The growth of mycobacterial cells requires successful coordination between elongation and septation. However, it is not clear which factors mediate this coordination. Here, we studied the function ...and post‐translational modification of an essential division factor, SepIVA, in Mycobacterium smegmatis. We find that SepIVA is arginine methylated, and that alteration of its methylation sites affects both septation and polar elongation of Msmeg. Furthermore, we show that SepIVA regulates the localization of MurG and that this regulation may impact polar elongation. Finally, we map SepIVA's two regulatory functions to different ends of the protein: the N‐terminus regulates elongation while the C‐terminus regulates division. These results establish SepIVA as a regulator of both elongation and division and characterize a physiological role for protein arginine methylation sites for the first time in mycobacteria.
In this work, we examine the function and regulation of the cell wall regulatory protein, SepIVA in Mycobacterium smegmatis. We show that SepIVA regulates both cell septation and elongation and that its effect on elongation may be mediated through the regulation of MurG. We also show that SepIVA's regulation of these two processes is affected by its arginine methylation by an unknown bacterial PRMT (Protein arginine methyltransferase).
Climate change may affect the foraging success of bowhead whales Balaena mysticetus by altering the diversity and abundance of zooplankton species available as food. However, assessing ...climate-induced impacts first requires documenting feeding conditions under current environmental conditions. We collected seasonal movement and dive-behaviour data from 25 Eastern Canada−West Greenland bowheads instrumented with time-depth telemetry tags and used state-space models to examine whale movements and dive behaviours. Zooplankton samples were also collected in Cumberland Sound (CS) to determine species composition and biomass. We found that CS was used seasonally by 14 of the 25 tagged whales. Area-restricted movement was the dominant behaviour in CS, suggesting that the tagged whales allocated considerable time to feeding. Prey sampling data suggested that bowheads were exploiting energy-rich Arctic copepods such as Calanus glacialis and C. hyperboreus during summer. Dive behaviour changed seasonally in CS. Most notably, probable feeding dives were substantially shallower during spring and summer compared to fall and winter. These seasonal changes in dive depths likely reflect changes in the vertical distribution of calanoid copepods, which are known to suspend development and overwinter at depth during fall and winter when availability of their phytoplankton prey is presumed to be lower. Overall, CS appears to be an important year-round foraging habitat for bowheads, but is particularly important during the late summer and fall. Whether CS will remain a reliable feeding area for bowhead whales under climate change is not yet known.
With an estimated 25% of the global population infected with
(
), tuberculosis (TB) remains a leading cause of death by infectious diseases. Humoral immunity following TB treatment is largely ...uncharacterized, and antibody profiling could provide insights into disease resolution. Here we focused on the distinctive TB-specific serum antibody features in active TB disease (ATB) and compared them with latent TB infection (LTBI) or treated ATB (txATB). As expected, di-galactosylated glycan structures (lacking sialic acid) found on IgG-Fc differentiated LTBI from ATB, but also discriminated txATB from ATB. Moreover, TB-specific IgG4 emerged as a novel antibody feature that correlated with active disease, elevated in ATB, but significantly diminished after therapy. These findings highlight 2 novel TB-specific antibody changes that track with the resolution of TB and may provide key insights to guide TB therapy.
Macrophages are a protective replicative niche for Mycobacterium tuberculosis (Mtb) but can kill the infecting bacterium when appropriately activated. To identify mechanisms of clearance, we compared ...levels of bacterial restriction by human macrophages after treatment with 26 compounds, including some currently in clinical trials for tuberculosis. All-
-retinoic acid (ATRA), an active metabolite of vitamin A, drove the greatest increase in Mtb control. Bacterial clearance was transcriptionally and functionally associated with changes in macrophage cholesterol trafficking and lipid metabolism. To determine how these macrophage changes affected bacterial control, we performed the first Mtb CRISPR interference screen in an infection model, identifying Mtb genes specifically required to survive in ATRA-activated macrophages. These data showed that ATRA treatment starves Mtb of cholesterol and the downstream metabolite propionyl coenzyme A (propionyl-CoA). Supplementation with sources of propionyl-CoA, including cholesterol, abrogated the restrictive effect of ATRA. This work demonstrates that targeting the coupled metabolism of Mtb and the macrophage improves control of infection and that it is possible to genetically map the mode of bacterial death using CRISPR interference.
Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, is a leading cause of death due to infectious disease. Improving the immune response to tuberculosis holds promise for fighting the disease but is limited by our lack of knowledge as to how the immune system kills M. tuberculosis. Our research identifies a potent way to make relevant immune cells more effective at fighting M. tuberculosis and then uses paired human and bacterial genomic methods to determine the mechanism of that improved bacterial clearance.
Abstract
The pathogenicity of Mycobacterium tuberculosis depends upon its ability to catabolize host cholesterol. Upregulation of the methylcitrate cycle (MCC) is required to assimilate and detoxify ...propionyl-CoA, a cholesterol degradation product. The transcription of key genes prpC and prpD in MCC is activated by MtPrpR, a member of a family of prokaryotic transcription factors whose structures and modes of action have not been clearly defined. We show that MtPrpR has a novel overall structure and directly binds to CoA or short-chain acyl-CoA derivatives to form a homotetramer that covers the binding cavity and locks CoA tightly inside the protein. The regulation of this process involves a 4Fe4S cluster located close to the CoA-binding cavity on a neighboring chain. Mutations in the 4Fe4S cluster binding residues rendered MtPrpR incapable of regulating MCC gene transcription. The structure of MtPrpR without the 4Fe4S cluster-binding region shows a conformational change that prohibits CoA binding. The stability of this cluster means it is unlikely a redox sensor but may function by sensing ambient iron levels. These results provide mechanistic insights into this family of critical transcription factors who share similar structures and regulate gene transcription using a combination of acyl-CoAs and 4Fe4S cluster.
To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis ...bacillus Calmette–Guérin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis- specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosinyladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesi s . Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis -prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis -prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis . Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis , and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.
Bacille Calmette-Guerin (BCG), the only approved Mycobacterium tuberculosis (Mtb) vaccine, provides limited durable protection when administered intradermally. However, recent work revealed that ...intravenous (i.v.) BCG administration yielded greater protection in macaques. Here, we perform a dose-ranging study of i.v. BCG vaccination in macaques to generate a range of immune responses and define correlates of protection. Seventeen of 34 macaques had no detectable infection after Mtb challenge. Multivariate analysis incorporating longitudinal cellular and humoral immune parameters uncovered an extensive and highly coordinated immune response from the bronchoalveolar lavage (BAL). A minimal signature predicting protection contained four BAL immune features, of which three remained significant after dose correction: frequency of CD4 T cells producing TNF with interferon γ (IFNγ), frequency of those producing TNF with IL-17, and the number of NK cells. Blood immune features were less predictive of protection. We conclude that CD4 T cell immunity and NK cells in the airway correlate with protection following i.v. BCG.
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•An i.v. Bacille Calmette-Guerin (BCG) dose-ranging study generated a range of protection against tuberculosis in macaques•Immune measurements from blood and BAL were integrated for correlates analysis•Multivariate analysis of BAL features revealed a highly coordinated immune network•Tuberculosis-specific CD4 Th1/Th17 and NK cells in the airway correlated with protection
Vaccination with intravenous Bacille Calmette-Guerin (i.v. BCG) protects macaques from tuberculosis. Using a dose-ranging i.v. BCG strategy, Darrah, Zeppa, Wang, et al. elicited graded immune responses and 50% protection in macaques. Multivariate analysis of longitudinal cellular and humoral parameters identified antigen-specific CD4 T cells and NK cells in the airway as strong correlates of protection.
The functional role of CD8+ lymphocytes in tuberculosis remains poorly understood. We depleted innate and/or adaptive CD8+ lymphocytes in macaques and showed that loss of all CD8α+ cells (using ...anti-CD8α antibody) significantly impaired early control of Mycobacterium tuberculosis (Mtb) infection, leading to increased granulomas, lung inflammation, and bacterial burden. Analysis of barcoded Mtb from infected macaques demonstrated that depletion of all CD8+ lymphocytes allowed increased establishment of Mtb in lungs and dissemination within lungs and to lymph nodes, while depletion of only adaptive CD8+ T cells (with anti-CD8β antibody) worsened bacterial control in lymph nodes. Flow cytometry and single-cell RNA sequencing revealed polyfunctional cytotoxic CD8+ lymphocytes in control granulomas, while CD8-depleted animals were unexpectedly enriched in CD4 and γδ T cells adopting incomplete cytotoxic signatures. Ligand-receptor analyses identified IL-15 signaling in granulomas as a driver of cytotoxic T cells. These data support that CD8+ lymphocytes are required for early protection against Mtb and suggest polyfunctional cytotoxic responses as a vaccine target.
Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how ...the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. A phylogenetic analysis of cwlM implies that localization in the cytoplasm drove the evolution of this factor. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance.
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