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Pathogens harbour a variety of strategies for channelizing the host molecular machinery to facilitate their entry and pathogenesis. Host cytoskeleton has emerged as most exploited host component, however the underlying mechanisms are less studied. Here using quantitative microscopy-based techniques, we revealed mycobacterial sulfoglycolipid, SL-1, to alter the interaction landscape between the host plasma membrane, phosphatidylinositol lipids, and F-actin. The changed abundance and dispersion of phosphoinositides and F-actin further impact their interactions with essential cytoskeletal proteins such as Arp2/3, N-WASP, cofilin, vinculin, and talin involved in actin assembly and disassembly. We show that SL-1 reduces cholesterol-rich plasma membrane domains and sequesters phosphoinositides into de novo lipid domains, boosting interactions with actin at the plasma membrane, but phosphoinositide shielding reduces interactions with actin-binding proteins. The most noteworthy is the diminished interactions between cofilin with both phosphoinositide lipid and F-actin coupled with reduction of ADP-ribosylation factor (Arf) GTPase-activating proteins thus initiating actin fragmentation in ASAP-1 dependent fashion. This phenotype fosters higher bacterial uptake within the host and modulates cell migration by altering actin alignment and filopodia dynamics. Collectively, our findings highlight potential key molecular events and players involved in host cytoskeleton remodelling upon exposure to mycobacterial lipids during tubercular infection that enhances bacterial uptake within the host.

In search for broad-spectrum antivirals, we discover a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrate selective inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Lipidomics analysis reveals alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and links its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We identify PIP4K2C's roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays, reveals that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced autophagic flux impairment. Promoting viral protein degradation by reversing autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual PIP4K2C and PIKfyve inhibition as a candidate strategy to combat emerging viruses.

Polyethyleneimine (PEI), a polymeric non‐viral vector for gene delivery, shows rich pH‐dependent behavior. This results in PEI exhibiting a proton‐sponge mechanism during transfection. Recent studies show PEI chains can themselves undergo a specific type of self‐assembly in low‐pH environment. Such assemblies can affect transfection specifically if PEI/PEI‐DNA polyplexes are subjected to such pH conditions. However, the understanding of the correlation between variations in solution pH in which the free PEI chains are conditioned and gene transfection remains limited. Here, it is shown that an interplay of pH‐driven protonation of amines and a pH‐specific hierarchical micro‐structuration in branched PEI alters gene transfection in a temporal fashion. While conditioning of PEI chains in an aqueous solution at pH 1.4 prior to DNA complexation at pH 7.4 renders ~ 2 times efficient transfection, PEI chains routed through pH 3.0 show spatiotemporal deterioration. The decreased transfection is attributed to the self‐assembly of PEI chains between pH 2.5–4.0 to form micron‐sized fibrils, leading to poor cellular uptake and diminished nuclear localization. This study, therefore, unfolds the pH landscape and conditioning times of free PEI chains before DNA complexation under that efficient/inefficient gene transfection can be achieved.

In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2C’s roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.

Tuberculosis is a challenging disease due to the intracellular residence of its pathogen, Mycobacterium tubercu-losis, and modulation of the host bactericidal responses. Lipids from Mycobacterium tuberculosis regulate macrophage immune responses dependent on the infection stage and intracellular location. We show that li-posomes constituted with immunostimulatory lipids from mycobacteria modulate the cellular immune response and synergize with sustained drug delivery for effective pathogen eradication. We evaluate the pH-dependent release of Rifampicin from the mycobacterial-lipid-derived liposomes intracellularly and in vitro, their cell viability, long-term stability, and antimicrobial efficacy. Intracellular drug levels were higher following liposome treatment compared with the free drug in a temporal fashion underlying a sustained release. The drug-encapsulated liposomes were taken up by clathrin-mediated endocytosis and elicited a robust pro-inflammatory immune response while localizing in the recycling and late endosomes. Notably, these were the same cellular compartments that contained the pathogen underlying localized intracellular targeting. Our results also imply a lipid-centric and species-specific selectivity of the liposomal drug formulations. This work provides a proof-of-concept for the dual-action of liposomes derived from the pathogen itself for their effective eradication, in conjunction with the attuned host immunomodulation.

Lipids represent a less explored class of virulence-associated pathogenic molecules in infectious diseases. Lipids are amphipathic molecules that as part of the cell membranes, orchestrate various cellular processes by modulating membrane biophysical properties such as fluidity, stiffness, packing, curvature, and organization. Changes in the aforementioned membrane properties, in turn, alter lipid/protein diffusion, localization, lipid–protein interactions, and finally their activity. Hence, lipids play critical roles in infectious diseases by intervening in cellular signaling, protein trafficking, membrane fusion, and protein functions. Mycobacterium tuberculosis (Mtb)—the single leading cause of tuberculosis—serves as an epitome of pathogens that use their glycolipids to fine-tune various lipid-dependent interactions with the host cell. The Mtb cell membrane is composed of three layers: inner membrane, arabino-peptidoglycan layer, and outer membrane, and it is the noncovalently attached glycolipids within the outer membrane that are primarily involved in pathogenesis. In this chapter, we give an up-to-date account of recent findings on the biophysical consequences of Mtb lipid-host cell membrane contact, while elucidating the complex cell membrane structure of Mtb, and the specific immunomodulatory properties of distinct Mtb lipids. The presented work showcases how Mtb uses its structurally diverse glycolipids to impact specific host processes including immune modulation during host contact, bestowing the pathogen with a lipid-centric virulence strategy to spatiotemporally regulate host cell interactions by modulating host membrane structure and function.

Cells can adopt both mesenchymal and amoeboid modes of migration through membrane protrusive activities, namely formation of lamellipodia and blebbing. How the molecular players control the transition between lamellipodia and blebs is yet to be explored. Here, we show that addition of the ROCK inhibitor Y27632 or low doses of blebbistatin, an inhibitor of non-muscle myosin II (NMII) ATPase activity and filament partitioning, induces blebbing to lamellipodia conversion (BLC), whereas addition of low doses of ML7, an inhibitor of myosin light chain kinase (MLCK), induces lamellipodia to blebbing conversion (LBC) in human MDA-MB-231 cells. Similarly, siRNA-mediated knockdown of ROCK and MLCK induces BLC and LBC, respectively. Interestingly, both blebs and lamellipodia membrane protrusions are able to maintain the ratio of phosphorylated to unphosphorylated regulatory light chain at cortices when MLCK and ROCK, respectively, are inhibited either pharmacologically or genetically, suggesting that MLCK and ROCK activities are interlinked in BLC and LBC. Such BLCs and LBCs are also inducible in other cell lines, including MCF7 and MCF10A. These studies reveal that the relative activity of ROCK and MLCK, which controls both the ATPase activity and filament-forming property of NMII, is a determining factor in whether a cell exhibits blebbing or lamellipodia.

Rationale: Cancer cells rely on glucose metabolism for fulfilling their high energy demands. We previously reported that monoethanolamine (Etn), an orally deliverable lipid formulation, reduced intracellular glucose and glutamine levels in prostate cancer (PCa). Glucose deprivation upon Etn treatment exacerbated metabolic stress in PCa, thereby enhancing cell death. Moreover, Etn was potent in inhibiting tumor growth in a PCa xenograft model. However, the precise mechanisms underlying Etn-induced metabolic stress in PCa remain elusive. The purpose of the present study was to elucidate the mechanisms contributing to Etn-mediated metabolic rewiring in PCa. Methods: Glucose transporters (GLUTs) facilitate glucose transport across the plasma membrane. Thus, we assessed the expression of GLUTs and the internalization of GLUT1 in PCa. We also evaluated the effects of Etn on membrane dynamics, mitochondrial structure and function, lipid droplet density, autophagy, and apoptosis in PCa cells. Results: Compared to other GLUTs, GLUT1 was highly upregulated in PCa. We observed enhanced GLUT1 internalization, altered membrane dynamics, and perturbed mitochondrial structure and function upon Etn treatment. Etn-induced bioenergetic stress enhanced lipolysis, decreased lipid droplet density, promoted accumulation of autophagosomes, and increased apoptosis. Conclusion: We provide the first evidence that Etn alters GLUT1 trafficking leading to metabolic stress in PCa. By upregulating phosphatidylethanolamine (PE), Etn modulates membrane fluidity and affects mitochondrial structure and function. Etn also induces autophagy in PCa cells, thereby promoting apoptosis. These data strongly suggest that Etn rewires cellular bioenergetics and could serve as a promising anticancer agent for PCa.

Microbial lipids play a critical role in the pathogenesis of infectious diseases by modulating the host cell membrane properties, including lipid/protein diffusion and membrane organization. Mycobacterium tuberculosis (Mtb) synthesizes various chemically distinct lipids that are exposed on its outer membrane and interact with host cell membranes. However, the effects of the structurally diverse Mtb lipids on the host cell membrane properties to fine-tune the host cellular response remains unknown. In this work, we employed membrane biophysics and cell biology to assess the effects of different Mtb lipids on cell membrane mechanics, lipid diffusion, and cytoskeleton of THP-1 macrophages. We found that Mtb lipids modulate macrophage membrane properties, actin cytoskeleton. Combined with the profiling of host biochemical processes using infrared ratioing of biomolecular signatures, we elucidate modulation of processes such as protein phosphorylation and lipid peroxidation, in a virulence lipid-selective manner. These results emphasize that Mtb can fine-tune its interactions with the host cells governed by modulating the lipid profile on its surface. We shall also shed on the previously unknown, involvement of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) lipid in Mtb lipid-host interaction landscape using tether force experiments and microscopy. These observations provide a novel lipid-centric paradigm of Mtb pathogenesis that is amenable to pharmacological inhibition and could promote the development of robust biomarkers of Mtb infection and pathogenesis.

Mycobacterium species, including Mycobacterium tuberculosis, employs atypical long (C60-90) and branched lipids to produce a complex cell wall and localizes these toward distinct spatial locations, inner membrane (IM) and outer membrane (OM), thus forming a robust permeability barrier. The properties and functional roles of these spatially orchestrated membrane platforms remain unknown. Herein, we report the distinctive lateral organization, fluidity, and lipid domain architecture of protein-free membranes reconstituted from IM and OM lipids in vitro from M. smegmatis (Msm) underscored by their lipid packing and lipid dynamics. We show that Msm OM, against common notion, is more dynamic and fluid compared with IM and reveal the role of cell wall-associated peptidoglycans and lipoarabinomannan on the Msm OM organization. Overall, these studies indicate that mycobacterial species may regulate their overall membrane functionality by regulating the synthesis of these complex arrays of lipids. Based on the structure-function relationship drawn here, documented alteration in the mycobacterial lipidome during cellular infection and/or drug treatment could reflect a mechanism to fine-tune M. tuberculosis membrane properties to its advantage. These findings are expected to inspire development of lipid-centric therapeutic approaches targeted toward its membrane.

Microbial lipids play a critical role in the pathogenesis of infectious diseases by modulating the host cell membrane properties, including lipid/protein diffusion and membrane organization. Mycobacterium tuberculosis (Mtb) synthesizes various chemically distinct lipids that are exposed on its outer membrane and interact with host cell membranes. However, the effects of the structurally diverse Mtb lipids on the host cell membrane properties to fine-tune the host cellular response remain unknown. In this study, we employed membrane biophysics and cell biology to assess the effects of different Mtb lipids on cell membrane mechanics, lipid diffusion, and the cytoskeleton of THP-1 macrophages. We found that Mtb lipids modulate macrophage membrane properties, actin cytoskeleton, and biochemical processes, such as protein phosphorylation and lipid peroxidation, in a virulence lipid-selective manner. These results emphasize that Mtb can fine-tune its interactions with the host cells governed by modulating the lipid profile on its surface. These observations provide a novel lipid-centric paradigm of Mtb pathogenesis that is amenable to pharmacological inhibition and could promote the development of robust biomarkers of Mtb infection and pathogenesis.

Lipids dictate membrane properties to modulate lateral membrane organization, lipid/protein diffusion and lipid-protein interactions, thereby underpinning proper functioning of cells. Mycobacterium Tuberculosis (Mtb) harnesses the power of its atypical cell wall lipids to impact immune surveillance machinery centered at the host cell membrane. However, to what extent and how the structurally diverse Mtb lipids modify the host cell membrane properties to fine-tune the host cellular processes remains unknown. Biological membranes are heterogeneousplatforms containing micro-domains that play crucial roles in the assembly of signallinghotspsots driving various cellular functions. Modulation of membrane domain dynamics is intimately involved in host-pathogen interactions and requires suitable methods to be applied to investigate the same. In this work, we combined cell biology and biophysics elucidating the nano-mechanical alteration of host cell membranes and actin cytoskeleton by Mtb lipids instructure-dictated manner. Membrane biophysical characterization using two-photon microscopy imaging of live macrophage cells revealed substantial dynamic changes in the host plasma membrane organization. Moreover, the remodeling of host cell membrane affected the underneath actin-cytoskeleton leading to re-distribution of at least three morphologicallydiscernable actin pools. The functional outcome of this perturbation was assayed by monitoring protein phosphorylation and lipid peroxidation and finally membrane-associated autophagy signaling, which was regulated in a virulence-selective manner. Collectively, our data shows that Mtb can fine-tune its interaction with host cell membranes and proteins governed by thenature of exposed lipid in its outer membrane. These findings will deepen the understanding of host-pathogen interactions and facilitate discovery of the host membrane-associated novel therapeutic targets.

Intracellular pH plays a significant role in many pathological and physiological processes. A series of quinoline‐pyrene probes were synthesized in one‐step fashion through an oxonium‐ion‐triggered alkyne carboamination sequence involving C−C, C−O and C−N bond formation for intracellular pH sensing. The quinoline‐pyrenes showed significant red shifts at low pH. Fluorescence lifetime decay measurements of the probes showed decreases in lifetime at pH 4. The probes showed excellent selectivity in the presence of various potential interfering agents such as amino acids and cations/anions. Furthermore, the probes were found to show completely reversible emission behaviour in the window between pH 4 and 7. A morpholine‐substituted quinoline‐pyrene probe efficiently stained lysosomes with high Pearson correlation coefficients (0.86) with Lysotracker Deep Red DND‐99 as a reference. A co‐localization study of the probe with Lysotracker DND‐99 showed selective intracellular targeting and a shift in fluorescence emission due to acidic lysosomal pH.

Mycobacterium tuberculosis (Mtb) serves as the epitome of how lipids-next to proteins-are utilized as central effectors in pathogenesis. It synthesizes an arsenal of structurally atypical lipids (C60-C90) to impact various membrane-dependent steps involved in host interactions. There is a growing precedent to support insertion of these exposed lipids into the host membrane as part of their mode of action. However, the vital role of specific virulence-associated lipids in modulating cellular functions by altering the host membrane organization and associated signaling pathways remain unanswered questions. Here, we combined chemical synthesis, biophysics, cell biology, and molecular dynamics simulations to elucidate host membrane structure modifications and modulation of membrane-associated signaling using synthetic Mycobacterium tuberculosis sulfoglycolipids (Mtb SL). We reveal that Mtb SL reorganizes the host cell plasma membrane domains while showing higher preference for fluid membrane regions. This rearrangement is governed by the distinct conformational states sampled by SL acyl chains. Physicochemical assays with SL analogues reveal insights into their structure-function relationships, highlighting specific roles of lipid acyl chains and headgroup, along with effects on autophagy and cytokine profiles. Our findings uncover a mechanism whereby Mtb uses specific chemical moieties on its lipids to fine-tune host lipid interactions and confer control of the downstream functions by modifying the cell membrane structure and function. These findings will inspire development of chemotherapeutics against Mtb by counteracting their effects on the host-cell membrane.

Virulence-associated glycolipids from Mycobacterium tuberculosis (Mtb) act as effector molecules during infection-in addition to proteins. Upon insertion, they alter the host cell's membrane properties modifying the host's functions to aid Mtb survival and disease course. Here we combine tether force experiments and microscopy to reveal previously unknown insights on the potential involvement of the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) lipid in the Mtb lipid-host interaction landscape. Our data shows that Mtb lipids, having different structural and chemical make-up, distinctly alter a host's PI(4,5)P2 membrane abundance/organization and PI(4,5)P2-actin colocalization, thus impacting the plasma membrane-cytoskeletal adhesion forces. Combined with our previous findings that underscore the role of exogenous Mtb lipids in remodeling host plasma membrane organization and mechanics, this work builds upon a lipid-centric view of tubercular infections. Dynamically changing a host's plasma membrane lipid content - in response to virulent lipids - might represent a so far unexplored mechanism invoked by Mtb to modulate the host cell's adhesive properties to escape immune surveillance. These findings will deepen our collective understanding of the functional role of Mtb lipids in hijacking the host cell processes amenable to pharmacological inhibition.

Lipids dictate membrane properties to modulate lateral membrane organization, lipid/protein diffusion and lipid-protein interactions, thereby underpinning proper functioning of cells. Mycobacterium tuberculosis harnesses the power of its atypical cell wall lipids to impact immune surveillance machinery centered at the host cell membrane. However, the role of specific virulent lipids in altering host cellular functions by modulating membrane organization and the associated signaling response are still pertinent unresolved questions. Here, combining membrane biophysics and cell biology, we elucidate how virulent Mtb sulfoglycolipids hijack the host cell membrane, affecting its order, fluidity, and stiffness along with manipulating the linked cytoskeleton. The functional outcome of this perturbation was assayed by monitoring membrane-associated autophagy signaling. These actions form a part of the overall response to commandeer host membrane-associated immune processes during infection. The findings on the mechanism of action of Mtb lipids on host cell membrane structure and downstream signaling will deepen the collective understanding of their functional aspects in membrane-dictated bacterial survival, pathogenesis and drug resistance and reveal suitable membrane driven-therapeutic intervention points and diagnostic tools.