ClgR regulation of chaperone and protease systems is essential for Mycobacterium tuberculosis parasitism of the macrophage

Chaperone and protease systems play essential roles in cellular homeostasis and have vital functions in controlling the abundance of specific cellular proteins involved in processes such as transcription, replication, metabolism and virulence. Bacteria have evolved accurate regulatory systems to control the expression and function of chaperones and potentially destructive proteases. Here, we have used a combination of transcriptomics, proteomics and targeted mutagenesis to reveal that the clp gene regulator (ClgR) of Mycobacterium tuberculosis activates the transcription of at least ten genes, including four that encode protease systems (ClpP1/C, ClpP2/C, PtrB and HtrA-like protease Rv1043c) and three that encode chaperones (Acr2, ClpB and the chaperonin Rv3269). Thus, M. tuberculosis ClgR controls a larger network of protein homeostatic and regulatory systems than ClgR in any other bacterium studied to date. We demonstrate that ClgR-regulated transcriptional activation of these systems is essential for M. tuberculosis to replicate in macrophages. Furthermore, we observe that this defect is manifest early in infection, as M. tuberculosis lacking ClgR is deficient in the ability to control phagosome pH 1 h post-phagocytosis

Demonstration of In vivo expression of a hypothetical open reading frame (ORF-14) encoded by the RD1 region of Mycobacterium tuberculosis

Previously we identified a novel antigenic open reading frame (ORF), designated as ORF-14, on the RD1 region of Mycobacterium tuberculosis that was not originally predicted by Mahairas or by annotation of the M. tuberculosis H37 Rv genome. Here we show that anti-ORF-14 antibodies either from mice immunized with recombinant ORF-14 protein or isolated from serum samples from tuberculosis patients, react with a protein in culture filtrate but not in cytoplasmic or cell wall fractions from M. tuberculosis. Our data indicate that the ORF-14 protein is expressed as a secreted protein, representing one more secreted protein antigen not previously identified by genomics

Identification of a novel protein antigen encoded by a Mycobacterium tuberculosis-specific RD1 region gene

A genomic DNA region, designated RD1, that is present in virulent and clinical strains of Mycobacterium tuberculosis and M. bovis, has been shown to be deleted in bacillus Calmette Guerin (BCG). The DNA segments corresponding to three open reading frames (ORFs: ORF-10, ORF-14 and ORF-15) of the RD1 region, that are deleted in BCG strains, were amplified from M. tuberculosis genomic DNA by polymerase chain reaction (PCR), subcloned into pGEX-4T vector system and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST). The recombinant proteins appeared as major cellular proteins in SDS-PAGE gels at the expected molecular mass. The identity of each fusion protein was confirmed by reactivity with anti-GST antibodies in Western immunoblots. When pooled human sera from 11 tuberculosis (TB) patients were used as the source of antibodies, only GST- ORF-14 fusion protein reacted in Western immunoblots. The protein corresponding to ORF-14 was then purified to near homogeneity and isolated free of its fusion partner (GST) by treating the purified GST-ORF-14 fusion protein with thrombin protease. In Western immunoblots, the purified ORF-14 protein reacted with antibodies in 26 of 57 human sera (46%) from TB patients while no reactivity was seen with 11 sera from M. bovis BCG-vaccinated healthy subjects. Interestingly, sera from nine of 15 (60%) long-term contacts of TB patients also had antibodies reactive to the ORF-14 protein. These results suggest that the ORF-14 protein in combination with other immunodominant proteins could be useful in the serodiagnosis of individuals infected with M. tuberculosis

New live mycobacterial vaccines: the Geneva consensus on essential steps towards clinical development

As the disease caused by Mycobacterium tuberculosis continues to be a burden, which the world continues to suffer, there is a concerted effort to find new vaccines to combat this problem. Of the various vaccines strategies, one viable option is the development of live mycobacterial vaccines. A meeting with researchers, regulatory bodies, vaccines developers and manufactures was held to consider the challenges and progress, which has been achieved with live mycobacterial vaccines (either modified BCG or attenuated M. tuberculosis). Discussion led to the production of a consensus document of the proposed entry criteria for Phase I clinical trials of candidate live mycobacterial vaccines. The vaccine must be characterised thoroughly to prove identity and consistency, as clinical trial lots are prepared. In pre-clinical studies, greater protective efficacy as well as improved safety potential relative to BCG should be considered when assessing potential vaccine candidates. A standard way to measure the protective efficacy to facilitate comparison between vaccine candidates was suggested. Additional safety criteria and verification of attenuation must be considered for attenuated M. tuberculosis. Two non-reverting independent mutations are recommended for such vaccines. When entering Phase I trials, enrolment should be based upon an acceptable characterisation of the study population regarding mycobacterium status and exclude HIV+ individuals. BCG could be used as a comparator for blinding during the trials and to properly assess vaccine-specific adverse reactions, while assays are being developed to assess immunogenicity of vaccines. The proposed criteria suggested in this consensus document may facilitate the movement of the most promising vaccine candidates to the clinic and towards control of tuberculosi

Serodiagnosis of <i>Mycobacterium avium</i> infections in pigs

The aim of this study is the development and evaluation of a serodiagnostic assay for Mycobacterium avium (MA). After screening MA lipid fractions in an ELISA format, a polar lipid fraction was selected as antigen because of its superior recognition by serum antibodies in experimentally infected pigs. The resulting MA-ELISA was evaluated as an alternative for detection of MA infection by traditional pathological examination of pig lymph nodes for granulomatous lesions by meat inspectors. By comparing with bacteriological examination, the MA-ELISA showed significantly better sensitivity (69%) as compared to pathological examination (31%) in experimentally infected pigs. The MA-ELISA also appeared significantly more specific in a set of serum samples from MA negative pigs: only 1 out of these 153 serum samples reacted positive, whereas 99 (65%) of these had displayed false positive results by detection of lymph nodes lesions that appeared not to be associated with MA (Komijn et al., 2007).The MA-ELISA was subsequently evaluated using serum samples from two farms with pigs known to be infected with MA. Bacteriological examination of the sub-maxillary and mesenteric lymph nodes showed that 56% (103/184) and 35% (41/117) of the pigs, respectively were positive for MA in these farms. In the first farm, 16% (29/184) of the pigs tested positive in MA-ELISA and 31% (57/184) by pathological examination. On the contrary, in the second farm, more pigs tested positive 17% (15/117) in MA-ELISA with 8% (9/117) positivity by pathological examination. Taking the results on both farms together, the sensitivity of the MA-ELISA was 14% and the specificity 83%, whereas the sensitivity of the pathological examination was 31% and the specificity 86%.For practical reasons use of a serological test as the MA-ELISA may be preferred over pathological or bacteriological examinations. Our studies in experimentally infected and negative “field” sera indicate that the MA-ELISA is significantly more specific and more sensitive than detection by classical pathological examination. However, the studies in two MA infected farms show a variable picture with pathological examination overall performing better. Study in a wider range of “positive” farms will be needed to provide a more comprehensive view of the quality of both tests for detection of MA in infected farms. At the same time further optimization of MA-ELISA with use of lipid antigens from a broader range of serotypes may improve its performance in the face of infections with different MA serotype

Specific lymphoproliferation, gamma interferon production, and serum immunoglobulin G directed against a purified 32 kDa mycobacterial protein antigen (P32) in patients with active tuberculosis

Twenty-one patients treated for active tuberculosis were examined for immune reactivity to purified protein derivative (PPD) and to a purified 32-kDa protein antigen (P32) from Mycobacterium bovis, strain BCG. Lymphoproliferation of peripheral blood leucocytes to PPD and P32 was positive in 95% and 71% of the patients respectively. A positive IFN-gamma response was detected in 62% against PPD and in 48% against P32. Low blastogenesis and IFN-gamma production were observed, especially in patients with poor general health and advanced tuberculous lesions. Twelve out of twelve (100%) of the tuberculin-positive healthy volunteers responded to PPD and P32 with mean lymphoproliferation and IFN-gamma values that were higher than in the patient group. Twelve tuberculin-negative control subjects were completely unreactive to PPD and P32 antigen. On the other hand, IgG antibodies in the serum were detected in 95% of the patients against PPD, in 77% of the patients against P32 but in none of the tuberculin-positive or negative healthy volunteers. The highest IgG levels against PPD were found in those patients with the lowest in vitro lymphoproliferation and IFN-gamma production (r = -0.54; P less than 0.05). Nonspecific interferon production following induction with Newcastle disease virus, Corynebacterium parvum, or phytohaemagglutinin was comparable in the control and patient groups. Finally, low IFN-alpha titres were detected in the serum of about 50% of the patients.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jFLWNAinfo:eu-repo/semantics/publishe