Biology subjects

Gennaro, M. L.

Publications and source records attributed to Gennaro, M. L..

8 recordsLinked to original sources

bioRxiv2026

The phage shock protein A (PspA) maintains membrane potential and supports NADH dehydrogenase function in mycobacteria

Maintenance of membrane integrity and proton motive force (PMF) is critical for bacterial survival. The phage shock protein (Psp) system, conserved across bacterial species, stabilizes the membrane, maintains PMF, and protects against envelope damage. However, how the conserved effector PspA contributes to PMF maintenance remains unclear. Here, using the mycobacterial Psp system as a genetically tractable model, we provide mechanistic insight into this process. We show that PspA and the accessory protein PspM jointly sustain membrane potential, with PspM required to maintain a ~70 kDa PspA isoform at the membrane during envelope stress. Loss of PspA increases susceptibility to thioridazine, which targets type II NADH dehydrogenase (NDH-2), and to Ro 48-8071, an inhibitor of menaquinone biosynthesis. Notably, hypersusceptibility to thioridazine is rescued by exogenous menaquinone. Consistent with these phenotypes, a pspA-deficient mutant exhibits impaired NADH dehydrogenase activity despite unchanged abundance of NDH-2 and menaquinone (MK-9). Together, these findings identify a functional link between PspA and NADH dehydrogenase-dependent respiration and suggest that PspA contributes to PMF maintenance by supporting respiratory electron transfer.

molecular biology↗

bioRxiv2026

Synergistic activation of TLR2 and Dectin-2 by mannose-capped lipoarabinomannan reprograms macrophage lipid metabolism in tuberculosis

How innate immune receptors integrate signals from complex microbial ligands remains poorly understood, yet this integration may offer new avenues for host-directed therapies. Here, we show that the architecture of a single pathogen-derived component can organize the coordinated engagement of multiple pattern-recognition receptors to reprogram host cell behavior. We find that the mycobacterial lipoglycan mannose-capped lipoarabinomannan (ManLAM) uses distinct structural features to engage two pattern-recognition receptors, Toll-like receptor 2 (TLR2) and Dectin-2, thereby driving macrophage lipid remodeling and lipid droplet accumulation, a process linked to foam cell formation and necrotizing lesion development in tuberculosis. Dual receptor engagement also potentiates NF-{kappa}B-dependent inflammatory signaling, while lipid droplet accumulation proceeds through an mTORC1-PPAR{gamma}-dependent pathway that is largely independent of NF-{kappa}B activation, indicating that metabolic and inflammatory programs are mechanistically separable. ManLAM-induced lipid remodeling closely mirrors that induced by Mycobacterium tuberculosis infection in both neutral lipid composition and pathway dependence. In contrast, other mycobacterial ligands that are lipogenic in vitro do not measurably contribute to lipid droplet accumulation during infection. These findings identify ManLAM as a major mycobacterial driver of lipid remodeling associated with foam cell formation and establish ligand architecture as a mechanism by which complex microbial ligands organize multi-receptor signaling to direct distinct host cell programs. Significance statementOur findings establish the principle that the architecture of a single microbial ligand can organize the co-engagement of multiple innate immune receptors to shape host cell responses. Using the mycobacterial lipoglycan mannose-capped lipoarabinomannan as a model, we show that distinct structural features within a single microbial component coordinate the co-engagement of TLR2 and Dectin-2 to reprogram macrophage lipid metabolism and promote lipid droplet formation, a process linked to foam cell formation and necrotizing tuberculosis lesions. These results identify a mechanism by which complex microbial ligands can integrate host sensing pathways through their molecular structure. By defining the receptor-signaling axes that control foam cell formation, this work highlights host pathways as candidate targets for host-directed intervention.

microbiology↗

bioRxiv2024

Pharmacological inhibition of macrophage triglyceride biosynthesis pathways does not improve Mycobacterium tuberculosis control in infected mice

Triglyceride rich macrophages (foam cells) are a hallmark of necrotic granulomas in tuberculosis, and multiple antimicrobial functions are down-regulated in these cells. In this study, we assessed the ability of two different compounds to reduce triglyceride content and intracellular burden in Mycobacterium tuberculosis (Mtb)-infected macrophages: A-922500 (DGATi), an inhibitor of diacylglycerol acyltransferase 1, an enzyme involved in triglyceride synthesis; and LY2584702 (p70S6Ki), an inhibitor of p70 S6 kinase, a serine/threonine kinase involved in mTORC-1dependent lipid biogenesis. Additionally, we evaluated the adjunctive activity of these inhibitors as host-directed therapies against chronic Mtb infection in C3HeB/FeJ mice. DGATi and p70S6Ki significantly reduced the lipid content and bacillary burden in Mtb-infected human monocyte-derived macrophages. In Mtb-infected mice, each inhibitor reduced the triglyceride content (P[≤] 0.0001) in cells from bronchoalveolar lavage samples. Adjunctive treatment of DGATi with isoniazid and p70S6Ki monotherapy reduced the lipid droplet content (P[≤] 0.05) within lung macrophages of Mtb-infected mice. However, neither inhibitor reduced the lung bacterial burden in Mtb-infected mice alone or in combination with isoniazid, and they did not alter lung inflammation. These findings provide further insights into the role of foam cells in tuberculosis pathogenesis and the utility of interventions targeting these cell populations as adjunctive host-directed therapies.

pharmacology and toxicology↗

bioRxiv2023

Heterogeneity of foam cell biogenesis across diseases

Foam cells are dysfunctional, lipid-laden macrophages associated with chronic inflammation of diverse origin. The long-standing paradigm that foam cells are cholesterol-laden derives from atherosclerosis research. We previously showed that, in tuberculosis, foam cells surprisingly accumulate triglycerides. Here, we utilized bacterial (Mycobacterium tuberculosis), fungal (Cryptococcus neoformans), and human papillary renal cell carcinoma (pRCC) models to address the need for a new explanation of foam cell biogenesis. We applied mass spectrometry-based imaging to assess the spatial distribution of storage lipids relative to foam-cell-rich areas in lesional tissues, and we characterized lipid-laden macrophages generated under corresponding in vitro conditions. The in vivo data and the in vitro findings showed that cryptococcus-infected macrophages accumulate triglycerides, while macrophages exposed to pRCC- conditioned-medium accumulated both triglycerides and cholesterol. Moreover, cryptococcus- and mycobacterium-infected macrophages accumulated triglycerides in different ways. Collectively, the data show that the molecular events underlying foam cell formation are specific to disease and microenvironment. Since foam cells are potential therapeutic targets, recognizing that their formation is disease-specific opens new biomedical research directions.

immunology↗

bioRxiv2021

Design and proof-of-concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain

Development of effective vaccines against Coronavirus Disease 2019 (COVID-19) is a global imperative. Rapid immunization of the world human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and many different vaccine approaches are being pursued to meet this task. Engineered filamentous bacteriophage (phage) have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the design, development, and initial evaluation of targeted phage-based vaccination approaches against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by using dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. Towards a unique phage- and AAVP-based dual-display candidate approach, we first performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein for display on the recombinant major capsid coat protein pVIII. Targeted phage particles carrying one of these epitopes induced a strong and specific humoral response. In an initial experimental approach, when these targeted phage particles were further genetically engineered to simultaneously display a ligand peptide (CAKSMGDIVC) on the minor capsid protein pIII, which enables receptor-mediated transport of phage particles from the lung epithelium into the systemic circulation (termed "dual-display"), they enhanced a systemic and specific spike (S) protein-specific antibody response upon aerosolization into the lungs of mice. In a second line of investigation, we engineered targeted AAVP particles to deliver the entire S protein gene under the control of a constitutive cytomegalovirus (CMV) promoter, which induced tissue-specific transgene expression stimulating a systemic S protein-specific antibody response. As proof-of-concept preclinical experiments, we show that targeted phage- and AAVP-based particles serve as robust yet versatile enabling platforms for ligand-directed immunization and promptly yield COVID-19 vaccine prototypes for further translational development. SignificanceThe ongoing COVID-19 global pandemic has accounted for over 2.5 million deaths and an unprecedented impact on the health of mankind worldwide. Over the past several months, while a few COVID-19 vaccines have received Emergency Use Authorization and are currently being administered to the entire human population, the demand for prompt global immunization has created enormous logistical challenges--including but not limited to supply, access, and distribution--that justify and reinforce the research for additional strategic alternatives. Phage are viruses that only infect bacteria and have been safely administered to humans as antibiotics for decades. As experimental proof-of-concept, we demonstrated that aerosol pulmonary vaccination with lung-targeted phage particles that display short epitopes of the S protein on the capsid as well as preclinical vaccination with targeted AAVP particles carrying the S protein gene elicit a systemic and specific immune response against SARS-CoV-2 in immunocompetent mice. Given that targeted phage- and AAVP-based viral particles are sturdy yet simple to genetically engineer, cost-effective for rapid large-scale production in clinical grade, and relatively stable at room temperature, such unique attributes might perhaps become additional tools towards COVID-19 vaccine design and development for immediate and future unmet needs.

microbiology↗

bioRxiv2020

Phage-shock-protein (Psp) Envelope Stress Response: Evolutionary History and Discovery of Novel Players

Bacterial phage shock protein (PSP) systems stabilize the bacterial cell membrane and protect against envelope stress. These systems have been associated with virulence, but despite their critical roles, PSP components are not well-characterized outside proteobacteria. Using comparative genomics and protein sequence-structure-function analyses, we systematically identified and analyzed PSP homologs, phyletic patterns, domain architectures, and gene neighborhoods. This approach underscored the evolutionary significance of the system, revealing that the core PspA gene (Snf7 in ESCRT outside bacteria) was present in the Last Universal Common Ancestor (LUCA), and that this ancestral functionality has since diversified into multiple novel, distinct PSP systems across life. Several novel partners of the PSP system were identified: (i) the Toastrack domain, likely facilitating assembly of sub-membrane stress-sensing and signaling complexes, (ii) the newly-defined HAAS-PadR-like transcriptional regulator pair system, and (iii) multiple independent associations with ATPase, CesT/Tir-like chaperone, and Band-7 domains in proteins thought to mediate sub-membrane dynamics. Our work also uncovered links between the PSP components and other domains, such as novel variants of SHOCT-like domains, suggesting roles in assembling membrane-associated complexes of proteins with disparate biochemical functions. Results are available at https://jravilab.org/psp. ImportancePhage shock proteins (PSP) are virulence-associated, cell membrane stress-protective systems. They have mostly been characterized in proteobacteria and firmicutes. We now show that PSP systems were present in the Last Universal Common Ancestor, and that homologs have evolved and diversified into newly identified functional contexts. Recognizing the conservation and evolution of PSP systems across bacterial phyla contributes to our understanding of stress response mechanisms in prokaryotes. Moreover, the newly discovered PSP modularity will likely prompt new studies of lineage-specific cell-envelope structures, lifestyles, and adaptation mechanisms. Finally, our results validate use of domain architecture and genetic context for discovery in comparative genomics.

evolutionary biology↗

bioRxiv2020

Chaperone-mediated stress-sensing in Mycobacterium tuberculosis enables fast activation and sustained response

Dynamical properties of gene-regulatory networks are tuned to ensure bacterial survival. In mycobacteria, MprAB-{sigma}E network responds to the presence of stressors, such as surfactants causing surface stress. Positive feedback loops in this network were previously predicted to cause hysteresis, i.e. different responses to identical stressor levels for pre-stressed and unstressed cells. Here we show that hysteresis does not occur in non-pathogenic Mycobacterium smegmatis but occurs in Mycobacterium tuberculosis. However, the observed rapid temporal response in M. tuberculosis is inconsistent with the model predictions. To reconcile these observations, we implement a recently proposed mechanism for stress-sensing: the release of MprB from the inhibitory complex with chaperone DnaK upon the stress exposure. Using modeling and parameter fitting, we demonstrate that this mechanism can accurately describe the experimental observations. Furthermore, we predict perturbations in DnaK expression that can strongly affect dynamical properties. Experiments with these perturbations agree with model predictions, confirming the role of DnaK in fast and sustained response. IMPORTANCEGene-regulatory networks controlling stress response in mycobacterial species have been linked to persistence switches enabling the bacterial dormancy within a host. However, the mechanistic basis of switching and stress sensing is not fully understood. In this paper, combining quantitative experiments and mathematical modeling, we uncover how interactions between two master regulators of stress response -- MprAB two-component system and alternative sigma factor{sigma} E - shape the dynamical properties of surface-stress network. The result show hysteresis (history dependence) in the response of pathogenic bacteria M. tuberculosis to surface stress and lack of the hysteresis in non-pathogenic M. smegmatis. Furthermore, to resolve the apparent contradiction between the existence of hysteresis and fast activation of the response, we utilize a recently proposed role of chaperone DnaK in stress-sensing. The results leads to a novel system-level understanding of bacterial stress-response dynamics.

systems biology↗

bioRxiv2020

The anti-tubercular activity of simvastatin is mediated by cholesterol-dependent regulation of autophagy via the AMPK-mTORC1-TFEB axis

Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. We investigated the mechanism of anti-tubercular action of simvastatin in Mycobacterium tuberculosis-infected human monocytic cells. We found that the anti-tubercular activity of statins was phenocopied by cholesterol-branch but not prenylation-branch inhibitors. Moreover, statin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Overall, our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, AMPK-mTORC1-TFEB axis, and intracellular infection control, and uncover new anti-tubercular therapy targets.

immunology↗