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Translation engineering and bioinformatics have accelerated the rate at which gene sequences can be improved to generate multi-epitope proteins. Strong antigenic proteins for tuberculosis diagnosis include individual ESAT6 and CFP10 proteins or derived peptides. Obtention of heterologous multi-component antigens in E. coli without forming inclusion bodies remain a biotechnological challenge. The gene sequence for ESAT6-CFP10 fusion antigen was optimized by codon bias adjust for high-level expression as a soluble protein. The obtained fusion protein of 23.7 kDa was observed by SDS-PAGE and Western blot analysis after Ni-affinity chromatography and the yield of expressed soluble protein reached a concentration of approximately 67 mg/L in shake flask culture after IPTG induction. Antigenicity was evaluated at 4 μg/mL in whole blood cultures from bovines, and protein stimuli were assessed using a specific in vitro IFN-γ release assay. The hybrid protein was able to stimulate T-cell specific responses of bovine TB suspects. The results indicate that improved E. coli codon usage is a good and cost-effective strategy to potentialize large scale production of multi-epitope proteins with sustained antigenic properties for diagnostic purposes., Competing Interests: Declaration of competing interest The authors confirm that this article content has no conflicts of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The Bacillus Calmette-Guerin vaccine is still widely used in the developing world. The vaccination prevents infant death not only from tuberculosis but also from unrelated infectious agents, especially respiratory tract infections and neonatal sepsis. It is proposed that these off-target protective effects of the BCG vaccine are mediated by the general long-term boosting of innate immune mechanisms, also termed "trained innate immunity". Recent studies indicate that both COVID-19 incidence and total deaths are strongly associated with the presence or absence of national mandatory BCG vaccination programs and encourage the initiation of several clinical studies with the expectation that revaccination with BCG could reduce the incidence and severity of COVID-19. Here, presented results from the bioinformatics analysis of the Mycobacterium bovis (strain BCG/Pasteur 1173P2) proteome suggests four immunodominant antigens that could induce an immune response against SARS-CoV-2.

Growth and division by most bacteria requires remodelling and cleavage of their cell wall. A byproduct of this process is the generation of free peptidoglycan (PG) fragments known as muropeptides, which are recycled in many model organisms. Bacteria and hosts can harness the unique nature of muropeptides as a signal for cell wall damage and infection, respectively. Despite this critical role for muropeptides, it has long been thought that pathogenic mycobacteria such as Mycobacterium tuberculosis do not recycle their PG. Herein we show that M. tuberculosis and Mycobacterium bovis BCG are able to recycle components of their PG. We demonstrate that the core mycobacterial gene lpqI, encodes an authentic NagZ β-N-acetylglucosaminidase and that it is essential for PG-derived amino sugar recycling via an unusual pathway. Together these data provide a critical first step in understanding how mycobacteria recycle their peptidoglycan.

Background: Vitamin B 1 (V B1 ) 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 V B1 in Mycobacterium tuberculosis remains to be fully understood., Results: In this study, the transcriptional and metabolic profiles of V B1 -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 V B1 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 V B1. 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 V B1 treatment., Conclusions: This study provides the molecular and metabolic bases to understand the impacts of V B1 on M.bovis BCG.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Macrophages (MΦ) are the most described and characterized target and host of mycobacteria. Like other cells of innate immunity MΦ have a wide range of receptor molecules which interact with different pathogen associated molecular patterns (PAMPs). Immunodominant proteins MPT63 and MPT83 that are synthesized in abundance by Mycobacterium bovis or Mycobacterium tuberculosis strains could be involved in development of tuberculosis infection. The aim of this study was to search for effects of these mycobacterial antigens on target cells. For this aim full-sized sequences of MPT83 (rMPT83full) and MPT63 antigens were cloned into plasmid pET24a(+). The increase of phagocytic activity of murine peritoneal macrophages was demonstrated, but not of macrophage-like cells from J774 cell line, which were treated by rMPT63 and rMPT83full proteins for 24 h. This effect of such antigens can be considered as a way to facilitate the consumption of mycobacterial cells by macrophages to avoid other effector mechanisms of innate and adaptive immunity.

The tuberculin skin test is the primary screening test for the diagnosis of bovine tuberculosis (TB), and use of this test has been very valuable in the control of this disease in many countries. However, the test lacks specificity when cattle have been exposed to environmental mycobacteria or vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG). Recent studies showed that the use of three or four recombinant mycobacterial proteins, including 6-kDa early secretory antigenic target (ESAT6), 10-kDa culture filtrate protein (CFP10), Rv3615c, and Rv3020c, or a peptide cocktail derived from those proteins, in the skin test greatly enhanced test specificity, with minimal loss of test sensitivity. The proteins are present in members of the pathogenic Mycobacterium tuberculosis complex but are absent in or not expressed by the majority of environmental mycobacteria and the BCG vaccine strain. To produce a low-cost skin test reagent, the proteins were displayed at high density on polyester beads through translational fusion to a polyhydroxyalkanoate synthase that mediates the formation of antigen-displaying inclusions in recombinant Escherichia coli. Display of the proteins on the polyester beads greatly increased their immunogenicity, allowing for the use of very low concentrations of proteins (0.1 to 3 μg of mycobacterial protein/inoculum) in the skin test. Polyester beads simultaneously displaying all four proteins were produced in a single fermentation process. The polyester beads displaying three or four mycobacterial proteins were shown to have high sensitivity for detection of M. bovis-infected cattle and induced minimal responses in animals exposed to environmental mycobacteria or vaccinated with BCG., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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., (© 2015 Institute of Microbial Technology.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Intrinsically disordered proteins (IDPs) or unstructured segments within proteins play an important role in cellular physiology and pathology. Low cellular concentration, multiple binding partners, frequent post-translational modifications and the presence of multiple conformations make it difficult to characterize IDP interactions in intact cells. We used peptide aptamers selected by using the yeast-two-hybrid scheme and in-cell NMR to identify high affinity binders to transiently structured IDP and unstructured segments at atomic resolution. Since both the selection and characterization of peptide aptamers take place inside the cell, only physiologically relevant conformations of IDPs are targeted. The method is validated by using peptide aptamers selected against the prokaryotic ubiquitin-like protein, Pup, of the mycobacterium proteasome. The selected aptamers bind to distinct sites on Pup and have vastly different effects on rescuing mycobacterial proteasome substrate and on the survival of the Bacille-Calmette-Guèrin, BCG, strain of M. bovis. This technology can be applied to study the elusive action of IDPs under near physiological conditions.

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-β, 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-β, IL-6, TNF-α, IL-8, IL-12, and IL-10. However, the PRL costimulation considerably decreased IL1-β, 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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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 Å resolution. 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 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.

Pathogenic strains of mycobacteria produce copious amounts of glutamine synthetase (GS) in the culture medium. The enzyme activity is linked to synthesis of poly-α-l-glutamine (PLG) in the cell walls. This study describes a glnA-1 mutant of Mycobacterium bovis that produces reduced levels of GS. The mutant was able to grow in enriched 7H9 medium without glutamine supplementation. The glnA-1 strain contained no detectable PLG in the cell walls and showed marked sensitivity to different chemical and physical stresses such as lysozyme, SDS and sonication. The sensitivity of the mutant to two antitubercular drugs, rifampicin and d-cycloserine, was also increased. The glnA-1 strain infected THP-1 cells with reduced efficiency and was also attenuated for growth in macrophages. A Mycobacterium smegmatis strain containing the M. bovis glnA-1 gene survived longer in THP-1 cells than the wild-type strain and also produced cell wall-associated PLG. The M. bovis mutant was not able to replicate in the organs of BALB/c mice and was cleared within 4-6 weeks of infection. Disruption of the glnA-1 gene adversely affected biofilm formation on polystyrene surfaces. The results of this study demonstrate that the absence of glnA-1 not only attenuates the pathogen but also affects cell surface properties by altering the cell wall chemistry of the organism via the synthesis of PLG; this may be a target for drug development.

Culture filtrate and cell extracts from Mycobacterium bovis cultures contain molecules which could promote protective immunity to tuberculosis in animals. Different protein fractions of M. bovis cultures were obtained by elution electrophoresis and were tested in experimentally infected cattle. The fractions that elicited gamma interferon (IFN-gamma) responses were resolved by two-dimensional gel electrophoresis, and individual proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The open reading frames were cloned, expressed as their recombinant forms, and retested with naturally and experimentally infected animals. Eleven protein fractions were highly reactive, from which the Rv1636, HspX, Rv0138, Rv2524, EsxI, and Rv3740 recombinant proteins were obtained. EsxI and HspX were the antigens most recognized by the IFN-gamma release assay. In summary, a proteomic approach allowed the identification of novel antigens useful for the diagnosis of bovine tuberculosis.

cAMP generation in bacteria is often stimulated by sudden, but lasting, changes in extracellular conditions, whereas intracellular cAMP concentrations quickly settle at new levels. As bacteria lack G-proteins, it is unknown how bacterial adenylate cyclase (AC) activities are modulated. Mycobacterium tuberculosis has 15 class III AC genes; therefore, we examined whether mycobacteria contain a factor that may regulate AC activities. We identified mycobacterial polyphosphates with a mean chain length of 72 residues as highly potent inhibitors of dimeric class IIIa, class IIIb and class IIIc ACs from M. tuberculosis and other bacteria. The identity of the inhibitor was established by phosphatase degradation, 31P-NMR, acid or base hydrolysis, PAGE and comparisons with commercial standards, and functional substitution by several polyphosphates. The data indicate that each AC dimer occupies 8-15 phosphate residues on a polyphosphate strand. Other polyionic polymers such as polyglutamate, polylysine and hyaluronic acid do not affect cyclase activity. Notably, the structurally unrelated class I AC Cya from Escherichia coli is unaffected. Bacterial polyphosphate metabolism is generally viewed in the context of stress-related regulatory networks. Thus, regulation of bacterial class III ACs by polyphosphates could be a component of the bacterial stress response.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been known for a long time to prevent tuberculosis (TB) worldwide since 1921. Nonetheless, we know little about BCG membrane proteome. In the present study, we utilized alkaline incubation and Triton X-114-based methods to enrich BCG membrane proteins and subsequently digested them using proteolytic enzyme. The recovered peptides were further separated by 2-D LC and identified by ESI-MS/MS. As a result, total 474 proteins were identified, including 78 integral membrane proteins (IMPs). Notably, 18 BCG IMPs were described for the first time in mycobacterium. Further analysis of the 78 IMPs indicated that the theoretical molecular mass distribution of them ranged from 8.06 to 167.86 kDa and pI scores ranged from 4.40 to 11.60. Functional classification revealed that a large proportion of the identified IMPs (67.9%, 53 out of 78) were involved in cell wall and cell processes functional group. In conclusion, here we reported a comprehensive profile of the BCG membrane subproteome. The present investigation may allow the identification of some valuable vaccine and drug target candidates and thus provide basement for future designing of preventive, diagnostic, and therapeutic strategies against TB.

Functional classification of proteins is central to comparative genomics. The need for algorithms tuned to enable integrative interpretation of analytical data is felt globally. The availability of a general,automated software with built-in flexibility will significantly aid this activity. We have prepared ARC (Automated Resource Classifier), which is an open source software meeting the user requirements of flexibility. The default classification scheme based on keyword match is agglomerative and directs entries into any of the 7 basic non-overlapping functional classes: Cell wall, Cell membrane and Transporters (C), Cell division (D), Information (I), Translocation (L), Metabolism (M), Stress(R), Signal and communication (S) and 2 ancillary classes: Others (O) and Hypothetical (H). The keyword library of ARC was built serially by first drawing keywords from Bacillus subtilis and Escherichia coli K12. In subsequent steps,this library was further enriched by collecting terms from archaeal representative Archaeoglobus fulgidus, Gene Ontology, and Gene Symbols. ARC is 94.04% successful on 6,75,663 annotated proteins from 348 prokaryotes. Three examples are provided to illuminate the current perspectives on mycobacterial physiology and costs of proteins in 333 prokaryotes. ARC is available at http://arc.igib.res.in.

Two-dimensional gel electrophoresis (2-DE) is currently a widely used analytical method for resolving complex mixtures of proteins. Sample preparation has a marked influence on 2-DE pattern. To reduce impurities and to increase the low-abundance proteins, protein precipitation is often used for the preparation of samples before 2-DE. In this study, we revealed that addition of SDS prior to TCA precipitation of mycobacterial cell extract proteins increases the resolution of the 2-D gel pattern.

Mycobacterial plasma membrane proteins play essential roles in many cellular processes, yet their comprehensive proteomic profiling remains challenging. This is mainly due to obstacles related to their extraction and solubilization. To tackle this problem, we have developed a novel procedure to selectively enrich mycobacterial plasma membrane proteins based on alkaline sodium carbonate washing of crude membranes followed by Triton X-114 phase partitioning. The present study assesses the efficiency of this method by proteome analysis of plasma membrane proteins from Mycobacterium bovis BCG. Extracted proteins were separated in parallel by 1-D SDS-PAGE and 2-DE and then analyzed by LC-MS/MS and MALDI-MS/MS. Our study revealed 125 proteins, of which 54 contained 1-14 predicted transmembrane domains (TMD) including nine novel proteins. The 1-D SDS-PAGE-based proteome analysis identified 81 proteins, of which 49 (60.5%) harbored TMD. This approach also revealed many hydrophobic membrane-associated/periplasmic proteins lacking TMD, but only few soluble proteins. The identified proteins were characterized with regard to biological functions and physicochemical properties providing further evidence for the high efficiency of the prefractionation method described herein.

This study pertains to analysis of the protein profile of different mycobacterial strains by two-dimensional gel electrophoresis (2DE). The strains were selected as they exhibit different phenotypic behaviour. TCA-acetone precipitated proteins were resolved by 2DE using immobilized pH gradient (IPG) strips. This study demonstrates that 2DE may be used as a tool for characterization of mycobacterial strains. Visual examination of the electrophoretograms was sufficient for characterization. Detailed characterization of specific proteins might lead to development of novel targets, diagnostic probes or sub-unit vaccine(s) against tuberculosis.

A comprehensive analysis of culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv was accomplished by combination of two-dimensional electrophoresis (2-DE), mass spectrometry, and N-terminal sequencing by Edman degradation. Analytical 2-DE gels resolved approximately 1250 protein spots from CSN of M. tuberculosis H37Rv, 381 of which were identified by mass spectrometry and/or Edman degradation. This study revealed 137 different proteins, 42 of which had previously been described as secreted. Comparative proteome analysis of CSN from virulent M. tuberculosis H37Rv and attenuated Mycobacterium bovis BCG Copenhagen identified 39 M. tuberculosis-specific spots containing 27 different proteins, representing candidate antigens for novel vaccines and diagnostics in tuberculosis. These included five proteins encoded by open reading frames absent from M. bovis BCG, e.g., early secretory antigen target (Esat6), as well as 22 novel differential proteins, such as acetyl-CoA C-acetyltransferase (Rv0243) and two putative Esat6-like proteins (Rv1198, Rv1793).

Two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins in the isoelectric point range 6-11 in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin (BCG). Twelve proteins were identified, three of which had not been described previously. The expression of the identified proteins was comparatively analyzed in culture filtrates of BCG in different growth phases and culture conditions. For some of these proteins, the relative protein abundance in the different culture filtrate preparations was significantly different. The differential expression of the identified proteins is discussed in relation to their putative localization and/or biological function.

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

Heat-shock proteins (Hsps) from various origins are known to share a conserved structure and are assumed to be key partners in the biogenesis of proteins. Fractionation of the mycobacterial Hsp60, a 65 kDa protein also called Cpn60, from Mycobacterium bovis BCG zinc-deficient culture filtrate on phenyl-Sepharose followed by Western blotting revealed the existence of four Hsp60-1 and Hsp60-2 forms, based on their hydrophobicity behaviour. Hsp60-2 species were further purified by ion-exchange chromatography and partial amino acid sequences of cyanogen bromide (CNBr) peptides of purified Hsp60-2 species showed identity with the amino acid sequence deduced from the hsp60-2 gene, indicating that the various Hsp60-2 forms are encoded by the same gene. In addition, the mycobacterial Hsp60-2 was overexpressed in E. coli using the pRR3Hsp60-2 plasmid and analysed on phenyl-Sepharose. The elution pattern of the recombinant Hsp60-2, as well as that of Escherichia coli GroEL, was similar to that of the native Hsp60-2 from the culture filtrate of M. bovis BCG and entirely different from that of the mycobacterial antigen 85. Extraction of mycobacterial Hsp60-2 forms, recombinant BCG Hsp60-2 and E. coli GroEL with organic solvents releases various amounts of non-covalently bound lipids. The presence of lipids on Hsp60-2 was confirmed by labelling M. bovis BCG with radioactive palmitate. The radioactivity was specifically associated with Hsp60 in the aqueous phase and the 19 and 38 kDa lipoproteins in the Triton X-114 phase. Analysis of the lipids extracted from purified Hsp60-2, recombinant BCG Hsp60-2 and E. coli GroEL by TLC showed the same pattern for all the samples. Acid methanolysis of the lipids followed by GC analysis led to the identification of C(16:0), C(18:0) and C(18:1) as the major fatty acyl constituents, and of methylglycoside in these proteins. Altogether, these data demonstrate that lipids are non-covalently bound to Hsp60-2 and homologous proteins.

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis. It consists of more than 100 denatured proteins in a culture filtrate of a heated culture of Mycobacterium tuberculosis. In two-dimensional electrophoretic analysis of PPDs from M. tuberculosis and M. bovis BCG, most proteins were diffusely separated and could not be seen as spots because of denaturation, whereas a few proteins showed relatively clear spots, indicating heat resistance. Two such proteins corresponded to ribosomal proteins L7 and L12. The mixture of these proteins L7/L2 induced a strong delayed-type hypersensitivity reaction. Another protein showing a clear spot was a GroES analogue, but this did not induce delayed-type hypersensitivity. There were a few other unidentified proteins. It is well known that L7 and L12 are encoded by the same gene and that they differ from each other only by an acetylic post-translational modification that occurs at the N-terminus of L12 converting it to L7 in Escherichia coli. L12, but not L7, was found in two-dimensional electrophoresis of BCG ribosomes, although we found two proteins corresponding to L7 and L12 in PPDs and a native culture filtrate of BCG. We compared the delayed-type hypersensitivity reaction elicited by L7/L12 derived from a culture filtrate of BCG and L12 derived from BCG ribosomes. L7/L12 from the culture filtrate could induce delayed-type hypersensitivity, but L12 from ribosomes could not, indicating that L7 was attributable to the induction of delayed-type hypersensitivity. The activity of L7/L12 was heat resistant. Neither glycosylation nor phosphorylation of L7/L12 from a culture filtrate could be detected. The acetylation at N-terminal of L12 was essential for the delayed-type hypersensitivity activity.

Based on our N -terminal amino acid sequence of MPT53 and a deduced DNA sequence, we searched for the corresponding gene in the Mycobacterium tuberculosis genomic sequence at the Sanger centre, localizing mpt53 close to mpt70 and mpt83. The gene was cloned and expressed, followed by purification of MPT53 to homogeneity from recombinant M. smegmatis culture fluid. In MPT53 there is 60 % identity with the active site of thioredoxin of M. tuberculosis (MPT46) with two cysteins in a CXXC motif, but MPT53 could not serve as an alternative substrate for thioredoxin reductase. Testing for IgM and IgG1 anti-MPT53 in cattle sera showed that MPT53 is immunogenic following natural and experimental infection with M. bovis. Cloning of mpt53 represents cloning of the last of the 10 proteins originally defined as "secreted proteins" of M. tuberculosis and M. bovis based on determination of their "Localization index" (LI) (J Gen Microbiol 1991;137 : 875-84). The need for a precise definition of the term "secreted protein" is discussed. So far we have observed full concordance between occurrence of an LI value indicating secretion of a protein and occurrence of a signal sequence in the corresponding gene. Signal sequence independent protein secretion in mycobacteria may occur for a limited number of proteins and remains to be established., (Copyright 1999 Academic Press.)

Mycobacterial polypeptides from 2 kDa to 14 kDa may be involved in host response to infection and be useful for new diagnostics and vaccines. Tris-tricine SDS-polyacrylamide gel electrophoresis separation of proteins in tuberculin purified protein derivative (PPD) and in 6-8-week culture filtrates of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and other Mycobacterium species demonstrated as many as 10 low molecular mass bands. Common and distinct bands were observed among different species and PPD. These low molecular mass culture filtrate proteins may represent potential diagnostic reagents and vaccines for Mycobacterium tuberculosis.

A novel DNA-binding protein expressed (8-10% in total protein) in Mycobacterium bovis bacillus Calmette-Guérin was observed. This protein was designated mycobacterial DNA-binding protein 1 (MDP1). MDP1 recognized bases, sugar moieties, phosphate-backbone on DNA and preferentially bound to DNA guanine and cytosine. In the gel retardation assay, MDP1 preferentially bound to closed circular plasmid DNA than open circular and linear form plasmid DNA and also bound to RNA. MDP1 formed a highly polymerized structure and localized not only in the nucleoid but also at the 50S ribosomal subunits and cell surface. MDP1 was conserved in Mycobacterium thus far examined and the expression was enhanced in stationary growth phases. These results will provide a reasonable basis for further study of the function of MDP1 in living mycobacteria.

Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates of Mycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

The aim of the study was examination of MPB70 protein production by different BCG substrains and testing this protein as reagent in allergenic skin test on BCG vaccinated guinea pigs. Three BCG substrains: Danish 1331 (D), Japanese 172 (J) and Polish BCG Moreau (P) were used for the study. The protein MPB70 received from dr Nagai from Osaka University was used as reference preparation. It has been shown that MPB70 protein of molecular weight about 21,500 Da was present only in BCG Moreau (P) and Japanese (J) substrains. In immunoblotting test monoclonal antibodies reacted additionally with 43,000 Da protein in all tested substrains. We suspect that the detected additional molecule was a dimer of MPB70 protein. The development of skin reactions to MPB70 protein was seen in guinea pigs vaccinated with J and P substrains. The peak of alergy to MPB70 protein was observed 9 weeks after vaccination. In sera of vaccinated animals antibodies against MPB70 protein were detected by ELISA method. The possibility of using MPB70 protein for diagnostic purpose is discussed.

Tuberculosis remains a major health problem throughout the world and the failure of the existing bacille Calmette-Guérin (BCG) vaccine in recent trials has prompted a search for potential replacements. Recent advances in molecular and cell biology have cast doubts on the ability of genetic analysis alone to predict polygenic human diseases and other complex phenotypes and have therefore redirected our attention to proteome studies to complement information obtained from DNA sequencing initiatives. Novel acidic (pH 2.3-5) and basic (pH 6-11) IPG gel gradients were employed in conjunction with commercially available pH 4-7 gradients to significantly increase (fourfold) the number of protein spots previously resolved on two-dimensional (2-D) gels of Mycobacterium species. A total of 772 and 638 protein spots were observed for M. bovis BCG and M. tuberculosis H37Rv, respectively, the latter corresponding to only the pH regions 4-7 and 6-11. Of interest was the bimodal distribution observed for proteins separated from M. bovis BCG across both M(r) and pH ranges. Some differences in protein expression were observed between these two organisms, contrary to what may have been expected considering the high degree of conservation in gene order and sequence similarity between homologous genes. Further work will be directed towards a more detailed analysis of these differences, so as to allow more accurate diagnosis between vaccination and active tuberculosis. The latter is of major importance to epidemiological studies and for patient management.

The distribution of protein antigens in purified subcellular fractions of Mycobacterium bovis bacillus Calmette-Guérin (BCG) was comparatively analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with specific monoclonal antibodies and polyclonal sera. The 19- and 38-kDa lipoproteins were mainly detected in the cell wall and cell membrane enriched fractions, and they were extracted from the former by Triton X-114 and Nonidet P-40. The 65-kDa heat-shock protein (hsp) was present in the cytoplasmic fraction and only trace amounts were found in the crude cell wall preparation. In contrast, the 14-kDa hsp was highly represented in the cell wall fraction, besides being present in cytoplasmic fraction. Both superoxide dismutase (SOD) and antigen 85 complex (Ag 85) were abundantly released in culture medium, and to a lower extent, they were present in the cell wall fraction; SOD was present in comparable amounts also in the cytoplasmic fraction, while Ag 85 was far less represented in the same. Sera from mice immunized with culture filtrate (CF) proteins of BCG recognized several antigens in CFs, which were not detectable in cell wall, cell membrane, and cytoplasmic fractions, indicating that CF proteins include secreted antigens which have not yet been identified.

The monoclonal antibody, mAb3C4, raised against sonicated Mycobacterium bovis (Mb) BCG (Tokyo strain 172) cells recognises a 23-kDa protein in the cell wall. The gene encoding this protein was cloned and sequenced and found to be 100% homologous to mpb83 and mpt83 and the putative protein to have a 76% sequence similarity to the secreted, Mb-specific protein, MPB70. MPB83 contains the amino acid (aa) sequence LAGC, which corresponds to the consensus sequence for bacterial lipoprotein modification and processing. MPB83 associated with the detergent phase when separated with Triton X-114 confirming that it is a lipoprotein. When the putative site of acylation, the Cys in the sequence LAGC, was substituted with Ser, the mutated MPB83 associated with the aqueous phase. The cloned gene was used to determine the distribution of mpb83 in various Mycobacterium species. The gene was present in the M. tuberculosis (Mt) complex organisms, as well as in M. kansasii. In addition, Southern blot analysis of Mb and Mt DNA indicated that the mpb83 and mpb70 genes are located close to each other on the genome. Western blot analysis of cell lysates of various Mycobacterium species indicated that only Mt H37Rv and H37Ra produced proteins which reacted with mAb3C4. Furthermore, only two out of six of the Mb field isolates produced detectable antigen, indicating that expression of the mpb83 gene is variable within the Mt complex organisms.

MPB64, a secretory protein of Mycobacterium bovis BCG Tokyo, was isolated from a culture filtrate of the bacteria in Sauton synthetic medium harvested on day 8. The protein was isolated by five steps; (i) concentration of the culture filtrate by cutting the molecules smaller than 5 kDa with the Millipore Pellicon Cassette system, (ii) affinity separation by a Phenyl Sepharose CL-4B column, (iii) separation with a DEAE-Sepharose CL-6B column with 3 M urea, (iv) separation with a Sephacryl S200HR column, and (v) separation with a DEAE-Sepharose column without urea. MPB64 in each fraction was determined by comparing the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with that of standard MPB64. The specificity of isolated MPB64 was tested by immunoblotting with anti-MPB64 antibody. The potency of isolated MPB64 in eliciting skin reaction in the BCG-sensitized guinea pigs was the same to that of standard MPB64. The method described herein is an improved one for isolating MPB64 from a large volume of culture filtrate of M. bovis BCG Tokyo. The technique should be applicable to isolation of other mycobacterial secretory proteins.

Mycobacterium bovis BCG vaccine strains were compared with Mycobacterium tuberculosis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 25-kDa protein observed in the BCG strains was absent in M. tuberculosis. Rabbit antibodies specific to the 25-kDa protein uniquely identified this protein in BCG strains but not in M. tuberculosis. It is suggested that the 25-kDa protein and polyclonal antibodies directed against this antigen can be exploited to distinguish BCG strains from M. tuberculosis.

Isolation of MPB83 from Mycobacterium bovis BCG Tokyo culture fluid is described. MPB70 and MPB83 have similar molecular mass as judged by SDS-PAGE but differ in isoelectric points. Peptides isolated after CNBr cleavage of MPB83 revealed extensive homology as well as distinct differences from corresponding parts of the amino acid sequence deduced from the mpb70 gene cloned by Terasaka et al. Antibodies produced by immunization with MPB70 and MPB83 had distinctly different fine specificity revealing cross-reactivity between the proteins. These findings indicate that two distinct, homologous genes code for these proteins. Sensitization with live BCG Tokyo also induced T cell responses to MPB83 with development of delayed type hypersensitivity in guinea pigs.

Correspondence between components of sensitins (avian, bovine, and johnin-PPD tuberculins) and the proteins of the corresponding microorganisms (Mycobacterium avium, Mycobacterium bovis and Mycobacterium paratuberculosis) was established. Most of these identified proteins are present in the three organisms, but the 38.3- and 48.8-kDa proteins of M. avium and the 54.0- and 58.7-kDa proteins of M. bovis are endowed with species-specific epitope(s).

Previous studies have demonstrated that mycobacteria attach to fibronectin (FN). The attachment of mycobacteria to FN is considered to be biologically important in Mycobacterium bovis BCG therapy for superficial bladder cancer, initiation of delayed hypersensitivity to mycobacterial antigens, and the phagocytosis of mycobacteria by epithelial cells. Therefore, we purified the mycobacterial receptor for FN. Culture supernatants from 3-week cultures of Mycobacterium vaccae, which contained proteins that bound FN and inhibited the attachment of both M. vaccae and BCG to FN, were used as a source of receptor. Lyophilized M. vaccae supernatants were reconstituted in 0.02 M bis-Tris (pH 6.0) and applied sequentially to an ACA 54 gel filtration column and a DEAE-Sephacel anion-exchange column. A purified inhibitory protein of 55 kDa (p55) was obtained. The purified p55 protein was observed to bind to FN and to inhibit 125I-FN binding to viable BCG in a dose-dependent manner. Polyclonal and monoclonal antibodies to the protein were generated. The resulting polyclonal antiserum blotted a single protein band at 55 kDa in crude M. vaccae supernatants, cross-reacted with a 55-kDa BCG protein by Western blot (immunoblot), and recognized a 55-kDa band that was associated with the BCG cell wall, which is consistent with its function as a FN receptor. A monoclonal immunoglobulin M(lambda) was isolated from mice immunized with purified M. vaccae p55 protein that was not functional in Western blots but inhibited the attachment of viable BCG to FN. These studies demonstrate that a protein or antigenically related proteins with M(r)s of 55,000 function as FN receptors for at least two distinct mycobacteria.