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.