Lipoproteins of Mycobacterium tuberculosis : an abundant and functionally diverse class of cell envelope components

Mycobacterium tuberculosis remains the predominant bacterial scourge of mankind. Understanding of its biology and pathogenicity has been greatly advanced by the determination of whole genome sequences for this organism. Bacterial lipoproteins are a functionally diverse class of membrane-anchored proteins. The signal peptides of these proteins direct their export and post-translational lipid modification. These signal peptides are amenable to bioinformatic analysis, allowing the lipoproteins encoded in whole genomes to be catalogued. This review applies bioinformatic methods to the identification and functional characterisation of the lipoproteins encoded in the M. tuberculosis genomes. Ninety nine putative lipoproteins were identified and so this family of proteins represents ca. 2.5% of the M. tuberculosis predicted proteome. Thus, lipoproteins represent an important class of cell envelope proteins that may contribute to the virulence of this major pathogen

Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. α-antigen promoter (in plasmid pJH222). Among 14 rBCG:HIV-1gp120 (pMV261) colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222) colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors

Screening vaccine formulations for biological activity using fresh human whole blood.

Understanding the relevant biological activity of any pharmaceutical formulation destined for human use is crucial. For vaccine-based formulations, activity must reflect the expected immune response, while for non-vaccine therapeutic agents, such as monoclonal antibodies, a lack of immune response to the formulation is desired. During early formulation development, various biochemical and biophysical characteristics can be monitored in a high-throughput screening (HTS) format. However, it remains impractical and arguably unethical to screen samples in this way for immunological functionality in animal models. Furthermore, data for immunological functionality lag formulation design by months, making it cumbersome to relate back to formulations in real-time. It is also likely that animal testing may not accurately reflect the response in humans. For a more effective formulation screen, a human whole blood (hWB) approach can be used to assess immunological functionality. The functional activity relates directly to the human immune response to a complete formulation (adjuvant/antigen) and includes adjuvant response, antigen response, adjuvant-modulated antigen response, stability, and potentially safety. The following commentary discusses the hWB approach as a valuable new tool to de-risk manufacture, formulation design, and clinical progression

Natural variation in immune responses to neonatal mycobacterium bovis bacillus calmette-guerin (BCG) vaccination in a cohort of Gambian infants

Background There is a need for new vaccines for tuberculosis (TB) that protect against adult pulmonary disease in regions where BCG is not effective. However, BCG could remain integral to TB control programmes because neonatal BCG protects against disseminated forms of childhood TB and many new vaccines rely on BCG to prime immunity or are recombinant strains of BCG. Interferon-gamma (IFN-) is required for immunity to mycobacteria and used as a marker of immunity when new vaccines are tested. Although BCG is widely given to neonates IFN- responses to BCG in this age group are poorly described. Characterisation of IFN- responses to BCG is required for interpretation of vaccine immunogenicity study data where BCG is part of the vaccination strategy. Methodology/Principal Findings 236 healthy Gambian babies were vaccinated with M. bovis BCG at birth. IFN-, interleukin (IL)-5 and IL-13 responses to purified protein derivative (PPD), killed Mycobacterium tuberculosis (KMTB), M. tuberculosis short term culture filtrate (STCF) and M. bovis BCG antigen 85 complex (Ag85) were measured in a whole blood assay two months after vaccination. Cytokine responses varied up to 10 log-fold within this population. The majority of infants (89-98% depending on the antigen) made IFN- responses and there was significant correlation between IFN- responses to the different mycobacterial antigens (Spearman’s coefficient ranged from 0.340 to 0.675, p=10-6-10-22). IL-13 and IL-5 responses were generally low and there were more non-responders (33-75%) for these cytokines. Nonetheless, significant correlations were observed for IL-13 and IL-5 responses to different mycobacterial antigens Conclusions/Significance Cytokine responses to mycobacterial antigens in BCG-vaccinated infants are heterogeneous and there is significant inter-individual variation. Further studies in large populations of infants are required to identify the factors that determine variation in IFN- responses

Identification and characterization of "Plasmodium falciparum" and "Mycobacterium ulcerans" antigens as potential vaccine components and targets for serological test and molecular typing methods

The increase of Plasmodium falciparum’s drug resistance and the resurgence of Mycobacterium ulcerans disease through environmental changes aggravate health problems caused by these pathogens. Buruli ulcer, caused by M. ulcerans, is after tuberculosis and leprosy the third most common mycobacterial human infection and the most poorly understood of these three diseases. It is characterized by chronic, necrotizing ulceration of subcutaneous tissues and the overlying skin. M. ulcerans is a slow-growing mycobacterium which multiplies extracellularly in Buruli ulcer lesions. There is a broad antigenic overlap between mycobacterial species, which complicates the analysis of adaptive immune responses and hampers the development of specific sero-diagnostic tests for M. ulcerans in areas where BCG vaccination has been implemented and tuberculosis is endemic. In an effort to identify immunodominant antigens of M. ulcerans, we have generated panels of monoclonal antibodies from mice immunized with this pathogen. Cross-reactivity studies with other mycobacterial species performed by Western blot and immunofluorescence assays have identified immunodominant epitopes with a limited cross-species distribution (18kDa and the 34-37kDa proteins). In contrast, the majority of antigens were spread widely amongst different mycobacterial species. One set of non-crossreactive monoclonal antibodies recognized an 18kDa protein of M. ulcerans that is associated with the cell-wall fraction, and expressed in Buruli ulcer lesions. The target protein was identified by massspectroscopy as the M. ulcerans orthologue of the M. leprae 18kDa small heat shock protein, which has no orthologues in the genomes of M. bovis and M. tuberculosis. Human anti-18kDa small heat shock protein antibodies were found in the serum of all Buruli ulcer patients tested, but not in sera from Europeans volunteers and only rarely in sera from Africans living in Buruli ulcer non-endemic regions. Reactivity of sera from a large proportion of people living in a Buruli ulcer endemic area and in contact with Buruli ulcer patients indicated that an 18kDa small heat shock protein-based serological test is suitable to detect exposure to M. ulcerans. Since M. ulcerans shows only very limited genetic diversity, standard multi-locus sequence typing of housekeeping genes is not a suitable tool for molecular epidemiological analysis of Buruli ulcer. Among the monoclonal antibodies exhibiting broad inter-species cross-reactivity, one group recognized the M. ulcerans orthologue of mycobacterial laminin-binding protein. DNA sequence analysis demonstrated that the corresponding hupB gene from M. ulcerans isolates of diverse geographical origin exhibited considerable diversity based both on insertional/deletional polymorphism and on single base exchanges. Dominance of non-conservative exchanges was indicative of a diversifying selection pressure. Sequences analysis of a set of such variable genes may develop into a new tool for genetic fingerprinting of isolates. There is great need to identify new malaria vaccine and drug targets. Monoclonal antibodies were used to characterize a novel conserved protein of P. falciparum designated D13. Western blot analysis demonstrated that D13 is stage-specifically expressed during schizogony in asexual blood stages of the parasite. It has a functionally essential role in parasite biology, since anti-D13 monoclonal antibodies have parasite growth inhibitory activity. The D13 protein may represent a suitable target for a malaria vaccine design. Immunofluorescence analysis with monoclonal antibodies specific for glyceraldehydes-3- phosphate dehydrogenase (pfGAPDH) and pfAldolase showed that pfGAPDH and pfAldolase colocalise in early stages of both liver and asexual blood stage parasite development. However, during schizogony, unlike pfAldolase, pfGAPDH was enriched in the apical region of the parasites. In addition, Western blot analyses demonstrate that pfGAPDH is in both the membrane-containing pellet and supernatant fractions. These results have provided evidence that pfGAPDH exerts non-glycolytic function(s) in P. falciparum; including possibly a role in vesicular transport and biogenesis of apical organelles. This data together with the limited amino acid sequence identity with human GAPDH suggest that the pfGAPDH could be a promising safe target for drug treatment

The Mycobacterium tuberculosis KatG gene : identification of a novel function and analysis of the regulation of expression

A clone containing the terminal third of the Mycobacterium tuberculosis katG gene was previously shown to confer resistance to ethyl methane sulfonate on DNA repair-deficient Escherichia coli cells. The first aim of this study, therefore, was to examine the role played by the M tuberculosis katG gene in DNA repair. The strategy used was overexpression of different regions of the gene in DNA repair-deficient mutants of E. coli, and examination of the sensitivities of the transformants to DNA damaging agents. Overexpression of the gene resulted in an increase in the survival of recA mutants exposed to ultraviolet (UV) light irradiation (254 run) and hydrogen peroxide, and uvr mutants exposed to mitomycin C. Both the 5' and 3' regions of the M tuberculosis KatG protein conferred the above effects, and this was independent of the catalase or peroxidase activity of the enzyme. The results suggest that the M tuberculosis katG gene may encode a novel function related to the repair of DNA damage, and this may have implications for the survival of M tuberculosis in the presence of DNA damaging agents, for example, in the macrophage. UV sensitivity tests on M intracellulare and M tuberculosis strains mutant in katG revealed that the katG gene product does not play a demonstrable role in the survival of repair-competent mycobacterial cells after exposure to UV irradiation. The second aim of this study was to examine the regulation of expression of the M tuberculosis katG gene. An E. coli-mycobacterial shuttle vector, pJCluc, containing the luciferase reporter gene, was constructed and used to examine the katG promoter sequences. The region required for optimal expression in M. smegmatis was localized to a 559 hp fragment immediately upstream of the gene. Two transcription start sites were mapped and putative -10 and -35 promoter sequences identified. It was demonstrated that expression from the promoter peaks during late exponential phase, and declines during stationary phase, and that the promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. An upstream element that increased expression from the M. tuberculosis katG and the M. paratuberculosis PAN promoters was identified, and shown to bind to one or more M smegma/is proteins. Similar results were obtained in M bovis BCG. Understanding the regulation of gene expression in mycobacteria is essential for determining the processes that govern interaction with the host. This study provides information on both the mycobacterial transcription signals and gene regulatory mechanisms

Molecular biology and immunology of the 60-kDa stress protein (65-kDa antigen) of Mycobacterium paratuberculosis

M. paratuberculosis infection in sheep causes a chronic granulomatous enteritis, characterised by a massive cellular infiltration of macrophages, epithelioid cells and lymphocytes. Like other mycobacterial infections the predominant immune response is cell-mediated and this response centres on a complex interaction between T cells and macrophages infected with M. paratuberculosis organisms. T cells have been identified as the predominant inducers of the cell mediated immune response to mycobacteria. Stress proteins have been shown to be immunodominant antigens in immune responses to other pathogenic mycobacteria. T cells reactive to stress proteins have been isolated from infected animals. For these reasons, the 60-kDa stress protein of M. paratuberculosis was chosen for the investigation of the immune response to M. paratuberculosis infection in sheep.In this thesis the DNA encoding the 60-kDa stress protein of M. paratuberculosis was amplified by PCR, sequenced and the ORF of this protein expressed as a fusion protein with glutathione S-transferase. Recombinant M. paratuberculosis 60-kDa stress protein was obtained by cleavage of the fusion protein with the proteolytic enzyme thrombin.Recombinant M. paratuberculosis 60-kDa stress protein was used to generate polyclonal T and B cell responses in sheep which were assessed by in vitro proliferation and enzyme-linked immunosorbent assays, respectively. In addition, the recombinant protein was used to generate a monoclonal antibody. The assays and reagents generated in this thesis were subsequently used to investigate the immune response to M. paratuberculosis 60-kDa stress protein in ovine paratuberculosis.This investigation provides preliminary evidence that paratuberculosis-infected animals have T cells that recognise and are capable of responding by proliferation to M. paratuberculosis 60-kDa stress protein in vitro, and that antibodies to this protein could be detected in sera from these animals. Furthermore, M. paratuberculosis 60-kDa stress protein was detected in tissues from infected animals by immunocytochemical analysis. These preliminary findings support a role for M. paratuberculosis 60-kDa stress protein in the ovine immune response to M. paratuberculosis infection