Role of succinyl substituents in the mannose-capping of lipoarabinomannan and control of inflammation in Mycobacterium tuberculosis infection.

The covalent modification of bacterial (lipo)polysaccharides with discrete substituents may impact their biosynthesis, export and/or biological activity. Whether mycobacteria use a similar strategy to control the biogenesis of its cell envelope polysaccharides and modulate their interaction with the host during infection is unknown despite the report of a number of tailoring substituents modifying the structure of these glycans. Here, we show that discrete succinyl substituents strategically positioned on Mycobacterium tuberculosis (Mtb) lipoarabinomannan govern the mannose-capping of this lipoglycan and, thus, much of the biological activity of the entire molecule. We further show that the absence of succinyl substituents on the two main cell envelope glycans of Mtb, arabinogalactan and lipoarabinomannan, leads to a significant increase of pro-inflammatory cytokines and chemokines in infected murine and human macrophages. Collectively, our results validate polysaccharide succinylation as a critical mechanism by which Mtb controls inflammation.

Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae).

Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of C. tobin is small (59 Mb), compact (∼ 40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two "red" RuBisCO activases that are shared across many algal lineages. The Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.

A Modified BPaL Regimen for Tuberculosis Treatment replaces Linezolid with Inhaled Spectinamides.

The Nix-TB clinical trial evaluated a new 6-month regimen containing three-oral-drugs; bedaquiline (B), pretomanid (Pa) and linezolid (L) (BPaL regimen) for treatment of tuberculosis (TB). This regimen achieved remarkable results as almost 90% of the multidrug resistant (MDR) or extensively drug resistant (XDR) TB participants were cured but many patients also developed severe adverse effects (AEs). The AEs were associated with the long-term administration of the protein synthesis inhibitor linezolid. Spectinamide 1599 (S) is also a protein synthesis inhibitor of Mycobacterium tuberculosis with an excellent safety profile but which lacks oral bioavailability. Here we hypothesize that inhaled spectinamide 1599, combined with BPa --BPaS regimen--has similar efficacy to that of BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the Balb/c and C3HeB/FeJ murine chronic TB efficacy models. After 4-weeks of treatment, both regimens promoted equivalent bactericidal effect in both TB murine models. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL treatment also decreased myeloid to erythroid ratio and increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. During therapy both regimens improved the lung lesion burden, reduced neutrophil and cytotoxic T cells counts while increased the number of B and helper and regulatory T cells. These combined data suggest that inhaled spectinamide 1599 combined with BPa is an effective TB regimen that avoids L-associated AEs. IMPORTANCE: Tuberculosis (TB) is an airborne infectious disease that spreads via aerosols containing Mycobacterium tuberculosis (Mtb), the causative agent of TB. TB can be cured by administration of 3-4 drugs for 6-9 months but there are limited treatment options for patients infected with multidrug (MDR) and extensively resistant (XDR) strains of Mtb. BPaL is a new all-oral combination of drugs consisting of Bedaquiline (B), Pretomanid (Pa) and Linezolid (L). This regimen was able to cure ∼90% of MDR and XDR TB patients in clinical trials but many patients developed severe adverse effects (AEs) associated to the long-term administration of linezolid. We evaluated a new regimen in which Linezolid in the BPaL regimen was replaced with inhaled spectinamide 1599. In the current study, we demonstrate that 4-weeks of treatment with inhaled spectinamide 1599 in combination with Bedaquiline and Pretomanid has equivalent efficacy to the BPaL drug combination and avoids the L-associated-AEs.

Mucosal exposure to non-tuberculous mycobacteria elicits B cell-mediated immunity against pulmonary tuberculosis.

Bacille Calmette-Guerin (BCG) is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease. However, BCG has limited efficacy, necessitating the development of better vaccines. Non-tuberculous mycobacteria (NTMs) are opportunistic pathogens present ubiquitously in the environment. TB endemic countries experience higher exposure to NTMs, but previous studies have not elucidated the relationship between NTM exposure and BCG efficacy against TB. Therefore, we develop a mouse model (BCG + NTM) to simulate human BCG immunization regime and continuous NTM exposure. BCG + NTM mice exhibit superior and prolonged protection against pulmonary TB, with increased B cell influx and anti-Mtb antibodies in serum and airways, compared with BCG alone. Notably, spatial transcriptomics and immunohistochemistry reveal that BCG + NTM mice formed B cell aggregates with features of germinal center development, which correlate with reduced Mtb burden. Our studies suggest a direct relationship between NTM exposure and TB protection, with B cells playing a crucial role.

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events.

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Behavioral and physiological changes during benthic-pelagic transition in the harmful alga, Heterosigma akashiwo: potential for rapid bloom formation.

Many species of harmful algae transition between a motile, vegetative stage in the water column and a non-motile, resting stage in the sediments. Physiological and behavioral traits expressed during benthic-pelagic transition potentially regulate the timing, location and persistence of blooms. The roles of key physiological and behavioral traits involved in resting cell emergence and bloom formation were examined in two geographically distinct strains of the harmful alga, Heterosigma akashiwo. Physiological measures of cell viability, division and population growth, and cell fatty acid content were made using flow cytometry and gas chromatography - mass spectrometry techniques as cells transitioned between the benthic resting stage and the vegetative pelagic stage. Video-based tracking was used to quantify cell-level swimming behaviors. Data show increased temperature and light triggered rapid emergence from the resting stage and initiated cell swimming. Algal strains varied in important physiological and behavioral traits, including survivorship during life-stage transitions, population growth rates and swimming velocities. Collectively, these traits function as "population growth strategies" that can influence bloom formation. Many resting cells regained the up-swimming capacity necessary to cross an environmentally relevant halocline and the ability to aggregate in near-surface waters within hours after vegetative growth supporting conditions were restored. Using a heuristic model, we illustrate how strain-specific population growth strategies can govern the timescales over which H. akashiwo blooms form. Our findings highlight the need for identification and quantification of strain-specific physiological and behavioral traits to improve mechanistic understanding of bloom formation and successful bloom prediction.