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Cross-Talks of Virulent Bacterial Lipid Biomolecules with Host Cell Signaling Pathways: A Quantitative Approach for Therapeutic Interventions

Cross-Talks of Virulent Bacterial Lipid Biomolecules with Host Cell Signaling Pathways:  A Quantitative Approach for Therapeutic Interventions.

Manjari Mishra and Shobhna Kapoor

Introduction

Lipids play critical roles in infectious diseases by intervening in cellular signaling, membrane trafficking, and protein function during host-pathogen interactions. Seminal contributions exemplify the role of lipids in altering cell membrane properties modulating lipid/protein diffusion, and membrane organization. Thus, changes in membrane properties control the proper functioning of cells, and are harnessed by pathogens for their survival and infection. Our work is centered on lipids from Mycobacterium tuberculosis (Mtb) which serves as an epitome of how lipids—next to proteins—are utilized as central effectors in pathogenesis. It synthesizes an arsenal of atypical lipids (C60-90) predisposed on its surface to interact with host membrane. Some of these lipid species such as trehalose dimycolate (TDM), diacylated sulfglycolipid (AGSL), and mannan-based lipoglycans (LAM) trigger an immuno-pathologic response, whereas, others such as phthiocerol dimycocerosate (PDIM), mycolic acids (MA), sulpholipid-1 (SL-1), and di-andpolyacyl trehalose (PATS) appear to dampen the immune responses. Thus, lipids are attractive targets exploited throughout evolution by pathogens. To unravel the mechanism of modulation of host cell membrane physical, mechanical and biochemical properties by structurally diverse Mycobcaterium lipids, we combine chemistry with lipid biology. This enables discovering of novel drug targets in infectious diseases and to develop novel lipid-based chemical tools.

Significance:

Chemistry with its arcade of exceptional methods, such as fluorescence, atomic force  and infrared spectroscopy as well as microscopy, has enabled inventorying the landscape of pathogenic lipid-induced effects on host cell properties. Using fluorescence, we show Mtb lipids and their synthetic analogues remodel the host cell membrane by changing the lipid domain phases and alter lipid diffusion. In addition,  they alter the membrane fluidity, and lipid packing differently at different bilayer depths. Next, using atomic force spectroscopy, we decipher changes in the nano-mechanical cell properties such as elasticity, adhesion energy, tether dynamics. Using microscopy, we reveal structural alteration of membrane-linked actin-cytoskeleton, which subsequently perturbs the membrane-associated phosphoinositide and autophagy signaling. Finally, using infrared spectroscopy, we show dysregulation of protein phosphorylation, and lipid peroxidation. Our work establishes infrared spectroscopy as a non invasive, cheap and label-free technique for studies in infection biology. 

In totality, all our findings deepen the understanding of host-pathogen interactions and lay the foundation for answering pertinent questions related to modulation of eukaryotic signaling in diseased states and foster discovery of novel lipid-centric therapeutic interventions.

Figure1: Mtb virulent lipids distinctly remodels the host membrane and nano-mechanical properties (A)  Pseudo-colored GP images of THP-1 cellular cells  in the presence of the indicated concentration of Mtb lipids for 4 h. (B) Global GP distribution from the stack of GP images (n = 90, N= 3) deconvoluted by fitting Gaussian distributions. (C) Membrane fluidity of THP-1 cellular membrane before and after treatment with mycobacterial lipids. D) Reduced membrane microviscosity of Mtb lipid treated THP-1 cell membrane. Control and Mtb lipid treated THP-1 cells were labeled with DPH (4 μM) and the fluorescence anisotropy were measured. (E) Comparison of the normalized area ratios for selected peaks of the FTIR spectra in control cells and cells treated with Mtb lipids (10 μg/mL) for 4h. (E) Lipid peroxidation (A1146/A2957) (F)  Protein phosphorylation (G) Elastic moduli distribution of THP-1 cells in the absence and presence of Mtb lipids (H) Relative frequency of membrane tether forces in control and cells treated with different Mtb lipids both were fitted with a log-normal distribution. (I) LC-3 expression was observed in Mtb lipid treated THP-1 cells and was absent in control cells.

Novelty/Application of this research work:

We have generated a framework to answer questions on how M. tuberculosis uses its lipids to alter various mechanical and biochemical functions of the host cells. A substantial amount of M. tuberculosis genome and cellular machinery is used to synthesize and transport structurally diverse and complex lipid molecules on its outer surface. These chemically unique Mtb lipids were widely believed to regulate the permeability of bacterial membranes, protecting them from host-mediated defense mechanisms and antibiotics. However, the role of lipids in hijacking the host molecular machinery to facilitate its pathogenesis and survival is becoming increasingly evident. Elucidating the mechanisms of Mtb lipid-mediated host cell membrane alterations could identify novel therapeutic targets. Our results identified a novel strategy adopted by Mtb to regulate the synthesis of its atypical virulent lipids at distinct stages of the bacterial life-cycle and its interactions with host components, dictating the final biological outcome. These observations provide a novel lipid-centric paradigm of Mtb pathogenesis amenable to pharmacological inhibition. We also believe that these findings will provide a reference for future studies exploring the host components affected by virulent bacterial lipids.