Characterization of two in vivo-expressesd methyltransferases of the Mycobacterium tuberculosis complex:Antigenicity and genetic regulation

Genome sequencing of Mycobacterium tuberculosis complex members has accelerated the search for new disease-control tools. Antigen mining is one area that has benefited enormously from access to genome data. As part of an ongoing antigen mining programme, we screened genes that were previously identified by transcriptome analysis as upregulated in response to an in vitro acid shock for their in vivo expression profile and antigenicity. We show that the genes encoding two methyltransferases, Mb1438c/Rv1403c and Mb1440c/Rv1404c, were highly upregulated in a mouse model of infection, and were antigenic in M. bovis-infected cattle. As the genes encoding these antigens were highly upregulated in vivo, we sought to define their genetic regulation. A mutant was constructed that was deleted for their putative regulator, Mb1439/Rv1404; loss of the regulator led to increased expression of the flanking methyltransferases and a defined set of distal genes. This work has therefore generated both applied and fundamental outputs, with the description of novel mycobacterial antigens that can now be moved into field trials, but also with the description of a regulatory network that is responsive to both in vivo and in vitro stimuli

Human and mouse essentiality screens as a resource for disease gene discovery

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery. Discovery of causal variants for monogenic disorders has been facilitated by whole exome and genome sequencing, but does not provide a diagnosis for all patients. Here, the authors propose a Full Spectrum of Intolerance to Loss-of-Function (FUSIL) categorization that integrates gene essentiality information to aid disease gene discovery

Revisiting host preference in the Mycobacterium tuberculosis complex:Experimental infection shows M. tuberculosis H37Rv to be avirulent in cattle

Experiments in the late 19th century sought to define the host specificity of the causative agents of tuberculosis in mammals. Mycobacterium tuberculosis, the human tubercle bacillus, was independently shown by Smith, Koch, and von Behring to be avirulent in cattle. This finding was erroneously used by Koch to argue the converse, namely that Mycobacterium bovis, the agent of bovine tuberculosis, was avirulent for man, a view that was subsequently discredited. However, reports in the literature of M. tuberculosis isolation from cattle with tuberculoid lesions suggests that the virulence of M. tuberculosis for cattle needs to be readdressed. We used an experimental bovine infection model to test the virulence of well-characterized strains of M. tuberculosis and M. bovis in cattle, choosing the genome-sequenced strains M. tuberculosis H37Rv and M. bovis 2122/97. Cattle were infected with approximately 10(6) CFU of M. tuberculosis H37Rv or M. bovis 2122/97, and sacrificed 17 weeks post-infection. IFN-γ and tuberculin skin tests indicated that both M. bovis 2122 and M. tuberculosis H37Rv were equally infective and triggered strong cell-mediated immune responses, albeit with some indication of differential antigen-specific responses. Postmortem examination revealed that while M. bovis 2122/97–infected animals all showed clear pathology indicative of bovine tuberculosis, the M. tuberculosis–infected animals showed no pathology. Culturing of infected tissues revealed that M. tuberculosis was able to persist in the majority of animals, albeit at relatively low bacillary loads. In revisiting the early work on host preference across the M. tuberculosis complex, we have shown M. tuberculosis H37Rv is avirulent for cattle, and propose that the immune status of the animal, or genotype of the infecting bacillus, may have significant bearing on the virulence of a strain for cattle. This work will serve as a baseline for future studies into the genetic basis of host preference, and in particular the molecular basis of virulence in M. bovis

Cell Envelope Protein PPE68 Contributes to Mycobacterium tuberculosis RD1 Immunogenicity Independently of a 10-Kilodalton Culture Filtrate Protein and ESAT-6

The protective efficacy of Mycobacterium bovis BCG can be markedly augmented by stable integration of Mycobacterium tuberculosis genomic region RD1. BCG complemented with RD1 (BCG::RD1) encodes nine additional proteins. Among them, 10-kDa culture filtrate protein (CFP-10) and ESAT-6 (6-kDa early secreted antigenic target) are low-molecular-weight proteins that induce potent Th1 responses. Using pools of synthetic peptides, we have examined the potential immunogenicity of four other RD1 products (PE35, PPE68, Rv3878, and Rv3879c). PPE68, the protein encoded by rv3873, was the only one to elicit gamma interferon (IFN-γ)-producing cells in C57BL/6 mice infected with M. tuberculosis. Anti-PPE68 T cells were predominantly raised against an epitope mapped in the N-terminal end of the protein. Importantly, inactivation of rv3873 in BCG::RD1 did not modify CFP-10 and ESAT-6 secretion. Moreover, the generation of IFN-γ responses to these antigens following immunization with BCG::RD1 was independent of PPE68 expression. Taken together, these results show that PPE68 is an immunogenic product of the RD1 region, which does not interfere with the secretion and immunogenicity of CFP-10 and ESAT-6

Correlation of ESAT-6-specific gamma interferon production with pathology in cattle following Mycobacterium bovis BCG vaccination against experimental bovine tuberculosis

Vaccine development and the understanding of the pathology of bovine tuberculosis in cattle would be greatly facilitated by the definition of immunological correlates of protection and/or pathology. To address these questions, cattle were vaccinated with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and were then challenged with virulent M. bovis. Applying a semiquantitative pathology-scoring system, we were able to demonstrate that BCG vaccination imparted significant protection by reducing the disease severity on average by 75%. Analysis of cellular immune responses following M. bovis challenge demonstrated that proliferative T-cell and gamma interferon (IFN-γ) responses towards the M. bovis-specific antigen ESAT-6, whose gene is absent from BCG, were generally low in vaccinated animals but were high in all nonvaccinated calves. Importantly, the amount of ESAT-6-specific IFN-γ measured by enzyme-linked immunosorbent assay after M. bovis challenge, but not the frequency of responding cells, correlated positively with the degree of pathology found 18 weeks after infection. Diagnostic reagents based on antigens not present in BCG, like ESAT-6 and CFP-10, were still able to distinguish BCG-vaccinated, diseased animals from BCG-vaccinated animals without signs of disease. In summary, our results suggest that the determination of ESAT-6-specific IFN-γ, while not a direct correlate of protection, constitutes nevertheless a useful prognostic immunological marker predicting both vaccine efficacy and disease severity

Minimum infective dose of Mycobacterium bovis in cattle

The aim of this work was to determine the minimum infective dose of Mycobacterium bovis necessary to stimulate specific immune responses and generate pathology in cattle. Four groups of calves (20 animals) were infected by the intratracheal route with 1,000, 100, 10, or 1 CFU of M. bovis. Specific immune responses (gamma interferon [IFN-γ] and interleukin-4 [IL-4] responses) to mycobacterial antigens were monitored throughout the study, and the responses to the tuberculin skin test were assessed at two times. Rigorous post mortem examinations were performed to determine the presence of pathology, and samples were taken for microbiological and histopathological confirmation of M. bovis infection. One-half of the animals infected with 1 CFU of M. bovis developed pulmonary pathology typical of bovine tuberculosis. No differences in the severity of pathology were observed for the different M. bovis doses. All animals that developed pathology were skin test positive and produced specific IFN-γ and IL-4 responses. No differences in the sizes of the skin test reactions, the times taken to achieve a positive IFN-γ result, or the levels of the IFN-γ and IL-4 responses were observed for the different M. bovis doses, suggesting that diagnostic assays (tuberculin skin test and IFN-γ test) can detect cattle soon after M. bovis infection regardless of the dose. This information should be useful in modeling the dynamics of bovine tuberculosis in cattle and in assessing the risk of transmission

<i>M. bovis</i> and <i>M. tuberculosis</i> infection induces comparable bovine cellular immune responses.

<p>Panels A and B; Blood was collected at regulated intervals from cattle following experimental infection with either <i>M. bovis</i> (n = 5) or <i>M. tuberculosis</i> (n = 5). PBMCs were isolated and stimulated with bovine-PPD (panel A) or a cocktail of peptides derived from ESAT-6 and CFP-10 (Panel B). The number of antigen-specific cells expressing IFN-γ was measured using an ELISPOT assay. Data for each time point is presented as the mean response ± SEM with the no-antigen response subtracted. The response in the <i>M. tuberculosis</i> infected cattle is shown as open circles with a dotted line, and for the <i>M. bovis</i> infected cattle as closed triangles with a solid line. Panel C; A tuberculin skin-test was performed at week 16 post-infection. The increase in skin induration was measured 72 hr after administration of bovine (black bars) or avian (grey bars) PPD and is presented as the mean ± SEM.</p

Enhancement of the sensitivity of the whole-blood gamma interferon assay for diagnosis of Mycobacterium bovis infections in cattle

In this study, we determined if the sensitivity of the currently available in vitro test to detect bovine tuberculosis could be enhanced by adding the following immunomodulators: interleukin-2 (IL-2); granulocyte-macrophage colony-stimulating factor (GM-CSF); antibodies neutralizing IL-10 and transforming growth factor β (TGF-β); mono-methyl-l-arginine, which blocks nitric oxide production; and l-methyl-tryptophan, which interferes with the indoleamine dioxygenase pathway. Blood was obtained from uninfected control cattle, experimentally infected cattle, cattle responding positively to the skin test in tuberculosis-free areas (false positives), and cattle naturally infected with Mycobacterium bovis from New Zealand and Great Britain. Gamma interferon (IFN-γ) responses to bovine purified protein derivative (PPD-b), avian purified protein derivative, and a fusion protein of ESAT-6 and CFP-10 were measured. Mono-methyl-l-arginine, l-methyl-tryptophan, or an antibody neutralizing TGF-β had minimal impact on IFN-γ production. IL-2 and GM-CSF promoted IFN-γ release whether antigen was present or not. In contrast, adding an antibody against IL-10 enhanced only antigen-specific responses. In particular, addition of anti-IL-10 to ESAT-6/CFP-10-stimulated blood cultures enhanced the test sensitivity. Furthermore, whole blood cells from field reactors produced substantial amounts of IL-10 upon stimulation with PPD-b or ESAT-6/CFP-10. Testing “false-positive” cattle from tuberculosis-free areas of New Zealand revealed that addition of anti-IL-10 did not compromise the test specificity. Therefore, the use of ESAT-6/CFP-10 with anti-IL-10 could be useful to detect cattle potentially infected with tuberculosis, which are not detected using current procedures

With the exception of animals 2757, post mortems were performed 16 weeks post infection.

<p>Post mortem for 2757 was performed at week 6 post infection for reasons of animal welfare. The number of individual lung lobes and respiratory tract associated lymph nodes with TB lesions is presented. The pathology score is based on a semi-quantitative assignment of disease severity within affected tissues as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008527#s2" target="_blank">material and methods</a>. Statistical difference between groups was determined using the Fisher Exact test (** p<0.01). Bacteriology score is based on the subtotal of a semi-quantitative ranking of total CFU for each individual tissue homogenate as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008527#s2" target="_blank">material and methods</a>. Statistical difference between groups was determined using the Mann-Whitney test (** p<0.01).</p