The liposome of trehalose dimycolate extracted from M. bovis BCG induces antitumor immunity via the activation of dendritic cells and CD8+ T cells.

Intravesical Bovis bacillus Calmette-Guérin (BCG) therapy is the most effective immunotherapy for bladder cancer, but it sometime causes serious side effects because of its inclusion of live bacteria. It is necessary to develop a more active but less toxic immunotherapeutic agent. Trehalose 6,6'-dimycolate (TDM), the most abundant hydrophobic glycolipid of the BCG cell wall, has been reported to show various immunostimulatory activities such as granulomagenesis and adjuvant activity. Here, we developed cationic liposomes incorporating TDM purified from Mycobacterium bovis BCG Connaught, and we investigated the antitumor effect of the cationic liposome TDM (Lip-TDM). Lip-TDM exerted an antitumor effect in bladder cancer, colon cancer, and melanoma-bearing mouse models that was comparable or even superior to that of BCG, with no body weight loss or granuloma formation. The antitumor effect of Lip-TDM disappeared in two types of mice: those with depletion of CD8+ T cells, and those with knockout of macrophage-inducible C-type lectin (Mincle) which recognize TDM. Lip-TDM treatment enhanced the maturation and migration of dendritic cells in the tumor microenvironment in a Mincle-dependent manner. Our results elucidate mechanisms that underlie Lip-TDM treatment and suggest that Lip-TDM has potential as a safe and effective treatment for various cancers.

Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry.

The limited availability of rapid and reliable flow cytometry-based assays for ex vivo quantification of autophagy has hampered their clinical applications for studies of diseases pathogenesis or for the implementation of autophagy-targeting therapies. To this aim, we modified and improved the protocol of a commercial kit developed for quantifying the microtubule-associated protein 1A/1B light chain 3B (LC3), the most reliable marker for autophagosomes currently available. The protocol modifications were set up measuring the autophagic flux in neoplastic (THP-1 cells) and primary cells (peripheral blood mononuclear cells; PBMC) of healthy donors. Moreover, PBMC of active tuberculosis (TB) patients were stimulated with the Mycobacterium tuberculosis purified protein derivatives or infected with live Mycobacterium bovis bacillus Calmette-Guerin (BCG). We found that the baseline median fluorescent intensity (MFI) of THP-1 cells changed depending on the time of sample acquisition to the flow cytometer. To solve this problem, a fixation step was introduced in different stages of the assay's protocol, obtaining more reproducible and sensitive results when a post-LC3 staining fixation was performed, in either THP1 or PBMC. Furthermore, since we found that results are influenced by the type and the dose of the lysosome inhibitor used, the best dose of Chloroquine for LC3 accumulation were set up in either THP-1 cells or PBMC. Finally, applying these experimental settings, we measured the autophagic flux in CD14+ cells from active TB patients' PBMC upon BCG infection. In conclusion, our data indicate that the protocol modifications here described in this work improve the stability and accuracy of a flow cytometry-based assay for the evaluation of autophagy, thus assuring more standardised cell analyses.

The prominent alteration in transcriptome and metabolome of Mycobacterium bovis BCG str. Tokyo 172 induced by vitamin B1.

BACKGROUND: Vitamin B1 (VB1) is a crucial dietary nutrient and essential cofactor for several key enzymes in the regulation of cellular and metabolic processes, and more importantly in the activation of immune system. To date, the precise role of VB1 in Mycobacterium tuberculosis remains to be fully understood. RESULTS: In this study, the transcriptional and metabolic profiles of VB1-treated Mycobacterium. bovis BCG were analyzed by RNA-sequencing and LC-MS (Liquid chromatography coupled to mass spectrometry). The selection of BCG strain was based on its common physiological features shared with M. tuberculosis. The results of cell growth assays demonstrated that VB1 inhibited the BCG growth rate in vitro. Transcriptomic analysis revealed that the expression levels of genes related to fatty acid metabolism, cholesterol metabolism, glycolipid catabolism, DNA replication, protein translation, cell division and cell wall formation were significantly downregulated in M. bovis BCG treated with VB1. In addition, the metabolomics LC-MS data indicated that most of the amino acids and adenosine diphosphate (ADP) were decreased in M. bovis BCG strain after VB1 treatment. CONCLUSIONS: This study provides the molecular and metabolic bases to understand the impacts of VB1 on M.bovis BCG.

Poncet's disease after the intravesical instillation of Bacillus Calmette-Guérin (BCG): a case report.

BACKGROUND: Poncet's disease is a rare syndrome characterized by articular impairment in a form of rare tuberculid. One of the theories of its cause involves an autoimmune response induced by the intravesical administration of the Calmette-Guerin Bacillus or the treatment of bladder carcinoma. Furthermore, there may be an appearance of oligoarticular or polyarticular arthritis, beginning 1-3 months after the start of therapy. Few physicians know the disease and the literature related to that syndrome is scarce and restricted to case reports, which contributes to its under diagnosis. CASE PRESENTATION: Female patient, 64 years old, Caucasian, in whom was noticed firstly dark urine, without haematuria or dysuria. Later felt also colic pain in the hypogastric region. Microscopically, the conclusive diagnosis was a high grade non-invasive papillary urothelial carcinoma. Thereupon, the treatment of the tumour began with transurethral resection technique and intravesical instillation of Calmette-Guérin Bacillus as adjuvant treatment. Eight months after the beginning of treatment, the lingering presence of the carcinoma was identified. Nevertheless, arthritis was identified through radiographs, after an increase in the clavicle capitation, right knee and left ankle in bone scintigraphy. Coinciding with the joint manifestations, the patient developed fever and purulent urethral discharge (culture was negative). Therefore, trying to investigate the cause of the arthritis, Purified Protein Derivate was taken, with reactive results. An increase of acute phase reactants was found, with other tests resulting normal: blood chemistry, Complete Blood Count, immunology and serology. Human Leukocyte Antigen typing by polymerase chain reaction revealed the presence of A24/AX, B44, B27, BW4/BW4, DQ7 and DQ5. Consequently, Poncet's disease was the diagnostic conclusion. The treatment with intravesical Calmette-Guérin Bacillus was immediately discontinued. The patient received corticosteroids associated with etoricoxib and isoniazid for 4 months, achieving disappearance of the inflammatory joint signs in 3 months. After 6 months, no joint pain recurrence or other manifestations suggesting active disease had been seen. CONCLUSIONS: Therefore, such diagnosis should be considered when confronted with an osteoarticular clinical picture in patients treated with intravesical Calmette-Guérin Bacillus, especially patients with HLA-B27 (+) and B7 (+), as Poncet's disease is a reactive arthritis.

BCG Increased Membrane Expression of TRIM59 Through the TLR2/ TLR4/IRF5 Pathway in RAW264.7 Macrophages.

OBJECTIVE: In our previous study, we showed that Bacillus Calmette-Guerin (BCG)- activated macrophages have the ability to directly kill tumor cells. One of the main properties of these macrophages is the high expression of tripartite motif family protein 59 (TRIM59). This study was conducted to investigate the mechanism of BCG-induced TRIM59 expression on macrophages and to identify the subcellular localization of TRIM59. METHODS: TRIM59 expression and TNF-α secretion were compared in RAW264.7 macrophage cells that were stimulated using BCG with or without Toll-like receptor 2/4 (TLR2/4)-neutralizing antibodies. Next, small interfering RNA (siRNA) was used to down-regulated interferon regulatory factor 5 (IRF5) gene expression in RAW264.7 cells. Transfected cells were stimulated with BCG, after which TRIM59 expression and TNF-α secretion were evaluated in cells pre-treated with siRNA or scramble control. After treatments, supernatants were co-cultured with MCA207, and cell viabilities were determined. Moreover, BCG-stimulated RAW264.7 cells were stained for TRIM59 and F4/80 expression. RESULTS: In this study, we showed that TRIM59 was expressed on the membrane of RAW264.7 cells. After blocking TLR2/4, treatment with BCG failed to induce the expression of TRIM59, IRF5, and TNF-α on RAW264.7 cells. In addition, down-regulation of IRF5 inhibited TRIM59 and TNF-α expression. CONCLUSION: Our study showed that TRIM59 is a membrane protein, and that BCG treatment upregulated TRIM59 expression on macrophages via TLR2/4 and IRF5 pathways.

Secretome profiling of highly virulent Mycobacterium bovis 04-303 strain reveals higher abundance of virulence-associated proteins.

Mycobacterium bovis is the causative agent of tuberculosis in farms, wildlife and causes sporadic disease in humans. Despite the high similitude in genome sequence between M. bovis strains, some strains like the wild boar 04-303 isolate show a highly virulent phenotype in animal models. Comparative studies will contribute to link protein expression with the virulence phenotype. In vitro, the 04-303 strain was more phagocytized by J774A.1 macrophages in comparison with 444 strain (a cow isolate with the same genotype) and BCG. The secretome of these strains showed a significant proportion of shared proteins (368 spots). Among the proteins only visualized in the secretome of the 04-303 strain, we identify the nine most abundant proteins by LC-MS/MS. The most relevant were EsxA and EsxB proteins, which are encoded in the RD1 region, deleted in BCG strains. These proteins are the major virulence factor of M. tuberculosis. The other proteins identified belong to functional categories of virulence, detoxification, and adaptation; lipid metabolism; and cell wall and cell processes. The relatively high proportion of proteins involved in the cell wall and cell process is consistent with the previously described variation among M. bovis genomes.

Layer-by-layer nanocoating of live Bacille-Calmette-Guérin mycobacteria with poly(I:C) and chitosan enhances pro-inflammatory activation and bactericidal capacity in murine macrophages.

Tuberculosis (TB) is a major disease burden globally causing more than 1.5 million deaths per year. The attenuated live vaccine strain Bacille Calmette-Guérin (BCG), although providing protection against childhood TB, is largely ineffective against adult pulmonary TB. A major aim therefore is to increase the potency of the BCG vaccine to generate stronger and more sustained immunity against TB. Here, we investigated the use of layer-by-layer (LbL) nanocoating of the surface of live BCG with several layers of polyinosinic-polycytidylic acid (poly(I:C)), a strong inducer of cell-mediated immunity, and the biodegradable polysaccharide chitosan to enhance BCG immunogenicity. Nanocoating of live BCG did not affect bacterial viability or growth in vitro but induced killing of the BCG in infected mouse bone marrow-derived macrophages and enhanced macrophage production of pro-inflammatory cytokines and expression of surface co-stimulatory molecules relative to uncoated BCG. In addition, poly(I:C) surface-coated BCG, but not BCG alone or together with soluble poly(I:C), induced high production of nitric oxide (NO) and IL-12. These results argue that BCG and surface absorbed poly(I:C) act in a synergistic manner to elicit pro-inflammatory macrophage activation. In conclusion, nanocoating of live BCG with the immunostimulatory agent poly(I:C) may be an appropriate strategy to enhance and modulate host responses to the BCG vaccine.

Structure, chain conformation, and immunomodulatory activity of the polysaccharide purified from Bacillus Calmette Guerin formulation.

A polysaccharide, coded as BDP, purified from the injection powder of Bacillus Calmette Guerin (BCG) polysaccharide and nucleic acid, was composed mainly of α-D-(1→4)-linked glucan with (1→6)-linked branches and trace amounts of fucose and mannose from the results of FT-IR, HPAEC-PAD and NMR spectrum. The Mw, Mn, Mz, and [Formula: see text] were determined to be 1.320×10(5)g/mol, 1.012×10(5)g/mol, 2.139×10(5)g/mol, and 21.8±3.2%nm by using HPSEC-MALLS, respectively. The ν value from [Formula: see text] was calculated to be 0.52±0.01, which firstly clarified that BDP existed as random coils in 0.9% NaCl aqueous solution. AFM and SEM combined with Congo-red test also revealed that the polysaccharide was irregular globular like or curly structure. Furthermore, in vitro tests on RAW264.7 murine macrophages cells revealed that BDP exhibited significant immunomodulatory activity.

Outcome after BCG treatment for urinary bladder cancer may be influenced by polymorphisms in the NOS2 and NOS3 genes.

PURPOSE: Bacillus Calmette-Guérin (BCG)-treatment is an established treatment for bladder cancer, but its mechanisms of action are not fully understood. High-risk non-muscle invasive bladder-cancer (NMIBC)-patients failing to respond to BCG-treatment have worse prognosis than those undergoing immediate radical cystectomy and identification of patients at risk for BCG-failure is of high priority. Several studies indicate a role for nitric oxide (NO) in the cytotoxic effect that BCG exerts on bladder cancer cells. In this study we investigated whether NO-synthase (NOS)-gene polymorphisms, NOS2-promoter microsatellite (CCTTT)n, and the NOS3-polymorphisms-786T>C (rs2070744) and Glu298Asp (rs1799983), can serve as possible molecular markers for outcome after BCG-treatment for NMIBC. MATERIALS AND METHODS: All NMIBC-patients from a well-characterized population based cohort were analyzed (n=88). Polymorphism data were combined with information from 15-years of clinical follow-up. The effect of BCG-treatment on cancer-specific death (CSD), recurrence and progression in patients with varying NOS-genotypes were studied using Cox proportional hazard-models and log rank tests. RESULTS: BCG-treatment resulted in significantly better survival in patients without (Log rank: p=0.006; HR: 0.12, p=0.048), but not in patients with a long version ((CCTTT)n ≧13 repeats) of the NOS2-promoter microsatellite. The NOS3-rs2070744(TT) and rs1799983(GG)-genotypes showed decreased risk for CSD (Log rank(TT): p=0.001; Log rank(GG): p=0.010, HR(GG): 0.16, p=0.030) and progression (Log rank(TT): p<0.001, HR(TT): 0.05, p=0.005; Log rank(GG): p<0.001, HR(GG): 0.10, p=0.003) after BCG-therapy compared to the other genotypes. There was also a reduction in recurrence in BCG-treated patients that was mostly genotype independent. Analysis of combined genotypes identified a subgroup of 30% of the BCG-treated patients that did not benefit from BCG-treatment. CONCLUSIONS: Our results suggest that the investigated polymorphisms influence patient response to BCG-treatment and thus may serve as possible markers for identification of BCG-failures.

A novel recombinant BCG-expressing pro-apoptotic protein BAX enhances Th1 protective immune responses in mice.

One-third of the world's population is infected with Mycobacterium tuberculosis (MTB). The protective efficacy of bacille Calmette Guérin (BCG) vaccine against tuberculosis (TB) in adults is highly controversial even though the BCG vaccine has been available for more than 90 years. Because BCG is effective against infantile tuberculosis meningitis and miliary tuberculosis in young children and provides cost-effective prevention from tuberculosis for developing countries, it would be desirable to modify the existing BCG vaccine to provide more comprehensive protection. In our study, we constructed a novel recombinant BCG strain expressing pro-apoptotic BAX (rBCG::BAX) and demonstrated that it significantly induced the apoptosis of macrophages infected with rBCG::BAX both in vitro and in vivo. In addition, it significantly enhanced Ag85B-specific IFN-γ enzyme-linked immunospot responses, IFN-γ secretion, IL-2 secretion and the ratio of Ag85B-specific IgG2b/IgG1, and it significantly decreased Ag85B-specific IL-4. Furthermore, it presumably facilitated antigen presentation by inducing a significant up-regulation in the expression of MHC-II and B7.1 (CD80) co-stimulatory molecules on macrophages. In conclusion, these results suggest that the rBCG::BAX strain elicited predominantly a Th1 protective immune responses and might be a potential tuberculosis vaccine candidate for further study.

Enhanced and durable protective immune responses induced by a cocktail of recombinant BCG strains expressing antigens of multistage of Mycobacterium tuberculosis.

Although Bacillus Calmette-Guérin (BCG) vaccine confers protection from Mycobacterium tuberculosis infection in children, its immune protection gradually wanes over time, and consequently leads to an inability to prevent the reactivation of latent infection of M. tuberculosis. Therefore, improving BCG for better control of tuberculosis (TB) is urgently needed. We thus hypothesized that recombinant BCG overexpressing immunodominant antigens expressed at different growth stages of M. tuberculosis could provide a more comprehensive protection against primary and latent M. tuberculosis infection. Here, a novel cocktail of recombinant BCG (rBCG) strains, namely ABX, was produced by combining rBCG::85A, rBCG::85B, and rBCG::X, which overexpressed respective multistage antigens Ag85A, Ag85B, and HspX of M. tuberculosis. Our results showed that ABX was able to induce a stronger immune protection than individual rBCGs or BCG against primary TB infection in C57BL/6 mice. Mechanistically, the immune protection was attributed to stronger antigen-specific CD4(+) Th1 responses, higher numbers of IFN-γ(+) CD4(+) TEM and IL-2(+) CD8(+) TCM cells elicited by ABX. These findings thus provide a novel strategy for the improvement of BCG efficacy and potentially a promising prophylactic TB vaccine candidate, warranting further investigation.

Prime-boost vaccination strategy with bacillus Calmette-Guérin (BCG) and liposomized alpha-crystalline protein 1 reinvigorates BCG potency.

Bacillus Calmette-Guérin (BCG) remains the only available and most widely administered vaccine against Mycobacterium tuberculosis (Mtb), yet it fails to protect vaccinated individuals either from primary infection or reactivation of latent tuberculosis (TB). Despite BCG's variable efficacy against TB, the fact remains that BCG imparts protection in children against the disease, indicating that BCG possesses a wide protective antigenic repertoire. However, its failure to impart protection in adulthood can be linked to its failure to generate long-lived memory response and elicitation of an inadequate immune response against latency-associated antigens. Therefore, to improve the protective efficacy of BCG, a novel vaccination strategy is required. Consequently, in the present study, we have exploited the vaccination potential of liposomized α-crystalline 1 (Acr1L), a latency-associated antigen to induce enduring protective immunity against Mtb in BCG-primed animals. It is noteworthy that an increase in the multi-functional [interferon (IFN)-γ(hi) /tumour necrosis factor (TNF)-α(hi) ] CD4 and CD8 T cells were observed in BCG-primed and Acr1L-boosted (BCG-Acr1L) animals, compared to BCG alone. Further, substantial expansion of both central memory (CD44(hi) /CD62L(hi) ) and effector memory (CD44(hi) /CD62L(lo) ) populations of CD4 and CD8 T cells was noted. Importantly, BCG-Acr1L exhibited significantly better protection than BCG, as evidenced by a reduction in the bacterial burden and histopathological data of the lungs. In essence, BCG-Acr1L could be a potent future vaccination strategy to reinvigorate BCG potency.

Prolactin modulates cytokine production induced by culture filtrate proteins of M. bovis through different signaling mechanisms in THP1 cells.

The immunomodulatory functions of prolactin (PRL) are well recognized. Augmented PRL plasma levels were observed in patients with advanced tuberculosis (TB). Recently, we have reported that LPS and Mycobacterium bovis (M. bovis) induced differential expression of PRL receptor (PRLR) isoforms in THP-1 cells and bovine macrophages, respectively. The aim of this work was to determine whether PRL should be considered as a potential modulator of the signaling pathways and cytokine synthesis, induced by culture filtrate protein (CFP) from M. bovis in THP-1 monocytes. The THP-1 cells were stimulated with PRL (20ng/mL), M. bovis CFP (50µg/mL). PRLR as well as phosphorylated STAT3, STAT5, Akt1/2/3, ERK1/2 and p38 expression were evaluated by Western blot. IL1-ß, TNF-α, IL-6, IL-12, IL-8, and IL-10 concentrations were measured by ELISA. Our results demonstrated that the expression pattern of PRLR short isoforms is induced by M. bovis CFP. M bovis CFP induced phosphorylation of Akt2, ERK1/2, p38, STAT3, and STAT5 pathways. In turn, PRL only activated the JAK2/STAT3-5 signaling pathway. However, when combined both stimuli, PRL significantly increased STAT3-5 phosphorylation and downregulated Akt2, ERK1/2, and p38 phosphorylation. As expected, M. bovis CFP induced substantial amounts of IL1-ß, IL-6, TNF-α, IL-8, IL-12, and IL-10. However, the PRL costimulation considerably decreased IL1-ß, TNF-α, and IL-12 secretion, and increased IL-10 production. This results suggest that up-regulation of IL-10 by PRL might be modulating the pro-inflammatory response against mycobacterial antigens through the MAPK pathway.

BCG-induced protection: effects on innate immune memory.

The Bacille Calmette-Guerin (BCG) vaccine is the only vaccine proved to be effective against tuberculosis and it remains the most commonly used vaccine worldwide. In addition to its effects on mycobacterial diseases, an increasing body of epidemiological evidence accumulated since its introduction in 1921 shows that BCG also exerts beneficial non-specific effects ranging from protection against non-mycobacterial diseases, decreased incidence of allergic diseases, and treatment of certain malignancies. The biological substrate of these effects is mediated partly by heterologous effects on adaptive immunity, but also on the potentiation of innate immune responses through epigenetic mechanisms, a process termed 'trained immunity'. The process of trained immunity may also play a role in the beneficial effects of BCG against tuberculosis and Mycobacterium tuberculosis infection, and this could have important consequences for our quest for improving vaccination strategies.

Crystal structure and functional implications of LprF from Mycobacterium tuberculosis and M. bovis.

The Gram-positive bacteria Mycobacterium tuberculosis and M. bovis are causative agents of tuberculosis in humans and cattle. The lipoprotein LprF is found in M. tuberculosis and M. bovis but not in the nonpathogenic M. smegmatis. To date, the role of LprF remains to be elucidated. In this study, the crystal structure of LprF has been determined at 1.1 Šresolution. The overall structure is similar to that of a homologue, LprG, with a central hydrophobic cavity that binds a triacylated glycolipid. LprF exhibited a central cavity structure similar to that of LprG, but with a smaller cavity that binds two alkyl chains. Consistently, subsequent mass-spectrometric analysis revealed that the bound ligand was a diacylated glycolipid, as found in the structure. Furthermore, an increased ratio of lipoarabinomannan to lipomannan in the mycobacterial cell wall was observed when lprF was introduced into M. smegmatis. These observations suggested that LprF transfers the diacylated glycolipid from the plasma membrane to the cell wall, which might be related to the pathogenesis of the bacteria.

The application of metabolite profiling to Mycobacterium spp.: determination of metabolite changes associated with growth.

In order to decipher the complex biological networks underlying biochemical and physiological processes, cellular regulation at all levels must be studied. The metabolites determined by metabolomics represent the end-point of cellular regulation and thus vital components of any integrative network. In the case of pathogenic agents such as Mycobacterium tuberculosis metabolomics offers an ideal opportunity to gain a better understanding of how this species adapts to environmental conditions and antimicrobial treatments. In the present study a metabolite profiling protocol for Mycobacterium including optimised quenching, extraction and analysis has been devised. These methods have been applied to three different Mycobacterium spp. demonstrating potential translation across the genus. Steady-state levels of metabolites during growth have been determined for Mycobacterium smegmatis, Mycobacterium phlei and Mycobacterium bovis BCG (Bacillus Calmette-Guérin). The changes of designated biomarkers emphasised phenotypical differences (e.g. nitrogen metabolism) and similarities (e.g. cysteine biosynthesis) between the bacteria. Each time point showed distinguishable metabolic characteristics from early lag to late stationary phase/beginning of non-replicating phase. The combination of the metabolic results with published "omics" data indicated that transcription appeared to be the most predominant mode of cellular regulation utilised by these bacteria studied.

The role of PTEN tumor suppressor pathway staining in carcinoma in situ of the bladder.

OBJECTIVES: The PI3k/Akt pathway has been associated with the development and progression of bladder tumors, with most studies focused on papillary or muscle-invasive tumors. We sought to characterize the expression patterns of the PI3K/Akt pathway in a large cohort of high-risk preinvasive carcinoma in situ (CIS) tumors of the bladder. Our goal was to understand whether PI3K/Akt pathway alterations associated with CIS resemble early- or late-stage bladder cancers. MATERIAL AND METHODS: We evaluated tissue specimens from 97 patients with CIS of the bladder, of which 14 had a concomitant papillary tumor. All patients were treated with intravesical bacillus Calmette-Guerin. All specimens were evaluated for PTEN, p-AKT, and p-S6 immunoreactivity. Markers were evaluated for percentage and intensity of staining and were scored using a 0 to 3+grading system. RESULTS: PTEN staining was noted as least intense in 67% of tumor specimens and 22% of normal urothelium. P-Akt and p-S6 had intense staining in 77% and 90% of tumor specimens vs. 44% and 68% in normal tissue, respectively. Low-intensity staining for PTEN at 12 months correlated with higher recurrence risk (P = 0.026). CONCLUSION: We describe a large cohort of CIS bladder tumors with decreased staining intensity of PTEN and increased staining intensity of p-AKT and p-S6, similar to high-grade and high-stage papillary tumors. Low-intensity staining of PTEN at 12 months was associated with an increased risk of recurrence.

Circulating Mycobacterium bovis peptides and host response proteins as biomarkers for unambiguous detection of subclinical infection.

Bovine tuberculosis remains one of the most damaging diseases to agriculture, and there is also a concern for human spillover. A critical need exists for rapid, thorough, and inexpensive diagnostic methods capable of detecting and differentiating Mycobacterium bovis infection from other pathogenic and environmental mycobacteria at multiple surveillance levels. In a previous study, Seth et al. (PLoS One 4:e5478, 2009, doi:10.1371/journal.pone.0005478) identified 32 host peptides that specifically increased in the blood serum of M. bovis-infected animals). In the current study, 16 M. bovis proteins were discovered in the blood serum proteomics data sets. A large-scale validation analysis was undertaken for selected host and M. bovis proteins using a cattle serum repository containing M. bovis (n = 128), Mycobacterium kansasii (n = 10), and Mycobacterium avium subsp. paratuberculosis (n = 10), cases exposed to M. bovis (n = 424), and negative controls (n = 38). Of the host biomarkers, vitamin D binding protein (VDBP) showed the greatest sensitivity and specificity for M. bovis detection. Circulating M. bovis proteins, specifically polyketide synthetase 5, detected M. bovis-infected cattle with little to no seroreactivity against M. kansasii- and M. avium subsp. paratuberculosis-infected animals. These data indicate that host and pathogen serum proteins can serve as reliable biomarkers for tracking M. bovis infection in animal populations.

Comparison of the membrane proteome of virulent Mycobacterium tuberculosis and the attenuated Mycobacterium bovis BCG vaccine strain by label-free quantitative proteomics.

The Mycobacterium tuberculosis membrane is rich in antigens that are potential targets for diagnostics and the development of new vaccines. To better understand the mechanisms underlying MTB virulence and identify new targets for therapeutic intervention, we investigated the differential composition of membrane proteomes between virulent M. tuberculosis H37Rv (MTB) and the Mycobacterium bovis BCG vaccine strain. To compare the membrane proteomes, we used LC-MS/MS analysis in combination with label-free quantitative proteomics, utilizing the area under the curve of the extracted ion chromatograms of peptides obtained from m/z and retention time alignment of MS1 features. With this approach, we obtained relative abundance ratios for 2203 identified membrane-associated proteins in high confidence. Of these proteins, 294 showed statistically significant differences of at least two fold in relative abundance between MTB and BCG membrane fractions. Our comparative analysis detected several proteins associated with known genomic regions of difference between MTB and BCG as being absent, which validated the accuracy of our approach. In further support of our label-free quantitative data, we verified select protein differences by immunoblotting. To our knowledge, we have generated the first comprehensive and high-coverage profile of comparative membrane proteome changes between virulent MTB and its attenuated relative BCG, which helps elucidate the proteomic basis of the intrinsic virulence of the MTB pathogen.

Lipoarabinomannan mannose caps do not affect mycobacterial virulence or the induction of protective immunity in experimental animal models of infection and have minimal impact on in vitro inflammatory responses.

Mannose-capped lipoarabinomannan (ManLAM) is considered an important virulence factor of Mycobacterium tuberculosis. However, while mannose caps have been reported to be responsible for various immunosuppressive activities of ManLAM observed in vitro, there is conflicting evidence about their contribution to mycobacterial virulence in vivo. Therefore, we used Mycobacterium bovis BCG and M. tuberculosis mutants that lack the mannose cap of LAM to assess the role of ManLAM in the interaction of mycobacteria with the host cells, to evaluate vaccine-induced protection and to determine its importance in M. tuberculosis virulence. Deletion of the mannose cap did not affect BCG survival and replication in macrophages, although the capless mutant induced a somewhat higher production of TNF. In dendritic cells, the capless mutant was able to induce the upregulation of co-stimulatory molecules and the only difference we detected was the secretion of slightly higher amounts of IL-10 as compared to the wild type strain. In mice, capless BCG survived equally well and induced an immune response similar to the parental strain. Furthermore, the efficacy of vaccination against a M. tuberculosis challenge in low-dose aerosol infection models in mice and guinea pigs was not affected by the absence of the mannose caps in the BCG. Finally, the lack of the mannose cap in M. tuberculosis did not affect its virulence in mice nor its interaction with macrophages in vitro. Thus, these results do not support a major role for the mannose caps of LAM in determining mycobacterial virulence and immunogenicity in vivo in experimental animal models of infection, possibly because of redundancy of function.