Host transcription in active and latent tuberculosis

A recent study has identified a transcriptional signature for active tuberculosis, suggesting that the distinction between active and latent forms may not be absolute

Cyclic AMP Regulation of Protein Lysine Acetylation in Mycobacterium Tuberculosis

Protein lysine acetylation networks can regulate central processes such as carbon metabolism and gene expression in bacteria. In Escherichia coli, cyclic-AMP (cAMP) regulates protein lysine acetyltransferase (PAT) activity at the transcriptional level, but in Mycobacterium tuberculosis, fusion of a cyclic-nucleotide binding domain to a Gcn5-like PAT domain enables direct cAMP control of protein acetylation. Here we describe the allosteric activation mechanism of M. tuberculosis PAT. The crystal structures of the auto-inhibited and cAMP-activated PAT reveal that cAMP binds to a cryptic site in the regulatory domain over 32 Å from the catalytic site. An extensive conformational rearrangement relieves auto-inhibition by a substrate-mimicking lid that covers the protein-substrate binding surface. A steric double latch couples the domains by harnessing a classic, cAMP-mediated, conformational switch. The structures suggest general features that enable the evolution of long-range communication between linked domains

Year-round foraging across large spatial scales suggest that bowhead whales have the potential to adapt to climate change

The ecological impact of environmental changes at high latitudes (e.g., increasing temperature, and decreased sea ice cover) on low-trophic species, such as bowhead whales, are poorly understood. Key to understanding the vulnerability of zooplanktivorous predators to climatic shifts in prey is knowing whether they can make behavioural or distributional adjustments to maintain sufficient prey acquisition rates. However, little is known about how foraging behaviour and associated environmental conditions fluctuate over space and time. We collected long-term movement (average satellite transmission days were 397 (± 204 SD) in 2012 and 484 (± 245 SD) in 2013) and dive behaviour data for 25 bowhead whales (Balaena mysticetus) equipped with time-depth telemetry tags, and used hierarchical switching-state-space models to quantify their movements and behaviours (resident and transit). We examined trends in inferred two-dimensional foraging behaviours based on dive shape of Eastern Canada-West Greenland bowhead whales in relation to season and sea ice, as well as animal sex and age via size. We found no differences with regards to whale sex and size, but we did find evidence that subsurface foraging occurs year-round, with peak foraging occurring in fall (7.3 hrs d-1 ± 5.70 SD; October) and reduced feeding during spring (2.7 hrs d-1 ± 2.55 SD; May). Although sea ice cover is lowest during summer foraging, whales selected areas with 65% (± 36.1 SD) sea ice cover. During winter, bowheads occurred in areas with 90% (± 15.5 SD) ice cover, providing some open water for breathing. The depth of probable foraging varied across seasons with animals conducting epipelagic foraging dives (< 200 m) during spring and summer, and deeper mesopelagic dives (> 400 m) during fall and winter that approached the sea bottom, following the seasonal vertical migration of lipid-rich zooplankton. Our findings suggest that, compared to related species (e.g., right whales), bowheads forage at relatively low rates and over a large geographic area throughout the year. This suggests that bowhead whales have the potential to adjust their behaviours (e.g., increased time allocated to feeding) and shift their distributions (e.g., occupy higher latitude foraging grounds) to adapt to climate-change induced environmental conditions. However, the extent to which energetic consumption may vary seasonally is yet to be determined

Bowhead whales use two foraging strategies in response to fine-scale differences in zooplankton vertical distribution

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Fortune, S. M. E., Ferguson, S. H., Trites, A. W., Hudson, J. M., & Baumgartner, M. F. Bowhead whales use two foraging strategies in response to fine-scale differences in zooplankton vertical distribution. Scientific Reports, 10(1), (2020): 20249, doi:10.1038/s41598-020-76071-9.As zooplanktivorous predators, bowhead whales (Balaena mysticetus) must routinely locate patches of prey that are energy-rich enough to meet their metabolic needs. However, little is known about how the quality and quantity of prey might influence their feeding behaviours. We addressed this question using a new approach that included: (1) multi-scale biologging and unmanned aerial system observations of bowhead whales in Cumberland Sound, Nunavut (Canada), and (2) an optical plankton counter (OPC) and net collections to identify and enumerate copepod prey species through the water column. The OPC data revealed two prey layers comprised almost exclusively of lipid-rich calanoid copepods. The deep layer contained fewer, but larger, particles (10% greater overall biomass) than the shallow prey layer. Dive data indicated that the whales conducted long deep Square-shaped dives (80% of dives; averaging depth of 260.4 m) and short shallow Square-shaped dives (16%; averaging depth of 22.5 m) to feed. The whales tended to dive proportionally more to the greater biomass of zooplankton that occurred at depth. Combining behavioural recordings with prey sampling showed a more complex feeding ecology than previously understood, and provides a means to evaluate the energetic balance of individuals under current environmental conditions.Funding was awarded to S.H.F and provided by: Fisheries and Oceans Canada (Emerging Fisheries), World Wildlife Fund Canada (Arctic Species Conservation Fund), Nunavut Wildlife Research Trust Fund, Nunavut General Monitoring Program, Ocean Tracking Network and ArcticNet Centre of Excellence. Personal support was awarded to S.M.E.F and provided by Natural Sciences and Engineering Research Council Canadian Graduate Scholarship, Northern Scientific Training Program (Canadian Polar Commission), The Molson Foundation and the W.Garfield Weston Foundation

Antibody Fc Glycosylation Discriminates Between Latent and Active Tuberculosis

Background. Mycobacterium tuberculosis remains a global health problem and clinical management is complicated by difficulty in discriminating between latent infection and active disease. While M. tuberculosis-reactive antibody levels are heterogeneous, studies suggest that levels of IgG glycosylation differ between disease states. Here we extend this observation across antibody domains and M. tuberculosis specificities to define changes with the greatest resolving power. Methods. Capillary electrophoretic glycan analysis was performed on bulk non-antigen–specific IgG, bulk Fc domain, bulk Fab domain, and purified protein derivative (PPD)- and Ag85A-specific IgG from subjects with latent (n = 10) and active (n = 20) tuberculosis. PPD-specific isotype/subclass, PPD-specific antibody-dependent phagocytosis, cellular cytotoxicity, and natural killer cell activation were assessed. Discriminatory potentials of antibody features were evaluated individually and by multivariate analysis. Results. Parallel profiling of whole, Fc, and Fab domain-specific IgG glycosylation pointed to enhanced differential glycosylation on the Fc domain. Differential glycosylation was observed across antigen-specific antibody populations. Multivariate modeling highlighted Fc domain glycan species as the top discriminatory features, with combined PPD IgG titers and Fc domain glycans providing the highest classification accuracy. Conclusions. Differential glycosylation occurs preferentially on the Fc domain, providing significant discriminatory power between different states of M. tuberculosis infection and disease

Modelling multi-scale state-switching functional data with hidden Markov models

Data sets comprised of sequences of curves sampled at high frequencies in time are increasingly common in practice, but they can exhibit complicated dependence structures that cannot be modelled using common methods of Functional Data Analysis (FDA). We detail a hierarchical approach which treats the curves as observations from a hidden Markov model (HMM). The distribution of each curve is then defined by another fine-scale model which may involve auto-regression and require data transformations using moving-window summary statistics or Fourier analysis. This approach is broadly applicable to sequences of curves exhibiting intricate dependence structures. As a case study, we use this framework to model the fine-scale kinematic movement of a northern resident killer whale (Orcinus orca) off the coast of British Columbia, Canada. Through simulations, we show that our model produces more interpretable state estimation and more accurate parameter estimates compared to existing methods.Comment: 23 pages, 8 figures, 2 tables. Supplementary material appended to submissio

Sterilization of granulomas is common in both active and latent tuberculosis despite extensive within-host variability in bacterial killing

Over 30% of the world’s population is infected with Mycobacterium tuberculosis (Mtb), yet only ~5–10% will develop clinical disease1. Despite considerable effort, we understand little about what distinguishes individuals who progress to active tuberculosis (TB) from those who remain latent for decades. The variable course of disease is recapitulated in cynomolgus macaques infected with Mtb2. Active disease in macaques is defined by clinical, microbiologic and immunologic signs and occurs in ~45% of animals, while the remaining are clinically asymptomatic2,3. Here, we use barcoded Mtb isolates and quantitative measures of culturable and cumulative bacterial burden to show that most lesions are likely founded by a single bacterium and reach similar maximum burdens. Despite common origins, the fate of individual lesions varies substantially within the same host. Strikingly, in active disease, the host sterilizes some lesions even while others progress. Our data suggest that lesional heterogeneity arises, in part, through differential killing of bacteria after the onset of adaptive immunity. Thus, individual lesions follow diverse and overlapping trajectories, suggesting critical responses occur at a lesional level to ultimately determine the clinical outcome of infection. Defining the local factors that dictate outcome will be important in developing effective interventions to prevent active TB

RNA Extraction from a Mycobacterium under Ultrahigh Electric Field Intensity in a Microfluidic Device

Studies of transcriptomes are critical for understanding gene expression. Release of RNA molecules from cells is typically the first step for transcriptomic analysis. Effective cell lysis approaches that completely release intracellular materials are in high demand especially for cells that are structurally robust. In this report, we demonstrate a microfluidic electric lysis device that is effective for mRNA extraction from mycobacteria that have hydrophobic and waxy cell walls. We used a packed bed of microscale silica beads to filter M. smegmatis out of the suspension. 4000–8000 V/cm field intensity was used to lyse M. smegmatis with long pulses (i.e., up to 30 pulses that were 5 s long each). Our quantitative reverse transcription (qRT)-PCR results showed that our method yielded a factor of 10–20 higher extraction efficiency than the current state-of-the-art method (bead beating). We conclude that our electric lysis technique is an effective approach for mRNA release from hard-to-lyse cells and highly compatible with microfluidic molecular assays

Loss of RNase J leads to multi-drug tolerance and accumulation of highly structured mRNA fragments in Mycobacterium tuberculosis

Despite the existence of well-characterized, canonical mutations that confer high-level drug resistance to Mycobacterium tuberculosis (Mtb), there is evidence that drug resistance mechanisms are more complex than simple acquisition of such mutations. Recent studies have shown that Mtb can acquire non-canonical resistance-associated mutations that confer survival advantages in the presence of certain drugs, likely acting as stepping-stones for acquisition of high-level resistance. Rv2752c/rnj, encoding RNase J, is disproportionately mutated in drug-resistant clinical Mtb isolates. Here we show that deletion of rnj confers increased tolerance to lethal concentrations of several drugs. RNAseq revealed that RNase J affects expression of a subset of genes enriched for PE/PPE genes and stable RNAs and is key for proper 23S rRNA maturation. Gene expression differences implicated two sRNAs and ppe50-ppe51 as important contributors to the drug tolerance phenotype. In addition, we found that in the absence of RNase J, many short RNA fragments accumulate because they are degraded at slower rates. We show that the accumulated transcript fragments are targets of RNase J and are characterized by strong secondary structure and high G+C content, indicating that RNase J has a rate-limiting role in degradation of highly structured RNAs. Taken together, our results demonstrate that RNase J indirectly affects drug tolerance, as well as reveal the endogenous roles of RNase J in mycobacterial RNA metabolism.Fil: Martini, María Carla. Worcester Polytechnic Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hicks, Nathan D.. Harvard University. Harvard School of Public Health; Estados UnidosFil: Xiao, Junpei. Worcester Polytechnic Institute; Estados UnidosFil: Alonso, Maria Natalia. Worcester Polytechnic Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barbier, Thibault. Harvard University. Harvard School of Public Health; Estados UnidosFil: Sixsmith, Jaimie. Harvard University. Harvard School of Public Health; Estados UnidosFil: Fortune, Sarah M.. Harvard University. Harvard School of Public Health; Estados UnidosFil: Shell, Scarlet S.. Worcester Polytechnic Institute; Estados Unido

Efferocytosis is an innate antibacterial mechanism

Mycobacterium tuberculosis persists within macrophages in an arrested phagosome and depends upon necrosis to elude immunity and disseminate. Although apoptosis of M. tuberculosis-infected macrophages is associated with reduced bacterial growth, the bacteria are relatively resistant to other forms of death, leaving the mechanism underlying this observation unresolved. We find that after apoptosis, M. tuberculosis-infected macrophages are rapidly taken up by uninfected macrophages through efferocytosis, a dedicated apoptotic cell engulfment process. Efferocytosis of M. tuberculosis sequestered within an apoptotic macrophage further compartmentalizes the bacterium and delivers it along with the apoptotic cell debris to the lysosomal compartment. M. tuberculosis is killed only after efferocytosis, indicating that apoptosis itself is not intrinsically bactericidal but requires subsequent phagocytic uptake and lysosomal fusion of the apoptotic body harboring the bacterium. While efferocytosis is recognized as a constitutive housekeeping function of macrophages, these data indicate that it can also function as an antimicrobial effector mechanism.Behar, Fortune, and Remold labs for reagents, helpful discussion, and insights. TIM4-blocking antibodies were a generous gift of Vijay Kuchroo. Members of the Harvard Electron Microscopy Core Facility helped in the preparation, staining, and operation of the electron microscope. The Small Animal Biocontainment (ABC) Suite is supported by CFAR 5P30AI060354. T.R.R and S.M.F were supported by CFAR 5P30AI060354, DP2-0d001378, and T32-AI07387. C.N.A. is the recipient of a fellowship from FCT. S.M.B and H.G.R. were supported by R56AI084161 and R01AI072143