed in other cell types upon pneumococcal infection. We report here that two bacterial molecules of S. pneumoniae, pneumolysin (PLY) and pneumococcal hydrogen peroxide (H2O2), are employed to target host stress and repair systems. From studies with different cell lines, it is now clear while PLY targets the ATF6-dependent UPR pathway, pneumococcal H2O2 targets the translation attenuation induced by the activation of the EIF2AK3 pathway. Using RNAi-mediated depletion of EIF2AK3 we demonstrated that activation of UPR is a prerequisite for the induction of autophagy marker.

We discovered that S. pneumoniae also induces a novel cellular phenotype, hitherto not recognized, in airway epithelial cells. We found that wild type S. pneumoniae but not its isogenic spxB derivative induces the formation of microvillus inclusion bodies (MVIB) in infected H441 cells. The lack of microvilli on surface of enterocytes, for example, is associated with persistent life-threatening watery diarrhea, a disease known as the microvillus inclusion disease (MVID). Both Type I and Type II alveolar epithelial cells as well as the club cells that line the bronchiolar epithelium contain microvilli on the apical site which increase the cell surface area and are required for secretion and absorption of metabolites. These cells contain channels on their apical site that are involved in ion transport. For example, the sodium channel ENaC regulates the sodium uptake. Another channel is the cystic fibrosis transmembrane conductance regulator CFTR which is involved in chloride transport to the lumen. Ion transport within the lung regulates fluid movement. Therefore, loss of microvilli on alveolar and bronchial epithelial cells would lead to fluid disturbance and perturbation of lung function.

In this proposal, we will examine the role of UPR focusing particularly on the effects of transient shutdown on protein translation and its protective effects. We will examine mechanisms and pathways leading to microvillus loss and analyze the functional consequences by examining for changes in electrogenic transport mediated by the sodium channel ENaC and the chloride dependent CFTR channel as well as monitoring edema formation in the mouse lung. In collaboration with the proposed Mercator Fellow we will focus on novel therapeutic approaches to ameliorate pneumococcal impairment of alveolar liquid clearance. Our project will provide new aspects of pathogen detection and will help to understand pneumonia pathogenesis.