ARL8B mediates lipid droplet contact and delivery to lysosomes for lipid remobilization.

Lipid droplets (LDs) play a crucial role in maintaining cellular lipid balance by storing and delivering lipids as needed. However, the intricate lipolytic pathways involved in LD turnover remain poorly described, hindering our comprehension of lipid catabolism and related disorders. Here, we show a function of the small GTPase ARL8B in mediating LD turnover in lysosomes. ARL8B-GDP localizes to LDs, while ARL8-GTP predominantly favors lysosomes. GDP binding induces a conformation with an exposed N-terminal amphipathic helix, enabling ARL8B to bind to LDs. By associating with LDs and lysosomes, and with its property to form a heterotypic complex, ARL8B mediates LD-lysosome contacts and efficient lipid transfer between these organelles. In human macrophages, this ARL8B-dependent LD turnover mechanism appears as the major lipolytic pathway. Our finding opens exciting possibilities for understanding the molecular mechanisms underlying LD degradation and its potential implications for inflammatory disorders.

Inhibition of Granuloma Triglyceride Synthesis Imparts Control of Mycobacterium tuberculosis Through Curtailed Inflammatory Responses.

Lipid metabolism plays a complex and dynamic role in host-pathogen interaction during Mycobacterium tuberculosis infection. While bacterial lipid metabolism is key to the success of the pathogen, the host also offers a lipid rich environment in the form of necrotic caseous granulomas, making this association beneficial for the pathogen. Accumulation of the neutral lipid triglyceride, as lipid droplets within the cellular cuff of necrotic granulomas, is a peculiar feature of pulmonary tuberculosis. The role of triglyceride synthesis in the TB granuloma and its impact on the disease outcome has not been studied in detail. Here, we identified diacylglycerol O-acyltransferase 1 (DGAT1) to be essential for accumulation of triglyceride in necrotic TB granulomas using the C3HeB/FeJ murine model of infection. Treatment of infected mice with a pharmacological inhibitor of DGAT1 (T863) led to reduction in granuloma triglyceride levels and bacterial burden. A decrease in bacterial burden was associated with reduced neutrophil infiltration and degranulation, and a reduction in several pro-inflammatory cytokines including IL1ß, TNFα, IL6, and IFNß. Triglyceride lowering impacted eicosanoid production through both metabolic re-routing and via transcriptional control. Our data suggests that manipulation of lipid droplet homeostasis may offer a means for host directed therapy in Tuberculosis.

Quantitative Lipid Droplet Proteomics Reveals Mycobacterium tuberculosis Induced Alterations in Macrophage Response to Infection.

Growing evidence suggests the importance of lipid metabolism in pathogenesis of tuberculosis. Neutral lipids form the majority of lipids in a caseous granuloma, a pathology characteristic of tuberculosis. Cytosolic lipid droplets (LDs) of macrophages form the store house of these lipids and have been demonstrated to contribute to the inflammatory response to infection. The proteome of lipid droplets reflects the mechanisms of lipid metabolism active under a condition. However, infection induced changes in the proteome of these dynamic organelles remains elusive. Here, we employed quantitative proteomics to identify alterations induced upon infection with live Mycobacterium tuberculosis (Mtb) in comparison with heat killed bacilli or uninfected macrophages. We found increased abundance of proteins coupled with lipid metabolism, protein synthesis, and vesicular transport function in LDs upon infection with live Mtb. Using biochemical methods and microscopy, we validated ADP-ribosyltransferase (Arf)-like 8 (ARL8B) to be increased on the lipid droplet surface of live Mtb infected macrophages and that ARL8B is a bonafide LD protein. This study provides the first proteomic evidence that the dynamic responses to infection also encompass changes at the level of LDs. This information will be important in understanding how Mtb manipulates lipid metabolism and defense mechanisms of the host macrophage.

Tuning the Mycobacterium tuberculosis Alternative Sigma Factor SigF through the Multidomain Regulator Rv1364c and Osmosensory Kinase Protein Kinase D.

Bacterial alternative sigma factors are mostly regulated by a partner-switching mechanism. Regulation of the virulence-associated alternative sigma factor SigF of Mycobacterium tuberculosis has been an area of intrigue, with SigF having more predicted regulators than other sigma factors in this organism. Rv1364c is one such predicted regulator, the mechanism of which is confounded by the presence of both anti-sigma factor and anti-sigma factor antagonist functions in a single polypeptide. Using protein binding and phosphorylation assays, we demonstrate that the anti-sigma factor domain of Rv1364c mediates autophosphorylation of its antagonist domain and binds efficiently to SigF. Furthermore, we identified a direct role for the osmosensor serine/threonine kinase PknD in regulating the SigF-Rv1364c interaction, adding to the current understanding about the intersection of these discrete signaling networks. Phosphorylation of SigF also showed functional implications in its DNA binding ability, which may help in activation of the regulon. In M. tuberculosis, osmotic stress-dependent induction of espA, a SigF target involved in maintaining cell wall integrity, is curtailed upon overexpression of Rv1364c, showing its role as an anti-SigF factor. Overexpression of Rv1364c led to induction of another target, pks6, involved in lipid metabolism. This induction was, however, curtailed in the presence of osmotic stress conditions, suggesting modulation of SigF target gene expression via Rv1364c. These data provide evidence that Rv1364c acts an independent SigF regulator that is sensitive to the osmosensory signal, mediating the cross talk of PknD with the SigF regulon.IMPORTANCEMycobacterium tuberculosis, capable of latently infecting the host and causing aggressive tissue damage during active tuberculosis, is endowed with a complex regulatory capacity built of several sigma factors, protein kinases, and phosphatases. These proteins regulate expression of genes that allow the bacteria to adapt to various host-derived stresses, like nutrient starvation, acidic pH, and hypoxia. The cross talk between these systems is not well understood. SigF is one such regulator of gene expression that helps M. tuberculosis to adapt to stresses and imparts virulence. This work provides evidence for its inhibition by the multidomain regulator Rv1364c and activation by the kinase PknD. The coexistence of negative and positive regulators of SigF in pathogenic bacteria reveals an underlying requirement for tight control of virulence factor expression.

Necrosis Driven Triglyceride Synthesis Primes Macrophages for Inflammation During Mycobacterium tuberculosis Infection.

Pulmonary tuberculosis (TB) exhibits granulomatous inflammation, a site of controlling bacterial dissemination at the cost of host tissue damage. Intrigued by the granuloma type-dependent expression of inflammatory markers in TB, we sought to investigate underlying metabolic changes that drive amplification of inflammation in TB. Here, we show an association of higher inflammation in necrotic granulomas with the presence of triglyceride (TG)-rich foamy macrophages. The conspicuous absence of these macrophages in solid granulomas identified a link between the ensuing pathology and the metabolic programming of foamy macrophages. Consistent with in vivo findings, in vitro infection of macrophages with Mycobacterium tuberculosis (Mtb) led to increase in TG synthesis only under conditions of ~60% necrosis. Genetic and pharmacologic intervention that reduced necrosis prevented this bystander response. We further demonstrate that necrosis independent of Mtb also elicits the same bystander response in human macrophages. We identified a role for the human enzyme involved in TG synthesis, diacylglycerol O-acyltransferase (DGAT1), in this phenomenon. The increased TG levels in necrosis-associated foamy macrophages promoted the pro-inflammatory state of macrophages to infection while silencing expression of diacylglycerol O-acyltransferase (DGAT1) suppressed expression of pro-inflammatory genes. Our data thus invoke a role for storage lipids in the heightened host inflammatory response during infection-associated necrosis. Our data provide a functional role to macrophage lipid droplets in host defense and open new avenues for developing host-directed therapies against TB.

Phospholipid homeostasis, membrane tenacity and survival of Mtb in lipid rich conditions is determined by MmpL11 function.

The mycobacterial cell wall is a chemically complex array of molecular entities that dictate the pathogenesis of Mycobacterium tuberculosis. Biosynthesis and maintenance of this dynamic entity in mycobacterial physiology is still poorly understood. Here we demonstrate a requirement for M. tuberculosis MmpL11 in the maintenance of the cell wall architecture and stability in response to surface stress. In the presence of a detergent like Tyloxapol, a mmpL11 deletion mutant suffered from a severe growth attenuation as a result of altered membrane polarity, permeability and severe architectural damages. This mutant failed to tolerate permissible concentrations of cis-fatty acids suggesting its increased sensitivity to surface stress, evident as smaller colonies of the mutant outgrown from lipid rich macrophage cultures. Additionally, loss of MmpL11 led to an altered cellular fatty acid flux in the mutant: reduced incorporation into membrane cardiolipin was associated with an increased flux into the cellular triglyceride pool. This increase in storage lipids like triacyl glycerol (TAG) was associated with the altered metabolic state of higher dormancy-associated gene expression and decreased sensitivity to frontline TB drugs. This study provides a detailed mechanistic insight into the function of mmpL11 in stress adaptation of mycobacteria.

The impact of consultant delivered service in emergency medicine: the Wrexham Model.

OBJECTIVE: Consultant based delivery of emergency service is perceived to add value. This study aims to demonstrate the impact of such a service model based on consultant working in a UK emergency department. METHODS: This retrospective study was based on the emergency department of a district general hospital. Activity data was analysed for 2009. Workload and admission rates were compared between consultants, middle grade doctors and senior house officers (SHOs). Admission rates were compared against two similar departments. Data from night shifts allowed consultant activity to be contrasted with middle grades and SHOs. Time spent in the department, admission rates, patients who left without treatment, discharged outright and clinic returns were used for comparison. RESULTS: Consultants often saw more patients than SHOs or middle grade doctors. This was on top of their traditional duties of senior opinion. On comparison of activity at night shifts, they admitted fewer (25.2% vs 30.3%, p=0.026), had fewer leaving without treatment (1.6% vs 5.1%, p<0.001), discharged more outright (59.8% vs 47.5%, p<0.001), referred fewer to clinic (5.7% vs 6.6%, p=0.49) and had a faster turnaround time (p<0.001: Priority 2, 3 and 4) for every triage category. Some of the comparisons were clinically but not statistically significant. CONCLUSION: A consultant based service delivery offers many advantages. These cannot be matched by either junior or middle grades. This would be in addition to the consultants' supervisory role. Consultant expansion is urgently required to achieve this sustainably. A further study evaluating the cost benefits of this service model is now underway.