Autophagy modulators to enhance host control against bacterial infections

Abstract

Macroautophagy (herein autophagy) is a cellular catabolic pathway in which cytoplasmic components are captured by vesicles (autophagosomes) that eventually fuse with lysosomes to degrade the cargo. Autophagy occurs in basal levels in all eukaryotic cells to maintain cellular homeostasis and at conditions of stress, superfluous organelles and proteins not essential for survival are degraded. The degraded products provide building blocks for cellular sustenance especially during starvation periods. Apart from these functions, cargos like aggregated proteins, damaged organelles and intracellular pathogens which are otherwise harmful to cells are also selectively captured by autophagy and destined for degradation. As expected, dysfunctional autophagy is linked to several human diseases such as Alzheimer’s disease, Parkinsons’s disease, Huntingtons’s disease where inability of autophagy machinery to clear aggregated proteins is one of the major causative agent for such neurodegenerative diseases. Apart from aggregate clearance, autophagy is also important in the context of infectious diseases where intracellular pathogens are cleared by a form of autophagy known as xenophagy. The process of xenophagy provides a broad spectrum of defense mechanism to capture bacterial, viral and protozoan pathogens. Many of the pathogens have evolved ways to subvert xenophagy and establish their intracellular niche for replication. In addition, mutation(s) in some of the autophagy genes are shown to cause pre-disposition towards intracellular infections. Chapter I introduces these concepts. Given the wide application of autophagy, its modulation by genetic or pharmacological means using small molecules could be a potential therapeutic approach. Although many small molecule autophagy inducers and inhibitors are known, there is still a need for more potent modulators that are functional in vivo and with better specificity rather than promiscuously disturbing many signaling pathways within cells. Yeast based high throughput screening done previously in lab have identified some potential autophagy inducers and inhibitors. I have tested the ability of these autophagy inducers to clear intracellular Salmonella population. The details of experimental assays that were carried out for this work have been explained in Chapter II. In this study we have identified a potential xenophagy inducer which shows intracellular pathogen clearance in different cell types and against candidate gram positive and gram negative bacterial pathogens. The results pertaining to screening and validation are summarized in Chapter III. The results also indicate that the potential xenophagy modulator could be helpful in targeting larger number of infectious pathogens in both epithelial and macrophage cell line. Further experiments were done to understand the mechanism of action of the compound whose results and interpretations are explained in Chapter IV. The results collectively suggest the involvement of autophagy and recruitment of xenophagy proteins to pathogens. Future studies will involve finding the exact mechanism through which compound work and finding its intracellular target.