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The cell envelope of Mycobacterium tuberculosis(Mtb) is complex and diverse; composed of proteins intermingled in a matrix of peptidoglycan, mycolic acids, lipids, and carbohydrates. Proteomic studies of the Mtbcell wall have been limited; nonetheless, the characterization of resident and secreted proteins associated with the cell wall are critical to understanding bacterial survival and immune modulation in the host. In this study, the cell wall proteome was defined in order to better understand its unique biosynthetic and secretion processes. Mtbcell wall was subjected to extraction with organic solvents to remove noncovalently bound lipids and lipoglycans and remaining proteins were solubilized with either SDS, Guanidine-HCl, or TX-114. These extracts were analyzed by two-dimensional gel electrophoresis and mass-spectrometry and resulted in the identification of 234 total proteins. The lipoproteome of Mtb, enriched in the TX-114 extract, was further resolved by multidimensional chromatography and mass spectrometry to identify an additional 294 proteins. A query of the 528 total protein identifications against Neural Network or Hidden Markov model algorithms predicted secretion signals in 87 proteins. Classification of these 528 proteins also demonstrated that 35% are involved in small molecule metabolism and 25% are involved in macromolecule synthesis and degradation building upon evidence that the Mtbcell wall is actively engaged in mycobacterial survival and remodeling.

Introduction:There are vast differences in the size, scope, and needs of institutions that conduct research involving biohazardous materials, thus resulting in vast differences among Institutional Biosafety Committees (IBCs) and biosafety programs.Methods:A benchmarking survey of IBC and biosafety programs was conducted in an effort to identify common practices in the field and compare this information with that of the other institutional bioethics committees, namely, Institutional Animal Care and Use Committees (IACUCs) and Institutional Review Boards (IRBs).Objectives:The primary objectives of the survey were to assess the organizational structure of IBC and biosafety programs, determine the scope of IBC review, and compare the size of IBC and biosafety programs with that of IACUCs and IRBs.Results:The survey results showed that IBCs most commonly reside under the same administrative unit as the IACUC and IRB, while the majority of institutions’ biosafety officers report to a different unit. The majority of respondents indicated their IBC reviews research utilizing biological hazards beyond what is required by the National Institutes of Health Guidelines. The survey data suggest that IBCs have fewer support staff than the other bioethics committees; 57% of institutions report one or more full-time employee (FTE) dedicated to support the IBC, compared to 86%, 85%, and 83% of institutions that reported one or more FTE to support the IACUC, the IRB, and the biosafety program, respectively.Conclusion:Data from the survey identified common practices among IBCs and provides institutions a tool to compare their program with others.

Introduction: There are vast differences in the size, scope, and needs of institutions that conduct research involving biohazardous materials, thus resulting in vast differences among Institutional Biosafety Committees (IBCs) and biosafety programs.Methods: A benchmarking survey of IBC and biosafety programs was conducted in an effort to identify common practices in the field and compare this information with that of the other institutional bioethics committees, namely, Institutional Animal Care and Use Committees (IACUCs) and Institutional Review Boards (IRBs).Objectives: The primary objectives of the survey were to assess the organizational structure of IBC and biosafety programs, determine the scope of IBC review, and compare the size of IBC and biosafety programs with that of IACUCs and IRBs.Results: The survey results showed that IBCs most commonly reside under the same administrative unit as the IACUC and IRB, while the majority of institutions’ biosafety officers report to a different unit. The majority of respondents indicated their IBC reviews research utilizing biological hazards beyond what is required by the National Institutes of Health Guidelines. The survey data suggest that IBCs have fewer support staff than the other bioethics committees; 57% of institutions report one or more full-time employee (FTE) dedicated to support the IBC, compared to 86%, 85%, and 83% of institutions that reported one or more FTE to support the IACUC, the IRB, and the biosafety program, respectively.Conclusion: Data from the survey identified common practices among IBCs and provides institutions a tool to compare their program with others.

Radioprotective 105 kDa (RP105, CD180) is a member of the Toll‐like receptor (TLR) family that interacts with TLR2 and facilitates recognition of mature lipoproteins expressed by Mycobacterium tuberculosisand Mycobacterium bovisBCG. In this study, we used synthetic lipopeptide analogs of the M. tuberculosis19 kDa lipoprotein to define structural characteristics that promote RP105‐mediated host cell responses. A tripalmitoylated lipopeptide composed of the first 16 N‐terminal amino acids of the M. tuberculosis19 kDa lipoprotein induced RP105‐dependent TNF and IL‐6 production by macrophages. Di‐ and tripalmitoylated variants of this lipopeptide elicited an equivalent RP105‐dependent response, indicating that while the lipid moiety is required for macrophage activation, it is not a determinant of RP105 dependency. Instead, substitution of two polar threonine residues at positions 7 and 8 with nonpolar alanine residues resulted in reduced RP105 dependency. These results strongly suggest that the amino acid composition of the M. tuberculosis19 kDa lipoprotein, and likely other mycobacterial lipoproteins, is a key determinant of RP105 agonism.

ABSTRACTγ9δ2T 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 γ9δ2T cells represent only a subset of the phosphoantigen {isopentenyl pyrophosphate [IPP] and (E)-4-hydroxy-3-methyl-but-2-enylpyrophosphate [HMBPP]}-responsive γ9δ2T cells, expressing an oligoclonal set of T cell receptor (TCR) sequences which more efficiently recognize and inhibit intracellular Mycobacterium tuberculosisinfection. Based on this premise, we have been searching for M. tuberculosisantigens specifically capable of inducing a unique subset of mycobacterium-protective γ9δ2T cells. Our screening strategy includes the identification of M. tuberculosisfractions that expand γ9δ2T cells with biological functions capable of inhibiting intracellular mycobacterial replication. Chemical treatments of M. tuberculosiswhole-cell lysates (MtbWL) ruled out protein, nucleic acid, and nonpolar lipids as the M. tuberculosisantigens inducing protective γ9δ2T cells. Mild acid hydrolysis, which transforms complex carbohydrate to monomeric residues, abrogated the specific activity of M. tuberculosiswhole-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.

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. tuberculosisATP-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 versusnormal growing cultures. These pathways represent a subset of proteins that may be relevant therapeutic targets for development of novel ATP-competitive antibiotics.

Purified protein derivative (PPD) is a widely used reagent for the diagnosis of Mycobacterium tuberculosis infection. Recently, the molecular composition of PPD was defined, with hundreds of mycobacterial protein representatives making up PPD. Which, if any, of these specific products drive the potency of PPD remains in question. In this study, two proteins (DnaK and GroEL2) previously identified as dominant proteins in PPD were tested for the capacity to induce delayed-type hypersensitivity (DTH) responses in H37Rv-infected or BCG-vaccinated guinea pigs. These two proteins were used in pull-down assays to identify interacting PPD products. Six proteins were identified as interacting partners with DnaK and GroEL2, i.e., Rv0009, Rv0475, Rv0569, Rv0685, Rv2626c, and Rv2632c. These six proteins were tested alone and in combination with DnaK and GroEL2 for the capacity to induce a DTH response in the guinea pig model. From these studies, two cocktails, DnaK/GroEL2/Rv0009 and DnaK/GroEL2/Rv0685, were found to induce DTH responses in H37Rv-infected or BCG-vaccinated guinea pigs that were indistinguishable from DTH responses driven by a PPD injection. The mechanism by which DTH responses were induced was elucidated by histologic examination, analysis of activated CD4+/CD8+T cells, and cytokine mRNA expression at the site of the DTH response. PPD and the protein cocktails tested induced strong DTH responses in H37Rv-infected guinea pigs. Ex vivo phenotyping of T cells at the DTH site indicated that this response is mediated by activated CD4+and CD8+T cells, with increases in gamma interferon and tumor necrosis factor alpha, but not interleukin-10, at the site of the DTH response. Our results demonstrate for the first time that the PPD response can be mimicked at the molecular level with defined protein cocktails. The use of this defined product will allow a more thorough understanding of the DTH response and may provide a platform for more rapid and sensitive second-generation skin test reagents for the diagnosis of M. tuberculosis infection.

ABSTRACTPurified protein derivative (PPD) is a widely used reagent for the diagnosis of Mycobacterium tuberculosisinfection. Recently, the molecular composition of PPD was defined, with hundreds of mycobacterial protein representatives making up PPD. Which, if any, of these specific products drive the potency of PPD remains in question. In this study, two proteins (DnaK and GroEL2) previously identified as dominant proteins in PPD were tested for the capacity to induce delayed-type hypersensitivity (DTH) responses in H37Rv-infected or BCG-vaccinated guinea pigs. These two proteins were used in pull-down assays to identify interacting PPD products. Six proteins were identified as interacting partners with DnaK and GroEL2, i.e., Rv0009, Rv0475, Rv0569, Rv0685, Rv2626c, and Rv2632c. These six proteins were tested alone and in combination with DnaK and GroEL2 for the capacity to induce a DTH response in the guinea pig model. From these studies, two cocktails, DnaK/GroEL2/Rv0009 and DnaK/GroEL2/Rv0685, were found to induce DTH responses in H37Rv-infected or BCG-vaccinated guinea pigs that were indistinguishable from DTH responses driven by a PPD injection. The mechanism by which DTH responses were induced was elucidated by histologic examination, analysis of activated CD4+/CD8+T cells, and cytokine mRNA expression at the site of the DTH response. PPD and the protein cocktails tested induced strong DTH responses in H37Rv-infected guinea pigs. Ex vivo phenotyping of T cells at the DTH site indicated that this response is mediated by activated CD4+and CD8+T cells, with increases in gamma interferon and tumor necrosis factor alpha, but not interleukin-10, at the site of the DTH response. Our results demonstrate for the first time that the PPD response can be mimicked at the molecular level with defined protein cocktails. The use of this defined product will allow a more thorough understanding of the DTH response and may provide a platform for more rapid and sensitive second-generation skin test reagents for the diagnosis of M. tuberculosisinfection.

ABSTRACTTuberculous meningitis (TBM) is the most common form of chronic infection of the central nervous system. Despite the magnitude of the problem, the general diagnostic outlook is discouraging. Specifically, there is no generally accepted early confirmative diagnosis protocol available for TBM. Various Mycobacterium tuberculosisantigens are now recognized as potential markers for diagnosis of TBM. However, their presence remains questionable, and many of these antigens are reported in the blood but not in the cerebrospinal fluid (CSF). This study identifies a specific protein marker in CSF which will be useful in early diagnosis of TBM. We have demonstrated the presence of a 30-kDa protein band in CSF of 100% (n= 5) of confirmed and 90% (n= 138) of suspected TBM patients out of 153 TBM patients. The 30-kDa band was excised from the gel, destained extensively, and digested with trypsin. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Partially purified proteins from CSF samples of TBM were analyzed by two-dimensional polyacrylamide gel electrophoresis and Western blotting. Immunoblotting and enzyme-linked immunosorbent assay (ELISA) were performed to confirm the presence of proteins in the 30-kDa protein band. The antigen 85 (Ag 85) complex was detected in CSF of TBM patients by indirect ELISA using antibodies against Ag 85 complex. The results of this study showed the 30-kDa protein band contained MTB proteins Rv3804c (Ag85A) and Rv1886c (Ag 85B), both members of the Ag85 complex. This was also confirmed by using immunotechniques such as indirect ELISA and the dot immunobinding assay. Detection of Ag85 complex was observed in CSF of 89% (71 out of 80) of suspected TBM patients that were 30-kDa protein positive. The observed 30-kDa protein in the CSF is comprised of the MTB Ag85 complex. This protein was earlier reported to be present in the blood of patients with extra-central nervous system tuberculosis. Therefore, this finding suggests that this protein can be used as a molecular marker for any type of tuberculous infection. It also provides a more sensitive immunoassay option for the early and confirmatory diagnosis of TBM.

Trends in increased tuberculosis infection and a fatality rate of ∼23% have necessitated the search for alternative biomarkers using newly developed postgenomic approaches. Here we provide a systematic analysis of Mycobacterium tuberculosis(Mtb) by directly profiling its gene products. This analysis combines high-throughput proteomics and computational approaches to elucidate the globally expressed complements of the three subcellular compartments (the cell wall, membrane, and cytosol) of Mtb. We report the identifications of 1044 proteins and their corresponding localizations in these compartments. Genome-based computational and metabolic pathways analyses were performed and integrated with proteomics data to reconstruct response networks. From the reconstructed response networks for fatty acid degradation and lipid biosynthesis pathways in Mtb, we identified proteins whose involvements in these pathways were not previously suspected. Furthermore, the subcellular localizations of these expressed proteins provide interesting insights into the compartmentalization of these pathways, which appear to traverse from cell wall to cytoplasm. Results of this large-scale subcellular proteome profile of Mtb have confirmed and validated the computational network hypothesis that functionally related proteins work together in larger organizational structures.

ABSTRACTMice that were transgenic for a T-cell receptor (TCR) specific for ovalbumin peptide323-339(DO11.10) were able to survive an infection with Mycobacterium tuberculosisfor approximately 80 days. This limited early control of infection was associated with gamma interferon production, inducible nitric oxide synthase expression within the lung, and an influx of clonotypic lymphocytes. The control of M. tuberculosiswas lost in DO11.10 mice bred in a ragmutant background, demonstrating that the immune responsiveness was recombinase dependent and likely to be associated with the expression of an alternative α TCR by DO11.10 mice. A characterization of the antigen specificity in DO11.10 TCR transgenic mice demonstrated that the specificity was limited and dominated by the 26-kDa (Rv1411c) lipoprotein of M. tuberculosis. This study identifies this lipoprotein as an important and potent inducer of protective T cells within the lungs of mice infected with M. tuberculosisand therefore as a possible target for vaccination.

ABSTRACTAn adipose cell (SW872) model was developed to observe cellular necrosis and apoptosis upon Mycobacterium ulceransinfection and treatment with mycobacterial exudate. Apoptosis was likely due to secreted proteins, while necrosis was likely due to mycolactone. Our data suggest that additional factors inM. ulceransmay be involved in Buruli ulcer pathogenesis.

ABSTRACTMycobacterium tuberculosisestablishes infection, progresses towards disease, and is transmitted from the alveolus of the lung. However, the role of the alveolar epithelium in any of these pathogenic processes of tuberculosis is unclear. In this study, lung epithelial cells (A549) were used as a model in which to examine cytotoxicity during infection with either virulent or avirulent mycobacteria in order to further establish the role of the lung epithelium during tuberculosis. Infection of A549 cells with M. tuberculosisstrains Erdman and CDC1551 demonstrated significant cell monolayer clearing, whereas infection with eitherMycobacterium bovisBCG or Mycobacterium smegmatisLR222 did not. Clearing of M. tuberculosis-infected A549 cells correlated to necrosis, not apoptosis. Treatment of M. tuberculosis-infected A549 cells with streptomycin, but not cycloheximide, demonstrated a significant reduction in the necrosis of A549 cell monolayers. This mycobacterium-induced A549 necrosis did not correlate to higher levels of intracellular or extracellular growth by the mycobacteria during infection. Staining of infected cells with propidium iodide demonstrated that M. tuberculosisinduced increased permeation of A549 cell membranes within 24 h postinfection. Quantitation of lactate dehydrogenase (LDH) release from infected cells further demonstrated that cell permeation was specific to M. tuberculosisinfection and correlated to A549 cellular necrosis. Inactivated M. tuberculosisor its subcellular fractions did not result in A549 necrosis or LDH release. These studies demonstrate that lung epithelial cell cytotoxicity is specific to infection by virulent mycobacteria and is caused by cellular necrosis. This necrosis is not a direct correlate of mycobacterial growth or of the expression of host cell factors, but is preceded by permeation of the A549 cell membrane and requires infection with live bacilli.

Identification of biomarkers for latent Mycobacterium tuberculosisinfection 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. tuberculosispeptides in serum extracellular vesicles from TB patients. We subsequently optimized this MRM-MS assay to selectively identify 40 M. tuberculosispeptides from 19 proteins that most commonly copurify with serum vesicles of patients with TB.