A two-step strategy for the complementation of M. tuberculosis mutants

The sequence of Mycobacterium tuberculosis, completed in 1998, facilitated both the development of genomic tools, and the creation of a number of mycobacterial mutants. These mutants have a wide range of phenotypes, from attenuated to hypervirulent strains. These phenotypes must be confirmed, to rule out possible secondary mutations that may arise during the generation of mutant strains. This may occur during the amplification of target genes or during the generation of the mutation, thus constructing a complementation strain, which expresses the wild-type copy of the gene in the mutant strain, becomes necessary. In this study we have introduced a two-step strategy to construct complementation strains using the Ag85 promoter. We have constitutively expressed dosR and have shown dosR expression is restored to wild-type level

Theoretical basis for reducing time-lines to the determination of positive Mycobacterium tuberculosis cultures using thymidylate kinase (TMK) assays

<p>Abstract</p> <p>Background</p> <p><it>In vitro </it>culture of pathogens on growth media forms a "pillar" for both infectious disease diagnosis and drug sensitivity profiling. Conventional cultures of <it>Mycobacterium tuberculosis </it>(M.<it>tb</it>) on Lowenstein Jensen (LJ) medium, however, take over two months to yield observable growth, thereby delaying diagnosis and appropriate intervention. Since DNA duplication during interphase precedes microbial division, "para-DNA synthesis assays" could be used to predict impending microbial growth. Mycobacterial thymidylate kinase (TMKmyc) is a phosphotransferase critical for the synthesis of the thymidine triphosphate precursor necessary for M.<it>tb </it>DNA synthesis. Assays based on high-affinity detection of secretory TMKmyc levels in culture using specific antibodies are considered. The aim of this study was to define algorithms for predicting positive TB cultures using antibody-based assays of TMKmyc levels <it>in vitro</it>.</p> <p>Methods and results</p> <p>Systems and chemical biology were used to derive parallel correlation of "M.<it>tb </it>growth curves" with "TMKmyc curves" theoretically in four different scenarios, showing that changes in TMKmyc levels in culture would in each case be predictive of M.<it>tb </it>growth through a simple quadratic curvature, |tmk| = at²+ bt + c, consistent with the "S" pattern of microbial growth curves. Two drug resistance profiling scenarios are offered: isoniazid (INH) resistance and sensitivity. In the INH resistance scenario, it is shown that despite the presence of optimal doses of INH in LJ to stop M.<it>tb </it>proliferation, bacilli grow and the resulting phenotypic growth changes in colonies/units are predictable through the TMKmyc assay. According to our current model, the areas under TMKmyc curves (AUC, calculated as the integral ∫(at²+ bt + c)dt or ~1/3 at³+ 1/2 bt²+ct) could directly reveal the extent of prevailing drug resistance and thereby aid decisions about the usefulness of a resisted drug in devising "salvage combinations" within resource-limited settings, where second line TB chemotherapy options are limited.</p> <p>Conclusion</p> <p>TMKmyc assays may be useful for reducing the time-lines to positive identification of <it>Mycobacterium tuberculosis </it>(M.<it>tb</it>) cultures, thereby accelerating disease diagnosis and drug resistance profiling. Incorporating "chemiluminiscent or fluorescent" strategies may enable "photo-detection of TMKmyc changes" and hence automation of the entire assay.</p

The Secreted Lipoprotein, MPT83, of Mycobacterium tuberculosis Is Recognized during Human Tuberculosis and Stimulates Protective Immunity in Mice

The long-term control of tuberculosis (TB) will require the development of more effective anti-TB vaccines, as the only licensed vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG), has limited protective efficacy against infectious pulmonary TB. Subunit vaccines have an improved safety profile over live, attenuated vaccines, such as BCG, and may be used in immuno-compromised individuals. MPT83 (Rv2873) is a secreted mycobacterial lipoprotein expressed on the surface of Mycobacterium tuberculosis. In this study, we examined whether recombinant MPT83 is recognized during human and murine M. tuberculosis infection. We assessed the immunogenicity and protective efficacy of MPT83 as a protein vaccine, with monophosphyl lipid A (MPLA) in dimethyl-dioctadecyl ammonium bromide (DDA) as adjuvant, or as a DNA vaccine in C57BL/6 mice and mapped the T cell epitopes with peptide scanning. We demonstrated that rMPT83 was recognised by strong proliferative and Interferon (IFN)-γ-secreting T cell responses in peripheral blood mononuclear cells (PBMC) from patients with active TB, but not from healthy, tuberculin skin test-negative control subjects. MPT83 also stimulated strong IFN-γ T cell responses during experimental murine M. tuberculosis infection. Immunization with either rMPT83 in MPLA/DDA or DNA-MPT83 stimulated antigen-specific T cell responses, and we identified MPT83127–135 (PTNAAFDKL) as the dominant H-2b-restricted CD8+ T cell epitope within MPT83. Further, immunization of C57BL/6 mice with rMPT83/MPLA/DDA or DNA-MPT83 stimulated significant levels of protection in the lungs and spleens against aerosol challenge with M. tuberculosis. Interestingly, immunization with rMPT83 in MPLA/DDA primed for stronger IFN-γ T cell responses to the whole protein following challenge, while DNA-MPT83 primed for stronger CD8+ T cell responses to MPT83127–135. Therefore MPT83 is a protective T cell antigen commonly recognized during human M. tuberculosis infection and should be considered for inclusion in future TB subunit vaccines

Differential expression of mycobacterial antigen MPT64, apoptosis and inflammatory markers in multinucleated giant cells and epithelioid cells in granulomas caused by Mycobacterium tuberculosis

The development of granulomas is a major histopathological feature of tuberculosis. Very little information is available concerning the physiology and functions of different cell types in the tuberculous granulomas. The aim of this study was to compare the epithelioid cells (ECs) and multinucleated giant cells (MGCs) in the granulomas caused by Mycobacterium tuberculosis complex organisms. Lymph node biopsies from 30 cases of lymphadenitis were studied for expression of the secreted mycobacterial protein MPT64, caspase 3 as a marker of apoptosis, apoptosis-related proteins (Fas Ligand, Fas and Bax) and inflammatory cytokines (interleukin-10, transforming growth factor-β (TGF-β), tumour necrosis factor-α and interferon-γ) by immunohistochemistry. MGCs more often contained M. tuberculosis secretory antigen MPT64 (p < 0.001) and expressed more TGF-β (p = 0.004) than ECs. The total number of apoptotic MGCs was higher than the number of apoptotic ECs (p = 0.04). Interestingly, there was a significant negative correlation between apoptosis and MPT64 expression in MGCs (r = −0.569, p = 0.003), but not in ECs, implying that the heavy antigen load would lead to inhibition of apoptosis in these cells. When compared with ECs, higher percentage of MGCs expressed Fas Ligand and Fas (p < 0.004). The role of MGCs may thus be different from surrounding ECs and these cells by virtue of higher mycobacterial antigen load, more TGF-β and reduced apoptosis may contribute towards persistence of infection

rBCG Induces Strong Antigen-Specific T Cell Responses in Rhesus Macaques in a Prime-Boost Setting with an Adenovirus 35 Tuberculosis Vaccine Vector

BACKGROUND: BCG vaccination, combined with adenoviral-delivered boosts, represents a reasonable strategy to augment, broaden and prolong immune protection against tuberculosis (TB). We tested BCG (SSI1331) (in 6 animals, delivered intradermally) and a recombinant (rBCG) AFRO-1 expressing perfringolysin (in 6 animals) followed by two boosts (delivered intramuscullary) with non-replicating adenovirus 35 (rAd35) expressing a fusion protein composed of Ag85A, Ag85B and TB10.4, for the capacity to induce antigen-specific cellular immune responses in rhesus macaques (Macaca mulatta). Control animals received diluent (3 animals). METHODS AND FINDINGS: Cellular immune responses were analyzed longitudinally (12 blood draws for each animal) using intracellular cytokine staining (TNF-alpha, IL-2 and IFN-gamma), T cell proliferation was measured in CD4(+), CD8alpha/beta(+), and CD8alpha/alpha(+) T cell subsets and IFN-gamma production was tested in 7 day PBMC cultures (whole blood cell assay, WBA) using Ag85A, Ag85B, TB10.4 recombinant proteins, PPD or BCG as stimuli. Animals primed with AFRO-1 showed i) increased Ag85B-specific IFN-gamma production in the WBA assay (median >400 pg/ml for 6 animals) one week after the first boost with adenoviral-delivered TB-antigens as compared to animals primed with BCG (<200 pg/ml), ii) stronger T cell proliferation in the CD8alpha/alpha(+) T cell subset (proliferative index 17%) as compared to BCG-primed animals (proliferative index 5% in CD8alpha/alpha(+) T cells). Polyfunctional T cells, defined by IFN-gamma, TNF-alpha and IL-2 production were detected in 2/6 animals primed with AFRO-1 directed against Ag85A/b and TB10.4; 4/6 animals primed with BCG showed a Ag85A/b responses, yet only a single animal exhibited Ag85A/b and TB10.4 reactivity. CONCLUSION: AFRO-1 induces qualitatively and quantitatively different cellular immune responses as compared with BCG in rhesus macaques. Increased IFN-gamma-responses and antigen-specific T cell proliferation in the CD8alpha/alpha+ T cell subset represents a valuable marker for vaccine-take in BCG-based TB vaccine trials

Community-based cross-sectional survey of latent tuberculosis infection in Afar pastoralists, Ethiopia, using QuantiFERON-TB Gold In-Tube and tuberculin skin test

<p>Abstract</p> <p>Background</p> <p>There is little information concerning community-based prevalence of latent tuberculosis infection (LTBI) using T-cell based interferon-γ (IFN-γ) release assays (IGRAs), particularly in TB endemic settings. In this study, the prevalence of LTBI in the Afar pastoral community was assessed using QuantiFERON-TB Gold In-Tube (QFTGIT) and tuberculin skin tests (TST).</p> <p>Methods</p> <p>A community-based cross-sectional survey of LTBI involving 652 apparently healthy adult pastoralists was undertaken in the pastoral community of Amibara District of the Afar Region between April and June 2010.</p> <p>Results</p> <p>The prevalence of LTBI was estimated as 63.7% (363/570) using QFTGIT at the cut-off point recommended by the manufacturer (≥ 0.35 IU/ml IFN-γ), while it was 74.9% (427/570) using a cut-off point ≥ 0.1 IU/ml IFN-γ. The QFTGIT-based prevalence of LTBI was not significantly associated with the gender or age of the study participants. However, the prevalence of LTBI was 31.2% (183/587) using TST at a cut-off point ≥ 10 mm of skin indurations, and it was higher in males than females (36.8% vs. 23.5%, X²= 11.76; p < 0.001). There was poor agreement between the results of the tests (k = 0.098, 95% CI, 0.08 - 0.13). However, there was a positive trend between QFTGIT and TST positivity (X²= 96.76, P < 0.001). Furthermore, individuals with skin indurations ≥ 10 mm were 13.6 times more likely to have positive results using QFTGIT than individuals with skin indurations of 0 mm (adjusted OR = 13.6; 95%CI, 7.5 to 24.7, p < 0.001).</p> <p>Conclusions</p> <p>There is currently no agreed gold standard for diagnosis of LTBI. However, the higher prevalence of LTBI detected using QFTGIT rather than TST suggests that QFTGIT could be used for epidemiological studies concerning LTBI at the community level, even in a population unreactive to TST. Further studies of adults and children will be required to assess the effects of factors such as malnutrition, non-tuberculosis mycobacterial infections, HIV and parasitic infections on the performance of QFTGIT.</p

T-cell and serological responses to Erp, an exported Mycobacterium tuberculosis protein, in tuberculosis patients and healthy individuals

<p>Abstract</p> <p>Background</p> <p>The identification of antigens able to differentiate tuberculosis (TB) disease from TB infection would be valuable. Cellular and humoral immune responses to Erp (Exported repetitive protein) – a recently identified <it>M. tuberculosis </it>protein – have not yet been investigated in humans and may contribute to this aim.</p> <p>Methods</p> <p>We analyzed the cellular and humoral immune responses to Erp, ESAT-6, Ag85B and PPD in TB patients, in BCG⁺individuals without infection, BCG⁺individuals with latent TB infection (LTBI) and BCG^-controls. We used lymphoproliferation, ELISpot IFN-γ, cytokine production assays and detection of specific human antibodies against recombinant <it>M. tuberculosis </it>proteins.</p> <p>Results</p> <p>We included 22 TB patients, 9 BCG⁺individuals without TB infection, 7 LTBI and 7 BCG^-controls. Erp-specific T cell counts were higher in LTBI than in the other groups. Erp-specific T cell counts were higher in LTBI subjects than TB patients (median positive frequency of 211 SFC/10⁶PBMC (range 118–2000) for LTBI subjects compared to 80 SFC/10⁶PBMC (range 50–191), p = 0.019); responses to PPD and ESAT-6 antigens did not differ between these groups. IFN-γ secretion after Erp stimulation differed between TB patients and LTBI subjects (p = 0.02). Moreover, LTBI subjects but not TB patients or healthy subjects produced IgG3 against Erp.</p> <p>Conclusion</p> <p>The frequencies of IFN-γ-producing specific T cells, the IFN-γ secretion and the production of IgG3 after Erp stimulation are higher in LTBI subjects than in TB patients, whereas PPD and ESAT-6 are not.</p

Systematic Genetic Nomenclature for Type VII Secretion Systems

CITATION: Bitter, W., et al. 2009. Systematic genetic nomenclature for type VII secretion systems. PLoS Pathogens, 5(10): 1-6, doi: 10.1371/journal.ppat.1000507.The original publication is available at http://journals.plos.org/plospathogensMycobacteria, such as the etiological agent of human tuberculosis, Mycobacterium tuberculosis, are protected by an impermeable cell envelope composed of an inner cytoplasmic membrane, a peptidoglycan layer, an arabinogalactan layer, and an outer membrane. This second membrane consists of covalently linked, tightly packed long-chain mycolic acids [1,2] and noncovalently bound shorter lipids involved in pathogenicity [3–5]. To ensure protein transport across this complex cell envelope, mycobacteria use various secretion pathways, such as the SecA1-mediated general secretory pathway [6,7], an alternative SecA2-operated pathway [8], a twin-arginine translocation system [9,10], and a specialized secretion pathway variously named ESAT-6-, SNM-, ESX-, or type VII secretion [11–16]. The latter pathway, hereafter referred to as type VII secretion (T7S), has recently become a large and competitive research topic that is closely linked to studies of host–pathogen interactions of M. tuberculosis [17] and other pathogenic mycobacteria [16]. Molecular details are just beginning to be revealed [18–22] showing that T7S systems are complex machineries with multiple components and multiple substrates. Despite their biological importance, there has been a lack of a clear naming policy for the components and substrates of these systems. As there are multiple paralogous T7S systems within the Mycobacteria and orthologous systems in related bacteria, we are concerned that, without a unified nomenclature system, a multitude of redundant and obscure gene names will be used that will inevitably lead to confusion and hinder future progress. In this opinion piece we will therefore propose and introduce a systematic nomenclature with guidelines for name selection of new components that will greatly facilitate communication and understanding in this rapidly developing field of research.http://journals.plos.org/plospathogens/article?id=10.1371%2Fjournal.ppat.1000507Publisher's versio

Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase.SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host-adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development