Assessment for Antibodies to Rifapentine and Isoniazid in Persons Developing Flu-like Reactions During Treatment of Latent Tuberculosis Infection.

BACKGROUND: Flu-like reactions can occur after exposure to rifampin, rifapentine, or isoniazid. Prior studies have reported the presence of antibodies to rifampin, but associations with underlying pathogenesis are unclear. METHODS: We evaluated PREVENT TB study participants who received weekly isoniazid + rifapentine for 3 months (3HP) or daily isoniazid for 9 months (9H) as treatment for M. tuberculosis infection. Flu-like reaction was defined as a grade ≥2 of any of flu-like symptoms. Controls (3HP or 9H) did not report flu-like reactions. We developed a competitive enzyme-linked immunosorbent assays (ELISA) to detect antibodies against rifapentine, isoniazid, rifampin, and rifapentine metabolite. RESULTS: Among 128 participants, 69 received 3HP (22 with flu-like reactions; 47 controls) and 59 received 9H (12 with flu-like reactions; 47 controls). In participants receiving 3HP, anti-rifapentine IgG was identified in 2/22 (9%) participants with flu-like reactions and 6/47 (13%) controls (P = 0.7), anti-isoniazid IgG in 2/22 (9%) participants with flu-like reactions and 4/47 (9%) controls (P = 0.9), and anti-rifapentine metabolite IgG in 2/47 (4%) controls (P = 0.9). Among participants receiving 9H, IgG and IgM anti-isoniazid antibodies were each present in 4/47 (9%) controls, respectively, but none among participants with flu-like reactions; anti-rifapentine IgG antibodies were not present in any participants with flu-like reactions or controls. CONCLUSIONS: We detected anti-rifapentine, anti-isoniazid, and anti-rifapentine metabolite antibodies, but the proportions of participants with antibodies were low, and did not differ between participants with flu-like reactions and those without such reactions. This suggests that flu-like reactions associated with 3HP and 9H were not antibody-mediated.

Towards a method for cryopreservation of mosquito vectors of human pathogens.

Mosquito-borne diseases are responsible for millions of human deaths every year, posing a massive burden on global public health. Mosquitoes transmit a variety of bacteria, parasites and viruses. Mosquito control efforts such as insecticide spraying can reduce mosquito populations, but they must be sustained in order to have long term impacts, can result in the evolution of insecticide resistance, are costly, and can have adverse human and environmental effects. Technological advances have allowed genetic manipulation of mosquitoes, including generation of those that are still susceptible to insecticides, which has greatly increased the number of mosquito strains and lines available to the scientific research community. This generates an associated challenge, because rearing and maintaining unique mosquito lines requires time, money and facilities, and long-term maintenance can lead to adaptation to specific laboratory conditions, resulting in mosquito lines that are distinct from their wild-type counterparts. Additionally, continuous rearing of transgenic lines can lead to loss of genetic markers, genes and/or phenotypes. Cryopreservation of valuable mosquito lines could help circumvent these limitations and allow researchers to reduce the cost of rearing multiple lines simultaneously, maintain low passage number transgenic mosquitoes, and bank lines not currently being used. Additionally, mosquito cryopreservation could allow researchers to access the same mosquito lines, limiting the impact of unique laboratory or field conditions. Successful cryopreservation of mosquitoes would expand the field of mosquito research and could ultimately lead to advances that would reduce the burden of mosquito-borne diseases, possibly through rear-and-release strategies to overcome mosquito insecticide resistance. Cryopreservation techniques have been developed for some insect groups, including but not limited to fruit flies, silkworms and other moth species, and honeybees. Recent advances within the cryopreservation field, along with success with other insects suggest that cryopreservation of mosquitoes may be a feasible method for preserving valuable scientific and public health resources. In this review, we will provide an overview of basic mosquito biology, the current state of and advances within insect cryopreservation, and a proposed approach toward cryopreservation of Anopheles stephensi mosquitoes.

Nontuberculous Mycobacteria Show Differential Infectivity and Use Phospholipids to Antagonize LL-37.

Comparisons of infectivity among the clinically important nontuberculous mycobacteria (NTM) species have not been explored in great depth. Rapid-growing mycobacteria, including Mycobacterium abscessus and M. porcinum, can cause indolent but progressive lung disease. Slow-growing members of the M. avium complex are the most common group of NTM to cause lung disease, and molecular approaches can now distinguish between several distinct species of M. avium complex including M. intracellulare, M. avium, M. marseillense, and M. chimaera. Differential infectivity among these NTM species may, in part, account for differences in clinical outcomes and response to treatment; thus, knowing the relative infectivity of particular isolates could increase prognostication accuracy and enhance personalized treatment. Using human macrophages, we investigated the infectivity and virulence of nine NTM species, as well as multiple isolates of the same species. We also assessed their capacity to evade killing by the antibacterial peptide cathelicidin (LL-37). We discovered that the ability of different NTM species to infect macrophages varied among the species and among isolates of the same species. Our biochemical assays implicate modified phospholipids, which may include a phosphatidylinositol or cardiolipin backbone, as candidate antagonists of LL-37 antibacterial activity. The high variation in infectivity and virulence of NTM strains suggests that more detailed microbiological and biochemical characterizations are necessary to increase our knowledge of NTM pathogenesis.

Identification of Mycobacterium tuberculosis Peptides in Serum Extracellular Vesicles from Persons with Latent Tuberculosis Infection.

Identification of biomarkers for latent Mycobacterium tuberculosis infection and risk of progression to tuberculosis (TB) disease are needed to better identify individuals to target for preventive therapy, predict disease risk, and potentially predict preventive therapy efficacy. Our group developed multiple reaction monitoring mass spectrometry (MRM-MS) assays that detected M. tuberculosis peptides in serum extracellular vesicles from TB patients. We subsequently optimized this MRM-MS assay to selectively identify 40 M. tuberculosis peptides from 19 proteins that most commonly copurify with serum vesicles of patients with TB. Here, we used this technology to evaluate if M. tuberculosis peptides can also be detected in individuals with latent TB infection (LTBI). Serum extracellular vesicles from 74 individuals presumed to have latent M. tuberculosis infection (LTBI) based on close contact with a household member with TB or a recent tuberculin skin test (TST) conversion were included in this study. Twenty-nine samples from individuals with no evidence of TB infection by TST and no known exposure to TB were used as controls to establish a threshold to account for nonspecific/background signal. We identified at least one of the 40 M. tuberculosis peptides in 70 (95%) individuals with LTBI. A single peptide from the glutamine synthetase (GlnA1) enzyme was identified in 61/74 (82%) individuals with LTBI, suggesting peptides from M. tuberculosis proteins involved in nitrogen metabolism might be candidates for pathogen-specific biomarkers for detection of LTBI. The detection of M. tuberculosis peptides in serum extracellular vesicles from persons with LTBI represents a potential advance in the diagnosis of LTBI.

Structural determinants in a glucose-containing lipopolysaccharide from Mycobacterium tuberculosis critical for inducing a subset of protective T cells.

Mycobacteria synthesize intracellular, 6-O-methylglucose-containing lipopolysaccharides (mGLPs) proposed to modulate bacterial fatty acid metabolism. Recently, it has been shown that Mycobacterium tuberculosis mGLP specifically induces a specific subset of protective γ9δ2 T cells. Mild base treatment, which removes all the base-labile groups, reduces the specific activity of mGLP required for induction of these T cells, suggesting that acylation of the saccharide moieties is required for γ9δ2 T-cell activation. On the basis of this premise, we used analytical LC/MS and NMR methods to identify and locate the acyl functions on the mGLP saccharides. We found that mGLP is heterogeneous with respect to acyl functions and contains acetyl, isobutyryl, succinyl, and octanoyl groups and that all acylations in mGLP, except for succinyl and octanoyl residues, reside on the glucosyl residues immediately following the terminal 3-O-methylglucose. Our analyses also indicated that the octanoyl residue resides at position 2 of an internal glucose toward the reducing end. LC/MS analysis of the residual product obtained by digesting the mGLP with pancreatic α-amylase revealed that the product is an oligosaccharide terminated by α-(1→4)-linked 6-O-methyl-d-glucosyl residues. This oligosaccharide retained none of the acyl groups, except for the octanoyl group, and was unable to induce protective γ9δ2 T cells. This observation confirmed that mGLP induces γ9δ2 T cells and indicated that the acylated glucosyl residues at the nonreducing terminus of mGLP are required for this activity.

Cytokine-Mediated Systemic Adverse Drug Reactions in a Drug-Drug Interaction Study of Dolutegravir With Once-Weekly Isoniazid and Rifapentine.

Background: Once-weekly isoniazid and rifapentine for 3 months is a treatment option in persons with human immunodeficiency virus and latent tuberculosis infection. This study aimed to examine pharmacokinetic drug-drug interactions between this regimen and dolutegravir, a first-line antiretroviral medication. Methods: This was a single-center, open-label, fixed-sequence, drug-drug interaction study in healthy volunteers. Subjects received oral dolutegravir 50 mg once daily alone (days 1-4) and concomitantly with once-weekly isoniazid 900 mg, rifapentine 900 mg, and pyridoxine 50 mg (days 5-19). Dolutegravir concentrations were measured on days 4, 14, and 19, and rifapentine, 25-desacetyl-rifapentine, and isoniazid concentrations were measured on day 19. Cytokines and antidrug antibodies to isoniazid and rifapentine were examined at select time points. Results: The study was terminated following the development of flu-like syndrome and elevated aminotransferase levels in 2 of 4 subjects after the third isoniazid-rifapentine dose. Markedly elevated levels of interferon-γ, CXCL10, C-reactive protein, and other cytokines were temporally associated with symptoms. Antidrug antibodies were infrequently detected. Dolutegravir area under the curve (AUC) was decreased by 46% (90% confidence interval, 27-110%; P = .13) on day 14. Rifapentine and 25-desacetyl rifapentine levels on day 19 were comparable to reference data, whereas isoniazid AUCs were approximately 67%-92% higher in the subjects who developed toxicities. Conclusions: The combined use of dolutegravir with once-weekly isoniazid-rifapentine resulted in unexpected and serious toxicities that were mediated by endogenous cytokine release. Additional investigations are necessary to examine the safety and efficacy of coadministering these medications. Clinical Trials Registration: NCT02771249.

Second generation multiple reaction monitoring assays for enhanced detection of ultra-low abundance Mycobacterium tuberculosis peptides in human serum.

BACKGROUND: Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease. TB diagnosis is performed by microscopy, culture or PCR amplification of bacterial DNA, all of which require patient sputum or the biopsy of infected tissue. Detection of mycobacterial products in serum, as biomarkers of diagnosis or disease status would provide an improvement over current methods. Due to the low-abundance of mycobacterial products in serum, we have explored exosome enrichment to improve sensitivity. Mtb resides intracellularly where its secreted proteins have been shown to be packaged into host exosomes and released into the bloodstream. Exosomes can be readily purified assuring an enrichment of mycobacterial analytes from the complex mix of host serum proteins. METHODS: Multiple reaction monitoring assays were optimized for the enhanced detection of 41 Mtb peptides in exosomes purified from the serum of individuals with TB. Exosomes isolated from the serum of healthy individuals was used to create and validate a unique data analysis algorithm and identify filters to reduce the rate of false positives, attributed to host m/z interference. The final optimized method was tested in 40 exosome samples from TB positive patients. RESULTS: Our enhanced methods provide limit of detection and quantification averaging in the low femtomolar range for detection of mycobacterial products in serum. At least one mycobacterial peptide was identified in 92.5% of the TB positive patients. Four peptides from the Mtb proteins, Cfp2, Mpt32, Mpt64 and BfrB, show normalized total peak areas significantly higher in individuals with active TB as compared to healthy controls; three of the peptides from these proteins have not previously been associated with serum exosomes from individuals with active TB disease. Some of the detected peptides were significantly associated with specific geographical locations, highlighting potential markers that can be linked to the Mtb strains circulating within each given region. CONCLUSIONS: An enhanced MRM method to detect ultra-low abundance Mtb peptides in human serum exosomes is demonstrated, highlighting the potential of this methodology for TB diagnostic biomarker development.

RP105 Engages Phosphatidylinositol 3-Kinase p110δ To Facilitate the Trafficking and Secretion of Cytokines in Macrophages during Mycobacterial Infection.

Cytokines are key regulators of adequate immune responses to infection with Mycobacterium tuberculosis. We demonstrate that the p110δ catalytic subunit of PI3K acts as a downstream effector of the TLR family member RP105 (CD180) in promoting mycobacteria-induced cytokine production by macrophages. Our data show that the significantly reduced release of TNF and IL-6 by RP105(-/-) macrophages during mycobacterial infection was not accompanied by diminished mRNA or protein expression. Mycobacteria induced comparable activation of NF-κB and p38 MAPK signaling in wild-type (WT) and RP105(-/-) macrophages. In contrast, mycobacteria-induced phosphorylation of Akt was abrogated in RP105(-/-) macrophages. The p110δ-specific inhibitor, Cal-101, and small interfering RNA-mediated knockdown of p110δ diminished mycobacteria-induced TNF secretion by WT but not RP105(-/-) macrophages. Such interference with p110δ activity led to reduced surface-expressed TNF in WT but not RP105(-/-) macrophages, while leaving TNF mRNA and protein expression unaffected. Activity of Bruton's tyrosine kinase was required for RP105-mediated activation of Akt phosphorylation and TNF release by mycobacteria-infected macrophages. These data unveil a novel innate immune signaling axis that orchestrates key cytokine responses of macrophages and provide molecular insight into the functions of RP105 as an innate immune receptor for mycobacteria.

Comparing isogenic strains of Beijing genotype Mycobacterium tuberculosis after acquisition of Isoniazid resistance: A proteomics approach.

We determined differences in the protein abundance among two isogenic strains of Mycobacterium tuberculosis (Mtb) with different Isoniazid (INH) susceptibility profiles. The strains were isolated from a pulmonary tuberculosis patient before and after drug treatment. LC-MS/MS analysis identified 46 Mtb proteins with altered abundance after INH resistance acquisition. Protein abundance comparisons were done evaluating the different bacterial cellular fractions (membrane, cytosol, cell wall and secreted proteins). MS data have been deposited to the ProteomeXchange with identifier PXD002986.

A Subset of Protective γ9δ2 T Cells Is Activated by Novel Mycobacterial Glycolipid Components.

γ9δ2 T cells provide a natural bridge between innate and adaptive immunity, rapidly and potently respond to pathogen infection in mucosal tissues, and are prominently induced by both tuberculosis (TB) infection and bacillus Calmette Guérin (BCG) vaccination. Mycobacterium-expanded γ9δ2 T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2 T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosis infection. Based on this premise, we have been searching for M. tuberculosis antigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2 T cells. Our screening strategy includes the identification of M. tuberculosis fractions that expand γ9δ2 T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosis whole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosis antigens inducing protective γ9δ2 T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosis whole-cell lysates, suggesting that a polysaccharide was required for biological activity. Extraction of MtbWL with chloroform-methanol-water (10:10:3) resulted in a polar lipid fraction with highly enriched specific activity; this activity was further enriched by silica gel chromatography. A combination of mass spectrometry and nuclear magnetic resonance analysis of bioactive fractions indicated that 6-O-methylglucose-containing lipopolysaccharides (mGLP) are predominant components present in this active fraction. These results have important implications for the development of new immunotherapeutic approaches for prevention and treatment of TB.

O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

A chemical proteomics approach to profiling the ATP-binding proteome of Mycobacterium tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the leading causes of death worldwide despite extensive research, directly observed therapy using multidrug regimens, and the widespread use of a vaccine. The majority of patients harbor the bacterium in a state of metabolic dormancy. New drugs with novel modes of action are needed to target essential metabolic pathways in M. tuberculosis; ATP-competitive enzyme inhibitors are one such class. Previous screening efforts for ATP-competitive enzyme inhibitors identified several classes of lead compounds that demonstrated potent anti-mycobacterial efficacy as well as tolerable levels of toxicity in cell culture. In this report, a probe-based chemoproteomic approach was used to selectively profile the M. tuberculosis ATP-binding proteome in normally growing and hypoxic M. tuberculosis. From these studies, 122 ATP-binding proteins were identified in either metabolic state, and roughly 60% of these are reported to be essential for survival in vitro. These data are available through ProteomeXchange with identifier PXD000141. Protein families vital to the survival of the tubercle bacillus during hypoxia emerged from our studies. Specifically, along with members of the DosR regulon, several proteins involved in energy metabolism (Icl/Rv0468 and Mdh/Rv1240) and lipid biosynthesis (UmaA/Rv0469, DesA1/Rv0824c, and DesA2/Rv1094) were found to be differentially abundant in hypoxic versus normal growing cultures. These pathways represent a subset of proteins that may be relevant therapeutic targets for development of novel ATP-competitive antibiotics.

Autophagy protects against active tuberculosis by suppressing bacterial burden and inflammation.

Autophagy is a cell biological pathway affecting immune responses. In vitro, autophagy acts as a cell-autonomous defense against Mycobacterium tuberculosis, but its role in vivo is unknown. Here we show that autophagy plays a dual role against tuberculosis: antibacterial and anti-inflammatory. M. tuberculosis infection of Atg5(fl/fl) LysM-Cre(+) mice relative to autophagy-proficient littermates resulted in increased bacillary burden and excessive pulmonary inflammation characterized by neutrophil infiltration and IL-17 response with increased IL-1α levels. Macrophages from uninfected Atg5(fl/fl) LysM-Cre(+) mice displayed a cell-autonomous IL-1α hypersecretion phenotype, whereas T cells showed propensity toward IL-17 polarization during nonspecific activation or upon restimulation with mycobacterial antigens. Thus, autophagy acts in vivo by suppressing both M. tuberculosis growth and damaging inflammation.

Investigating the lipid profile of Anopheles stephensi mosquitoes across developmental life stages.

Holometabolous insects undergo a distinct transition in their development, tightly correlated with shifting feeding patterns from larval stages and some adult phases to non-feeding phases as pupae and during other adult phases. Furthermore, the intricate life cycle of mosquitoes involves a sequence of developmental stages influenced by aquatic and terrestrial factors, demanding precise energy resource orchestration. Lipids serve multifaceted roles, encompassing energy storage, membrane structure, and participation in signal transduction and molecular recognition processes. A significant gap in the current research landscape is the need for a comprehensive study exploring the lipid repertoire throughout the developmental stages of Anopheles stephensi mosquitoes. We undertook an analysis of the An. stephensi metabolome across all life stages. We hypothesized that An. stephensi mosquitoes will have unique lipid metabolite markers for each life stage. A specific extraction and LC-MS based lipidomic approach was used to test this hypothesis. Our findings demonstrated that our methods were successful, with lipids comprising 62.15 % of the analyzed metabolome. Additionally, phospholipids (PL), lysophospholipids (LPL), sphingomyelin (SM), and triglycerides (TG) were abundant and dynamic across all life stages. Interestingly, comparison between the L1 and L2 lipidome revealed a dominant pattern of specific TGs in decreased abundance between these two life stages. Lastly, 20-hydroxyecdysone (20E), was found to be present in similar abundance across all 4 larval stages. These data indicate that there may be lipid metabolome pathways serving unique roles during mosquito development that may be used to explore laboratory management of colonies, parasite resistance, and environmental adaptation.

Modulation of riboflavin biosynthesis and utilization in mycobacteria.

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism and physiology, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins, and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for future studies investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria. IMPORTANCE: The pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen, Mycobacterium tuberculosis (Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal-associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation, and TB infection and disease.

Correction: Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0274415.].

Modulation of riboflavin biosynthesis and utilization in mycobacteria.

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism, physiology and MAIT cell recognition, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria.

The Many Hosts of Mycobacteria 9 (MHM9): A conference report.

The Many Hosts of Mycobacteria (MHM) meeting series brings together basic scientists, clinicians and veterinarians to promote robust discussion and dissemination of recent advances in our knowledge of numerous mycobacterial diseases, including human and bovine tuberculosis (TB), nontuberculous mycobacteria (NTM) infection, Hansen's disease (leprosy), Buruli ulcer and Johne's disease. The 9th MHM conference (MHM9) was held in July 2022 at The Ohio State University (OSU) and centered around the theme of "Confounders of Mycobacterial Disease." Confounders can and often do drive the transmission of mycobacterial diseases, as well as impact surveillance and treatment outcomes. Various confounders were presented and discussed at MHM9 including those that originate from the host (comorbidities and coinfections) as well as those arising from the environment (e.g., zoonotic exposures), economic inequality (e.g. healthcare disparities), stigma (a confounder of leprosy and TB for millennia), and historical neglect (a confounder in Native American Nations). This conference report summarizes select talks given at MHM9 highlighting recent research advances, as well as talks regarding the historic and ongoing impact of TB and other infectious diseases on Native American Nations, including those in Southwestern Alaska where the regional TB incidence rate is among the highest in the Western hemisphere.

Deciphering the proteome of the in vivo diagnostic reagent "purified protein derivative" from Mycobacterium tuberculosis.

Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.