Biology subjects

Fines, D. M.

Publications and source records attributed to Fines, D. M..

3 recordsLinked to original sources

bioRxiv2023

Mycobacterial formation of intracellular lipid inclusions is a dynamic process associated with rapid replication

Intracellular lipid inclusions (ILI) are triacylglyceride rich organelles produced by mycobacteria thought to serve as energy reservoirs. It is believed that ILI are formed as a result of a dosR mediated transition from replicative growth to non-replicating persistence (NRP). ILI rich Mycobacterium tuberculosis (Mtb) bacilli have been reported during infection and in sputum, establishing their importance in Mtb pathogenesis. Studies conducted in mycobacteria such as Mycobacterium smegmatis, Mycobacterium abscessus, or lab Mtb strains have demonstrated ILI formation in the presence of hypoxic, nitric oxide, nutrient limitation, or low nitrogen stress, conditions believed to emulate the host environment within which Mtb resides. Here, we show that M. marinum and clinical Mtb isolates make ILI during active replication in axenic culture independent of environmental stressors. By tracking ILI formation dynamics we demonstrate that ILI are quickly formed in the presence of fresh media or exogenous fatty acids but are rapidly depleted while bacteria are still actively replicating. We also show that the cell envelope is an alternate site for neutral lipid accumulation observed during stationary phase. In addition, we screen a panel of 60 clinical isolates and observe variation in ILI production during early log phase growth between and among Mtb lineages. Finally, we show that dosR expression level does not strictly correlate with ILI accumulation in fresh clinical isolates. Taken together, our data provide evidence of an active ILI formation pathway in replicating mycobacteria cultured in the absence of stressors, suggesting a decoupling of ILI formation from NRP.

microbiology↗

bioRxiv2021

IFN-γ-independent control of M. tuberculosis requires CD4 T cell-derived GM-CSF and activation of HIF-1α

The prevailing model of protective immunity to tuberculosis is that CD4 T cells produce the cytokine IFN-{gamma} to activate bactericidal mechanisms in infected macrophages. Recent evidence has expanded this model, and it is now clear that CD4 T cells can control M. tuberculosis infection in the absence of IFN-{gamma} production. To identify factors and pathways involved in IFN-{gamma}-independent control, we developed a co-culture model using CD4 T cells isolated from the lungs of infected mice and M. tuberculosis-infected murine bone marrow-derived macrophages (BMDMs). We show that IFN-{gamma}-independent control is primarily mediated by CD4 T cell production of the cytokine GM-CSF and requires activation of the macrophage transcription factor HIF-1. HIF-1 activation drives a metabolic shift toward aerobic glycolysis and leads to the production of lipid droplets, both of which support host defense against infection. Surprisingly, recombinant GM-CSF is insufficient to rescue the absence of control by GM-CSF-deficient CD4 T cells during co-culture with BMDMs. In peritoneal macrophages, GM-CSF is sufficient to control growth, induces lipid droplet biogenesis, and requires HIF-1 expression for control. While HIF-1-mediated control following IFN-{gamma} stimulation requires nitric oxide, we find that HIF-1 activation by CD4 T cells and recombinant GM-CSF is nitric oxide-independent, implying a distinct pathway of activation. In addition to GM-CSF, CD4 T cells produce a factor that helps maintain phagosome membrane integrity during infection and blocks bacterial access to host lipids, a primary nutrient source. These results advance our understanding of CD4 T cell-mediated immunity to M. tuberculosis, clarify the role of nitric oxide as primarily immunomodulatory during M. tuberculosis infection, and reveal a novel mechanism for the activation of HIF-1. Furthermore, we establish a previously unknown functional link between GM-CSF and HIF-1 and provide evidence that CD4 T cell-derived GM-CSF is a potent bactericidal effector.

immunology↗

bioRxiv2020

Screening a library of FDA-approved and bioactive compounds for antiviral activity against SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 80 million cases and 1.7 million deaths to date while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.

microbiology↗