|dc.description.abstract||Introduction: Mycobacterium tuberculosis (Mtb), the principal etiological agent of pulmonary tuberculosis (TB), continues to co-evolve with the human population making itself one of the most potent infectious killer in the modern-day world. The primary site of TB infection is the lung, wherein the tubercle bacillus is taken up by phagocytic cells such as macrophages, dendritic cells as well as neutrophils. Emerging studies have also indicated that Mtb immigrates into non-myelocytic cells to ensure itself a safe niche from the concerted immune onslaught of the host. In course of infecting the host, the bacterium attunes distinct cellular processes that would otherwise contribute to bacterial clearance viz. apoptosis, autophagy, antigen presentation, cytokine responses and lipid accumulation. Interestingly, Mtb, has been shown to initiate ectopic expression of a gamut of cellular signaling pathways, by virtue of which the immune activities of the host are tempered. The current work brings to light critical signaling nodes that are deregulated upon mycobacterial infection and demonstrates key epigenetic molecules that act downstream to these signaling intermediates to aid Mtb pathogenesis. Importantly, the study builds upon the critical role(s) of NOTCH and WNT signaling pathways during Mtb infection as well as reports the significance of SLIT-ROBO pathway that was hitherto unexplored in the context of mycobacterial pathogenesis.
PART I: Elucidation of the regulation of an E3 ubiquitin ligase by Mtb-elicited epigenetic modifier.
Mtb adopts diverse strategies to impede the host-mediated immunological offenses. As one of its many shrewd strategies, Mtb generates lipid-laden cells (foamy macrophages-FMs) that offer a favorable shelter for its persistence. FMs are formed by the complex regulation of influx, metabolism, storage, and mobilization of lipid molecules. The excess accumulation of lipids is often attributed to the surge in the expression of lipid influx genes with a concomitant decrease in the lipid efflux genes. In this part, we delineated a post-translational regulation of proteins involved in lipid accrual during Mtb infection. Notably, we observed significant reduction in the expression of a specific E3 ubiquitin ligase ITCH, that allowed the sustenance of key lipid accretion molecules (ADRP and CD36), by curbing their proteasomal turnover. Further, we found the repression of ITCH to be dependent on the concerted action of the bifunctional transcription factor YY1 and an arginine methyl transferase PRMT5. NOTCH signaling pathway was identified as a master-regulator of YY1 expression, and in vitro Mtb CFU analysis using small molecule inhibitors against NOTCH and PRMT5 revealed critical roles for these molecules in regulating innate responses during mycobacterial infection.
PART II: Molecular insights into the regulation of lipid peroxidation in macrophages upon mycobacterial infection.
During infection, Mtb mediates reactive oxygen species (ROS) generation within the host cellular milieu as an auxiliary manifestation of the host defence system. ROS accumulation can contribute to myriad events within a cell, including lipid peroxidation,
which could contribute to programmed cell necrosis. Recently, it was reported that ferroptosis, a form of cell death brought about by iron overload and lipid peroxidation, contributes to cellular necrosis in macrophages upon virulent mycobacterial infection. In line with the reported observations, we evaluated the expression of key molecules implicated during lipid peroxidation in Mtb-infected macrophages. To our interest, ACSL4 and ALOX15 displayed robust upregulation upon Mtb infection in a WNT-signaling dependent manner. As a result, enhanced lipid peroxidation leading to the accumulation of copious amount of a bioactive lipid alkenal, 4-Hydroxynonenal (4-HNE), was observed in infected macrophages. Accumulation of 4-HNE bears significance as it has been shown to modulate several processes by forming covalent adducts with the nucleophilic functional groups of proteins. Besides, Gpx4, the principal selenoprotein responsible for mitigating lipid peroxidation, was found to be downregulated, consequent upon the Mtb-mediated diminished expression of SIRT1. Thus, we uncover molecular details underlying the critical process of Mtb-induced lipid peroxidation in infected macrophages and demonstrate their impact on mycobacterial survival.
PART III: Delineation of the role for SLIT-ROBO signaling during mycobacterial infection.
Continuing with our line of investigation in implicating the role of distinct signaling pathways during mycobacterial pathogenesis, we chose to scrutinize the possible role of SLIT-ROBO pathway in Mtb-infected macrophages. It might be appreciated that a seminal report suggested the role of SLIT-ROBO signaling in LPS induced endothelial inflammation and endotoxemia. Our experiments revealed that Mtb-infection, both in vitro and in vivo, enhanced the expression of SLIT2 ligand and cognate ROBO2 receptor. The elevated levels of SLIT2 was found to be associated with an enhanced phosphorylation at the Serine 28 residue of Histone3, and a consequent reduction in the repressive histone methylation signature at the Slit2 promoter. We found that the activation of SLIT-ROBO pathway contributed to an enhanced expression of VNN1, a cellular pantetheinase involved in regulating oxidative stress, in an AHR dependent manner. in vitro CFU analysis revealed compromised mycobacterial survival upon perturbation of VNN1 or AHR, thereby, indicating the important role(s) for these mediators in contributing towards mycobacterial survival. Together, we uncover additional functions for NOTCH and WNT signaling pathways in regulating cellular processes during Mtb infection. Also, we report the intricate mechanism of the activation of SLIT-ROBO pathway during mycobacterial infection and indicate its potential role in modulating crucial inflammatory mediators during Mtb pathogenesis.||en_US