Sustainable manure management technologies are needed, and combining anaerobic digestion (AD) for energy generation and aerobic composting (AC) to stabilize digestate and remove emerging contaminants (ECs), including veterinary pharmaceuticals and steroid hormones, is promising. This study identified post-AD, AC operating conditions that maximized degradation of study ECs, expected to be present in cattle manure digested using treated municipal wastewater as the water source. Study ECs included sulfamethoxazole (SMX), chlortetracycline (CTC), oxytetracycline (OTC), estrone (E1), and naproxen (NPX). Composting conditions were simulated in bench-scale reactors, with microorganisms from digestate produced in an AD system (25L scale), by varying temperatures, pH, and carbon source compositions (representing food waste/manure co-digestion with different residence times). Results indicate maximum SMX biodegradation occurred at 35°C, pH 7, and with high levels of easily degradable carbon (≥99%, 99%, and 98%), and maximum E1 biodegradation occurred at 35°C, and with low levels of easily degradable carbon (≥97% and 99%). Abiotic degradation was responsible for the nearly complete removal of tetracyclines under all conditions and for partial degradation of NPX (between 20% and 48%). Microorganisms originating from the AD system putatively capable of SMX and E1 biodegradation, or of contributing to biodegradation during the AC phase, were identified, including phylotypes previously shown to biodegrade SMX (Brevundimonas and Alcaligenes).

Barth syndrome (BTHS) is an X-linked disorder that results from mutations in the TAFAZZIN gene, which encodes a phospholipid transacylase responsible for generating the mature form of cardiolipin in inner mitochondrial membranes. BTHS patients develop early onset cardiomyopathy and a derangement of intermediary metabolism consistent with mitochondrial disease, but the precise alterations in cardiac metabolism that distinguish BTHS from idiopathic forms of cardiomyopathy are unknown. We performed the first metabolic analysis of myocardial tissue from BTHS cardiomyopathy patients compared to age- and sex-matched patients with idiopathic dilated cardiomyopathy (DCM) and nonfailing controls. Results corroborate previous evidence for deficiencies in cardiolipin content and its linoleoyl enrichment as defining features of BTHS cardiomyopathy, and reveal a dramatic accumulation of hydrolyzed (monolyso-) cardiolipin molecular species. Respiratory chain protein deficiencies were observed in both BTHS and DCM, but a selective depletion of complex I was seen only in BTHS after controlling for an apparent loss of mitochondrial density in cardiomyopathic hearts. Distinct shifts in the expression of long-chain fatty acid oxidation enzymes and the tissue acyl-CoA profile of BTHS hearts suggest a specific block in mitochondrial fatty acid oxidation upstream of the conventional matrix beta-oxidation cycle, which may be compensated for by a greater reliance upon peroxisomal fatty acid oxidation and the catabolism of ketones, amino acids, and pyruvate to meet cardiac energy demands. These results provide a comprehensive foundation for exploring novel therapeutic strategies that target the adaptive and maladaptive metabolic features of BTHS cardiomyopathy.

Background: Antimicrobial resistance is a major challenge in treating infectious diseases. Therapeutic drug monitoring (TDM) can optimize and personalize antibiotic treatment. Previously, antibiotic concentrations in tissues were extrapolated from skin blister studies, but sweat analyses for TDM have not been conducted. Objective: To investigate the potential of sweat analysis as a non-invasive, rapid, and potential bedside TDM method. Methods: We analyzed sweat and blood samples from 13 in-house patients treated with intravenous cefepime, imipenem, or flucloxacillin. For cefepime treatment, full pharmacokinetic sampling was performed (five subsequent sweat samples every 2 h) using ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry. The ClinicalTrials.gov registration number is NCT03678142. Results: In this study, we demonstrated for the first time that flucloxacillin, imipenem, and cefepime are detectable in sweat. Antibiotic concentration changes over time demonstrated comparable (age-adjusted) dynamics in the blood and sweat of patients treated with cefepime. Patients treated with standard flucloxacillin dosage showed the highest mean antibiotic concentration in sweat. Conclusions: Our results provide a proof-of-concept that sweat analysis could potentially serve as a non-invasive, rapid, and reliable method to measure antibiotic concentration and as a surrogate marker for tissue penetration. If combined with smart biosensors, sweat analysis may potentially serve as the first lab-independent, non-invasive antibiotic TDM method.

Hibernators have adapted a physiological mechanism allowing them to undergo long periods of inactivity without experiencing bone loss. However, the biological mechanisms that prevent bone loss are unknown. Previous studies found meaningful changes, between active and hibernating marmots, in the endocannabinoid system of many tissues, including bone. Cannabinoid receptors (CB1 and CB2) have divergent localization in bone. CB1 is predominately found on sympathetic nerve terminals, while CB2 is more abundant on bone cells and their progenitors. This study aimed to determine the contribution of innervation on endocannabinoid regulation of bone properties in hibernating (during torpor) and non-hibernating yellow-bellied marmots. Neurectomy, a model for disuse osteoporosis, was performed unilaterally in both hibernating and active marmots. Endocannabinoid concentrations were measured in bone marrow, cortical, and trabecular regions from fourth metatarsals of both hindlimbs using microflow chromatography-tandem quadrupole mass spectrometry. Trabecular bone architectural properties of fifth metatarsals were evaluated using micro-computed tomography. There were ligand-specific increases with neurectomy in active, but not hibernating, marmots. Trabecular bone architectural properties were not affected by neurectomy during hibernation, but did show some minor negative changes in active marmots. These findings suggest protection from bone loss in hibernating rodents is peripherally rather than centrally regulated. Furthermore, findings suggest even active marmots with normal metabolism are partially protected from disuse induced bone loss compared to laboratory rodents. Understanding the mechanism hibernators use to maintain bone density may guide development for novel bone loss prevention therapies.

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV) is an economically important disease of sugar beet. Management includes the use of single resistance (Rz) genes. However, strains with the ability to overcome the widely used Rz1 resistance gene have already been reported. The mechanisms associated with these genes are unknown, therefore increased understanding of the host defense response may lead to more durable sources of rhizomania resistance. Non-targeted metabolome profiling, coupled with targeted phytohormone quantification, was applied to characterize the metabolic and signaling pathways associated with BNYVV infections in sugar beet. Clear differences in metabolites were identified following pathogen infection independent of plant genotype particularly those associated with host lipid composition or other membrane associated functions.

Cannabidiol (CBD) is a dietary supplement with numerous purported health benefits and an expanding commercial market. Commercially available CBD preparations range from tinctures, oils, and powders, to foods and beverages. Despite widespread use, information regarding bioavailability of these formulations is limited. The purpose of this study was to test the bioavailability of two oral formulations of CBD in humans and explore their potential acute anti-inflammatory activity. We conducted a pilot randomized, parallel arm, double-blind study in 10 healthy adults to determine differences in pharmacokinetics of commercially available water and lipid-soluble CBD powders. Participants consumed a single 30 mg dose, which is within the range of typical commercial supplement doses, and blood samples were collected over 6 hr and analyzed for CBD concentrations. Peripheral blood mononuclear cells (PBMCs) were collected at baseline andT= 90 min, cultured and stimulated with bacterial lipopolysaccharide (LPS) to induce an inflammatory response. Cell supernatants were assayed for IL-10 and TNF, markers of inflammation, using enzyme-linked immunosorbent assays. The water-soluble powder hadC(max)= 2.82 ng/ml,T-max= 90 min, and was approximately x4.5 more bioavailable than the lipid-soluble form. TNF was decreased in LPS-stimulated PBMCs collected 90 min after CBD exposure relative to cells collected at baseline. This study provides pilot data for designing and powering future studies to establish the anti-inflammatory potential and bioavailability of a larger variety of commercial CBD products consumed by humans.

Self-reporting methods for dietary exposure are error-prone and have had limited impact to identify food components that mitigate disease risk. The purpose of this study was to use non-targeted and targeted metabolomics from feeding trials with rice bran and navy beans for the identification of dietary biomarkers across the lifespan. Prepared meals/snacks, and biological samples from randomized-controlled trials performed in 50 infants, 38 children and 49 adults were utilized in this study. Diet groups were placebo control, rice bran, cooked navy bean powder, or a combination of rice bran/navy beans with increasing daily doses by age group and for duration of 4, 12 or 24 weeks per protocol. Plasma/dried blood spots, urine or stool samples were collected at a baseline, midpoint and endpoint. Non-targeted profiling was performed with UPLC-MS/MS, and metabolite quantification by LC-triple-quadropole (LC-QQQ-MS). A linear mixed model to compare between time points in each group was performed using SAS. The plasma/blood metabolomes contained between 771–1001 metabolites and showed variation in ∼20–30% of the profile following intervention. Fold changes over time and fold-differences in metabolite abundance were assessed by age (P < 0.05). There were 10–20 candidate identified from metabolomics across studies and with relevance to rice bran and/or navy bean were applied for targeted assay development. Food metabolomes confirmed metabolite origins and the host and microbial metabolism. Candidate metabolites included pipecolate, S-methlycysteine, S-methylcysteine sulfoxide, trigonelline, N-methyl-pipecolate, pyridoxal, 2-hydroxyhippurate, apigenin, xanthurenate, chiro-inositol, and salicylate. Inter-individual variation was reported across studies, ages and dietary patterns. Dietary biomarkers for rice bran and/or navy bean intake merit additional selection criteria from non-targeted metabolomics. Targeted assays will need validation in larger cohort investigations using cross-over study designs and diverse dietary patterns. This work was supported by a grant from the National Institute of Foods and Agriculture-U.S Department of Agriculture (NIFA-USDA).

Common wheat (Triticum aestivum L.) is a global staple crop, and insect pests can impact grain yield. The wheat stem sawfly (Cephus cinctus, WSS) is a major wheat pest, and while partial resistance has been deployed by breeding for a solid-stem trait, this trait is affected by environment. Here, a proteomics and metabolomics study was performed on four wheat cultivars to characterize a molecular response to WSS infestation. The cultivars Hatcher (hollow-stem partially tolerant), Conan (semisolid-stem-resistant), and Denali and Reeder (hollow-stem-susceptible) were infested with WSS, and changes in stem proteins and metabolites were characterized using liquid chromatography-mass spectrometry. The proteome was characterized as 1830 proteins that included five major biological processes, including metabolic processes and response to stimuli, and the metabolome (1823 metabolites) spanned eight chemical super-classes, including alkaloids, benzenoids, and lipids. All four varieties had a molecular response to WSS following infestation. Hatcher had the most distinct changes, whereby 62 proteins and 29 metabolites varied in metabolic pathways involving enzymatic detoxification, proteinase inhibition, and antiherbivory compound production via benzoxazinoids, neolignans, and phenolics. Taken together, these data demonstrate variation in the wheat stem molecular response to WSS infestation and support breeding for molecular resistance in hollow-stem cultivars.

Permethrin-treated clothing is available as consumer products to prevent bites by tick and insect pests. We used bioassays to examine the impact of wearing and washing/drying of permethrin-treated shirts, pants, and socks, and wearing of treated shoes, on their contact irritancy and toxicity for nymphal Ixodes scapularis Say (Acari: Ixodidae) ticks, the primary vectors in the eastern United States of the causative agents of Lyme disease, human anaplasmosis, and human babesiosis. Pristine permethrin-treated clothing displayed strong contact irritancy and toxicity toward I. scapularis nymphs, with 0-30% of ticks across clothing types and tick sources displaying normal movement 1 h after forced contact for 30-120 s with treated textile. Following 16 d of wear and 16 rounds of machine washing and drying, we recorded reduced concentrations (by 50-90%) of permethrin, compared with pristine treated clothing, from shirts, pants, and socks. This loss of permethrin was associated with reduced contact irritancy and toxicity for ticks after forced contact with worn and washed/dried treated clothing: 31-67% of ticks displayed normal movement 1 h after contact. Nevertheless, the worn and washed/dried treated clothing was still superior to nontreated textile, for which 90-100% of ticks displayed normal movement. Treated shoes, which were worn but not washed, remained as toxic to the ticks as pristine treated shoes. We caution that these laboratory bioassay results should not be interpreted as being directly indicative of the outcome of using washed/worn permethrin-treated clothing in daily life. Although wear and washing/drying did reduce the irritancy and toxicity of permethrin-treated clothing for I. scapularis nymphs more than we had expected, the remaining effect might still reduce the risk of tick bites in a real-life scenario.

Plant development, growth, and adaptation to stress are regulated by phytohormones, which can influence physiology even at low concentrations. Phytohormones are chemically grouped according to both structure and function as auxins, cytokinins, abscisic acid, jasmonates, salicylates, gibberellins, and brassinosteroids, among others. This chemical diversity and requirement for highly sensitive detection in complex matrices create unique challenges for comprehensive phytohormone analysis. Here, we present a robust and efficient quantitative UPLC-MS/MS assay for 17 phytohormones, including jasmonates, salicylates, abscisic acid, gibberellins, cytokinins, and auxins. Using this assay, 12 phytohormones were detected and quantified in sorghum plant tissue without the need for solid phase extraction (SPE) or liquid-liquid extraction. Variation of phytohormone profiles was explored in both root and leaf tissues between three genotypes, harvested at two different developmental time points. The results highlight the importance of tissue type, sampling time, and genetic factors when designing experiments that involve phytohormone analysis of sorghum. This research lays the groundwork for future studies, which can combine phytohormone profiling with other datasets such as transcriptome, soil microbiome, genome, and metabolome data, to provide important functional information about adaptation to stress and other environmental variables.

Estrogen decline during menopause is associated with altered metabolism, weight gain and increased risk of cardiometabolic diseases. The gut microbiota also plays a role in the development of cardiometabolic dysfunction and is also subject to changes associated with age-related hormone changes. Phytoestrogens are plant-based estrogen mimics that have gained popularity as dietary supplements for the treatment or prevention of menopause-related symptoms. These compounds have the potential to both modulate and be metabolized by the gut microbiota. Hops (Humulus lupulus L.) contain potent phytoestrogen precursors, which rely on microbial biotransformation in the gut to estrogenic forms. We supplemented ovariectomized (OVX) or sham-operated (SHAM) C57BL/6 mice, with oral estradiol (E2), a flavonoid-rich extract from hops, or a placebo carrier oil, to observe effects on adiposity, inflammation, and gut bacteria composition. Hops extract (HE) and E2 protected against increased visceral adiposity and liver triglyceride accumulation in OVX animals. Surprisingly, we found no evidence of OVX having a significant impact on the overall gut bacterial community structure. We did find differences in the abundance of Akkermansia muciniphila, which was lower with HE treatment in the SHAM group relative to OVX E2 treatment and to placebo in the SHAM group.

Metabolic responses to hypoxia play important roles in cell survival strategies and disease pathogenesis in humans. However, the homeostatic adjustments that balance changes in energy supply and demand to maintain organismal function under chronic low oxygen conditions remain incompletely understood, making it difficult to distinguish adaptive from maladaptive responses in hypoxia-related pathologies. We integrated metabolomic and proteomic profiling with mitochondrial respirometry and blood gas analyses to comprehensively define the physiological responses of skeletal muscle energy metabolism to 16 days of high-altitude hypoxia (5260 m) in healthy volunteers from the AltitudeOmics project. In contrast to the view that hypoxia down-regulates aerobic metabolism, results show that mitochondria play a central role in muscle hypoxia adaptation by supporting higher resting phosphorylation potential and enhancing the efficiency of long-chain acylcarnitine oxidation. This directs increases in muscle glucose toward pentose phosphate and one-carbon metabolism pathways that support cytosolic redox balance and help mitigate the effects of increased protein and purine nucleotide catabolism in hypoxia. Muscle accumulation of free amino acids favor these adjustments by coordinating cytosolic and mitochondrial pathways to rid the cell of excess nitrogen, but might ultimately limit muscle oxidative capacity in vivo. Collectively, these studies illustrate how an integration of aerobic and anaerobic metabolism is required for physiological hypoxia adaptation in skeletal muscle, and highlight protein catabolism and allosteric regulation as unexpected orchestrators of metabolic remodeling in this context. These findings have important implications for the management of hypoxia-related diseases and other conditions associated with chronic catabolic stress.

Marine bony fish live in a hyperosmotic environment and maintain osmotic homeostasis by drinking seawater, and absorbing salt and water across their gastrointestinal tract. Although the ion and water transport mechanisms in the intestine have been the subject of much study, numerous questions remain unanswered. To address some of these questions, a shotgun proteomics methodology employing isobaric tandem mass tags (TMT) was used to interrogate the anterior intestine, posterior intestine, and intestinal fluid proteomes of Gulf toadfish (Opsanus beta) acclimated to normal (35 ppt) or hypersaline (60 ppt) seawater. Relative protein abundance between tissues was also investigated using label free quantitation. Protein products from nearly 3000 unique toadfish loci were identified and quantified between the tissues, and pathway analysis was performed to gain insight into biological significance. Numerous proteins potentially involved in ion transport, digestion, nutrient absorption, and intestinal CaCO3 precipitation were found to respond to changing salinity, providing additional insight into the molecular mechanisms behind these processes. Intestinal protein heterogeneity was also observed with proteins involved in ion transport responding to hypersalinity exposure primarily in the anterior intestine, and proteins involved in digestion and nutrient absorption showing higher abundance in the anterior intestine, regardless of salinity.

Hibernation is a naturally occurring model for studying diseases such as obesity and osteoporosis. Hibernators, marmots (Marmota flaviventris) among them, are able to nearly double their body mass by increasing fat stores prior to hibernation without the negative consequences of obesity. They are also physically inactive for extended periods of time without experiencing negative effects on the skeleton. The endocannabinoid system is involved in modulating neural signaling, circannual rhythms, behavior, appetite, thermogenesis, and bone and energy metabolism. These systems are also altered to maintain homeostasis during hibernation. This study aims to better understand the involvement of the endocannabinoid system in the regulation of physiological processes during hibernation by quantifying the seasonal variation of endocannabinoids and endocannabinoid-like ligands in both active and hibernating marmots. We hypothesized that there would be significant changes in endocannabinoid concentrations at the tissue level in marmots between active and hibernating states. Concentrations were measured in brain, serum, brown adipose tissue, white adipose tissue, bone marrow, cortical bone, and trabecular bone using microflow chromatography coupled with tandem quadrupole mass spectrometry. Significant changes were found, such as a 30-fold decrease in 2-arachidonoyl glycerol (2-AG) in cortical bone during hibernation. Many endocannabinoid and endocannabinoid-like ligands decreased in brown adipose tissue, white adipose tissue, and cortical bone, while several ligands increased in bone marrow. This result supports our hypothesis and suggests the possibility of a peripherally controlled shift in energy metabolism, reduction in bone metabolism, and suppression of the immune system during hibernation.

The economical production of chemicals and fuels by microbial processes remains an intense area of interest in biotechnology. A key limitation in such efforts concerns the availability of key co-factors, in this case NADPH, required for target pathways. Many of the strategies pursued for increasing NADPH availability in Escherichia coli involve manipulations to the central metabolism, which can create redox imbalances and overall growth defects. In this study we used a reactive oxygen species based selection to search for novel methods of increasing NADPH availability. We report a loss of function mutation in the gene hdfR appears to increase NADPH availability in E. coli. Additionally, we show this excess NADPH can be used to improve the production of 3HP in E. coli.

ABSTRACT Bacteria in a biofilm community have increased tolerance to antimicrobial therapy. To characterize the role of biofilms in equine endometritis, six mares were inoculated with lux -engineered Pseudomonas aeruginosa strains isolated from equine uterine infections. Following establishment of infection, the horses were euthanized and the endometrial surfaces were imaged for luminescence to localize adherent lux -labeled bacteria. Samples from the endometrium were collected for cytology, histopathology, carbohydrate analysis, and expression of inflammatory cytokine genes. Tissue-adherent bacteria were present in focal areas between endometrial folds (6/6 mares). The Pel exopolysaccharide (biofilm matrix component) and cyclic di-GMP (biofilm-regulatory molecule) were detected in 6/6 mares and 5/6 mares, respectively, from endometrial samples with tissue-adherent bacteria ( P < 0.05). A greater incidence ( P < 0.05) of Pel exopolysaccharide was present in samples fixed with Bouin's solution (18/18) than in buffered formalin (0/18), indicating that Bouin's solution is more appropriate for detecting bacteria adherent to the endometrium. There were no differences ( P > 0.05) in the number of inflammatory cells in the endometrium between areas with and without tissue-adherent bacteria. Neutrophils were decreased ( P < 0.05) in areas surrounding tissue-adherent bacteria compared to those in areas free of adherent bacteria. Gene expression of interleukin-10, an immune-modulatory cytokine, was significantly ( P < 0.05) increased in areas of tissue-adherent bacteria compared to that in endometrium absent of biofilm. These findings indicate that P. aeruginosa produces a biofilm in the uterus and that the host immune response is modulated focally around areas with biofilm, but inflammation within the tissue is similar in areas with and without biofilm matrix. Future studies will focus on therapeutic options for elimination of bacterial biofilm in the equine uterus.

Infection with Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis, remains a global health concern. Both classically and non-classically restricted cytotoxic CD8+ T cells are important to the control of Mtb infection. We and others have demonstrated that the non-classical MHC I molecule HLA-E can present pathogen-derived peptides to CD8+ T cells. In this manuscript, we identified the antigen recognized by an HLA-E-restricted CD8+ T cell clone isolated from an Mtb latently infected individual as a peptide from the Mtb protein, MPT32. Recognition by the CD8+ T cell clone required N-terminal O-linked mannosylation of MPT32 by a mannosyltransferase encoded by the Rv1002c gene. This is the first description of a post-translationally modified Mtb-derived protein antigen presented in the context of an HLA-E specific CD8+ T cell immune response. The identification of an immune response that targets a unique mycobacterial modification is novel and may have practical impact in the development of vaccines and diagnostics.

In the present investigation the structural proteins associated with MAC-1 bacteriophage have been characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); tandem mass spectrometry of protein bands from SDS-PAGE gel; from the open reading frames (ORFs) deduced from MAC-1 genome sequence and amino acid sequence homology searches from the Uniprot database (up000002418). Results have led to the identification of at least three structural proteins associated with MAC-1 phage genome. They are: capsid protein (~55,000-daltons); spike protein (~22,000-daltons) and a low molecular weight DNA binding protein (~4000-dal- tons). In addition, two other minor proteins were tentatively identified as replicative and scaffold proteins based on two to three unique peptides from mass spectrometry data. However, other proteins coded (ORFs) by phage genome remain to be identified.

PURPOSE:Excessive sweating is a common symptom of the disease and an unexplored biofluid for TB diagnosis; we conducted a proof-of-concept study to identify potential diagnostic biomarkers of active TB in eccrine sweat.EXPERIMENTAL DESIGN:We performed a global proteomic profile of eccrine sweat sampled from patients with active pulmonary TB, other lung diseases (non-TB disease), and healthy controls. A comparison of proteomics between Active-TB, Non-TB, and Healthy Controls was done in search for potential biomarkers of active TB.RESULTS:Sweat specimens were pooled from 32 active TB patients, 27 patients with non-TB diseases, and 24 apparently healthy controls, all were negative for HIV. Over 100 unique proteins were identified in the eccrine sweat of all three groups. Twenty-six proteins were exclusively detected in the sweat of patients with active TB while the remaining detected proteins overlapped between three groups. Gene ontology evaluation indicated that the proteins detected uniquely in sweat of active TB patients were involved in immune response and auxiliary protein transport. Gene products for cellular components (e.g. ribosomes) were detected only in active TB patients. Data are available via ProteomeXchange with identifier PXD003224.CONCLUSIONS AND CLINICAL RELEVANCE:Proteomics of sweat from active TB patients is a viable approach for biomarker identification, which could be used to develop a nonsputum-based test for detection of active TB.

Tuberculosis (TB) is the deadliest infectious disease worldwide. One obstacle hindering the elimination of TB is our lack of understanding of host-pathogen interactions. Exosomes, naturally loaded with microbial molecules, are circulating markers of TB. Changes in the host protein composition of exosomes from Mycobacterium tuberculosis (Mtb)-infected cells have not been described, can contribute to our understanding of the disease process, and serve as a direct source of biomarkers or as capture targets to enrich for exosomes containing microbial molecules. Here, the protein composition of exosomes from Mtb-infected and uninfected THP-1-derived macrophages was evaluated by tandem-mass-spectrometry and differences in protein abundances were assessed. Our results show that infection with Mtb leads to significant changes in the protein composition of exosomes. Specifically, 41 proteins were significantly more abundant in exosomes from Mtb-infected cells; 63% of these were predicted to be membrane associated. Thus, we used a novel biotinylation strategy to verify protein localization, and confirmed the localization of some of these proteins in the exosomal membrane. Our findings reveal another important scenario where Mtb could be influencing changes in host cells that unveil new features of the host-pathogen interaction and may also be exploited as a source of biomarkers for TB.