Differential release of tumor necrosis factor-α from murine peritoneal macrophages stimulated with virulent and avirulent species of mycobacteria

Abstract The ability of Mycobacterium tuberculosis H37Rv and H37Ra, M. bovis BCG and M. smegmatis to induce the secretion of tumor necrosis factor-α (TNF-α) by cultured murine peritoneal macrophages is inversely related to their virulence. The avirulent species of mycobacteria which were unable to persist in macrophages were capable of inducing significant levels of TNF-α compared to that formed in cultures infected with the virulent M. tuberculosis H37Rv. This difference was also associated with an inherent toxicity by live H37Rv for macrophage cultures. Heat-killed H37Rv was non-toxic and induced significant levels of TNF-α; in contrast, live and heat-killed suspensions of avirulent mycobacteria had an equivalent ability to trigger TNF-α secretion. The TNF-α response was dose-dependent, related directly to the percentage of infected cells, and peaked 6–12 h post-infection. An early and vigorous TNF-α response appears to be a marker of macrophage resistance, while the downregulation of this response seems associated with macrophage toxicity and unrestricted mycobacterial growth.     

Survival of virulent Mycobacterium tuberculosis involves preventing apoptosis induced by Bcl-2 upregulation and release resulting from necrosis in J774 macrophages

Macrophage apoptosis plays a role in mycobacterial infection. To define the mechanism by which virulent Mycobacterium tuberculosis escapes apoptosis and killing in macrophages, J774 macrophages were infected with virulent M. tuberculosis H37Rv and attenuated H37Ra strains. H37Rv induced less apoptosis than H37Ra, and caspase 3 was activated in H37Ra- and H37Rv-infected macrophages. Intracellular H37Rv bacilli were released at a higher rate into the supernatant than were H37Ra by the sixth day of infection, and this was simultaneously accompanied by the increased necrosis of infected cells showing lactate dehydrogenase (LDH) release. Fas mRNA expression was downregulated and FasL was upregulated in H37Ra- and H37Rv-infected macrophages, while Bcl-2 was upregulated in H37Rv-infected macrophages but downregulated in H37Ra-infected macrophages as seen by real-time PCR. These results indicate that M. tuberculosis H37Ra and H37Rv proliferate in macrophages by preventing them from inducing apoptosis during the early phase of infection, and that M. tuberculosis H37Rv-infected macrophages are found to express Bcl-2 mRNA, which leads to anti-apoptotic activity, and that relatively distinct necrosis might occur during the later phase of infection.     

Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophages

Human alveolar macrophages (AMphi) undergo apoptosis following infection with Mycobacterium tuberculosis in vitro. Apoptosis of cells infected with intracellular pathogens may benefit the host by eliminating a supportive environment for bacterial growth. The present study compared AMphi apoptosis following infection by M. tuberculosis complex strains of differing virulence and by Mycobacterium kansasii. Avirulent or attenuated bacilli (M. tuberculosis H37Ra, Mycobacterium bovis bacillus Calmette-Guérin, and M. kansasii) induced significantly more AMphi apoptosis than virulent strains (M. tuberculosis H37Rv, Erdman, M. tuberculosis clinical isolate BMC 96.1, and M. bovis wild type). Increased apoptosis was not due to greater intracellular bacterial replication because virulent strains grew more rapidly in AMphi than attenuated strains despite causing less apoptosis. These findings suggest the existence of mycobacterial virulence determinants that modulate the apoptotic response of AMphi to intracellular infection and support the hypothesis that macrophage apoptosis contributes to innate host defense in tuberculosis.     

Early secreted antigen ESAT-6 of Mycobacterium tuberculosis promotes protective T helper 17 cell responses in a toll-like receptor-2-dependent manner

Despite its relatively poor efficacy, Bacillus Calmette-Guérin (BCG) has been used as a tuberculosis (TB) vaccine since its development in 1921. BCG induces robust T helper 1 (Th1) immune responses but, for many individuals, this is not sufficient for host resistance against Mycobacterium tuberculosis (M. tb) infection. Here we provide evidence that early secreted antigenic target protein 6 (ESAT-6), expressed by the virulent M. tb strain H37Rv but not by BCG, promotes vaccine-enhancing Th17 cell responses. These activities of ESAT-6 were dependent on TLR-2/MyD88 signalling and involved IL-6 and TGF-β production by dendritic cells. Thus, animals that were previously infected with H37Rv or recombinant BCG containing the RD1 region (BCG::RD1) exhibited improved protection upon re-challenge with virulent H37Rv compared with mice previously infected with BCG or RD1-deficient H37Rv (H37RvΔRD1). However, TLR-2 knockout (TLR-2^-/-) animals neither showed Th17 responses nor exhibited improved protection in response to immunization with H37Rv. Furthermore, H37Rv and BCG::RD1 infection had little effect on the expression of the anti-inflammatory microRNA-146a (miR146a) in dendritic cells (DCs), whereas BCG and H37RvΔRD1 profoundly induced its expression in DCs. Consistent with these findings, ESAT-6 had no effect on miR146a expression in uninfected DCs, but dramatically inhibited its upregulation in BCG-infected or LPS-treated DCs. Collectively, our findings indicate that, in addition to Th1 immunity induced by BCG, RD1/ESAT-6-induced Th17 immune responses are essential for optimal vaccine efficacy.     

Genes of Mycobacterium tuberculosis H37Rv downregulated in the attenuated strain H37Ra are restricted to M. tuberculosis complex species

By comparing gene expression of virulent Mycobacterium tuberculosis H37Rv and attenuated strain H37Ra, we previously detected six genes that appear to be markedly downregulated in the attenuated strain compared with the virulent one. Three of these genes, i.e. Rv1345, Rv2770c, and Rv0288, code for proteins that can be predictively associated to immunological or pathogenetic aspects of M. tuberculosis infection; the other genes, i.e. Rv2336, Rv1320c, and Rv2819c, code for proteins with unknown functions (Rindi et al., 1999). In this paper we searched for the above mentioned genes in Pvu II-digested genomic DNA of a number of mycobacterial species by southern blot analysis employing PCR-generated probes in high-stringency conditions. Hybridization signals were only found in species belonging to the M. tuberculosis complex, i.e., M. tuberculosis, M. bovis, including the BCG strain, and M. microti, but not in other mycobacterial species, including M. avium, M. intracellulare, M. malmoense, M. xenopi, M. kansasii, M. simiae, M. marinum, M. scrofulaceum, M. gordonae, M. fortuitum, and M. smegmantis. These results indicate that genes Rv1345, Rv2770c, Rv0288, Rv2336, Rv1320c, and Rv2819c are associated with the most virulent mycobacteria and further support their potential role in M. tuberculosis virulence.     

PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis: a novel immunodominant antigen of Mycobacterium tuberculosis induces humoral and cellular immune responses in mice

Subtractive DNA hybridization of pathogenic M. bovis and BCG, and comparative genome-wide DNA microarray analysis of M. tuberculosis H37Rv and BCG identified several RD, designated as RD1 to RD16, between M. tuberculosis and M. bovis on the one hand and BCG on the other. These regions cover 108 ORF of M. tuberculosis H37Rv, and are deleted from all 13 BCG sub-strains currently used as anti-tuberculosis vaccines in different parts of the world. In this study, we evaluated cellular and humoral immune response in C57BL/6 mice immunized with the PPE protein Rv3425, encoded by an ORF found in RD11 of M. tuberculosis. Rv3425 protein induced an increased Th1/Th2 type immune response in mice, characterized by an elevated concentration of IFN-gamma in antigen stimulated splenocyte culture and a strong IgG(1) antibody response. These results provide evidence on the immunogenicity of the PPE protein Rv3425 which, together with its reported immunodominant characteristics, imply that it may be a candidate for development of a vaccine for the control of TB.     

NK1.1+ cells and IL-22 regulate vaccine-induced protective immunity against challenge with Mycobacterium tuberculosis

We previously found that human NK cells lyse Mycobacterium tuberculosis-infected monocytes and alveolar macrophages and upregulate CD8(+) T cell responses. We also found that human NK cells produce IL-22, which inhibits intracellular growth of M. tuberculosis, and that NK cells lyse M. tuberculosis-expanded CD4(+)CD25(+)FOXP3(+) T regulatory cells (Tregs). To determine the role of NK cells during the protective immune response to vaccination in vivo, we studied the NK cell and T cell responses in a mouse model of vaccination with bacillus Calmette-Guérin (BCG), followed by challenge with virulent M. tuberculosis H37Rv. BCG vaccination enhanced the number of IFN-γ-producing and IL-22-producing NK cells. Depletion of NK1.1(+) cells at the time of BCG vaccination increased the number of immunosuppressive Tregs (CD4(+)CD25(hi), 95% Foxp3(+)) after challenge with M. tuberculosis H37Rv, and NK1.1(+) cells lysed expanded but not natural Tregs in BCG-vaccinated mice. Depletion of NK1.1(+) cells at the time of BCG vaccination also increased the bacillary burden and reduced T cell responses after challenge with M. tuberculosis H37Rv. IL-22 at the time of vaccination reversed these effects and enhanced Ag-specific CD4(+) cell responses in BCG-vaccinated mice after challenge with M. tuberculosis H37Rv. Our study provides evidence that NK1.1(+) cells and IL-22 contribute to the efficacy of vaccination against microbial challenge.     

Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens

In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS-PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2-DE database (http://www.mpiib-berlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.     

Immunogenicity of Cell Walls from Various Mycobacteria Against Airborne Tuberculosis in Mice

Protective potency of oil-treated cell walls of various mycobacteria against airborne infection of mice with a few cells of Mycobacterium tuberculosis H37Rv was compared with that of viable BCG. Although less potent than BCG cell walls, the cell walls of atypical mycobacteria of Runyon's groups I to IV protected against challenge by aerosol to some degree. Protection afforded by cell walls of H37Rv and of the avirulent mutants H37Ra and Washington II was comparable to that provided by BCG cell walls. However, cell walls of a highly virulent strain of M. bovis (Bovinus I) provided the best protection yet achieved. Present evidence suggests that protective substances are shared by all mycobacteria but in differing amounts; the relationship between virulence and immunogenicity has yet to be clarified.     

Survival of Mycobacterium tuberculosis in host macrophages involves resistance to apoptosis dependent upon induction of antiapoptotic Bcl-2 family member Mcl-1

Mcl-1 protein expression was found to be up-regulated during infection with virulent Mycobacterium tuberculosis strain H37Rv. Mcl-1 induction in THP-1 cells was optimal at a multiplicity of infection of 0.8-1.2 bacilli per macrophage and was independent of opsonin coating of the bacteria. Mcl-1 expression was elevated as early as 4 h, peaked at 5.8-fold above control cells at 24 h, and remained elevated at 48 h after infection. In THP-1 cells, mMcl-1 mRNA was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. In THP-1 cells and monocyte-derived macrophages (MDMs), Mcl-1 protein was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. Treatment of uninfected, H37Ra-infected, and H37Rv-infected THP-1 cells and MDMs with antisense oligonucleotides to mcl-1 reduced Mcl-1 expression by >84%. This resulted in an increase in apoptosis of both MDMs and THP-1 cells that were infected with H37Rv, but not cells that were uninfected or infected with H37Ra. Increased apoptosis correlated with a decrease in M. tuberculosis CFUs recovered from antisense-treated, H37Rv-infected cells at 4 and 7 days after infection. In contrast, CFU recoveries from sense-treated, H37Rv-infected cells or from antisense- or sense-treated, H37Ra-infected cells were unchanged from controls. Thus, the antiapoptotic effect of the induction of Mcl-1 expression in H37Rv-infected macrophages promotes the survival of virulent M. tuberculosis.     

Inhibition of bfl-1/A1 by siRNA inhibits mycobacterial growth in THP-1 cells by enhancing phagosomal acidification

Virulent tubercle bacilli inhibit apoptosis to establish a safe environment within the host cells. Here, we report that NF-kappaB dependent antiapoptotic protein bfl-1/A1 plays an important role in this process. Both virulent and avirulent mycobacteria bearing THP-1 cells expressed considerable amount of bfl-1/A1 after 4 h of infection. However, after 48 h of infection, bfl-1/A1 expression was evident only in Mycobacterium tuberculosis H37Rv but not in M. tuberculosis H37Ra infected cells. When parallel experiments were performed with Human monocyte-derived macrophages (MDMs), differential expression of bfl-1/A1 mRNA was observed in case of M. tuberculosis H37Rv and M. tuberculosis H37Ra infection. siRNA mediated inhibition of bfl-1/A1 induced apoptosis in M. tuberculosis H37Rv infected THP-1 and MDMs. Reduction in intracellular mycobacterial growth was observed in bfl-1/A1 siRNA transfected, M. tuberculosis H37Rv infected THP-1 cells. Enhancement of phagosome-lysosome fusion was observed in bfl-1/A1 siRNA treated and M. tuberculosis H37Rv infected THP-1 cells. These results clearly indicated that differential expression of bfl-1/A1 in M. tuberculosis H37Rv and M. tuberculosis H37Ra infected THP-1 cells probably account for the difference in infection outcome.     

The genetic regulation of host response to inoculation with Mycobacterium bovis (BCG) and Mycobacterium tuberculosis H37RV

Vaccination with M. bovis (BCG) essentially prolonged survival time (ST) of several strain mice, with the exception, of CBA/N, infected with M. tuberculosis H37Rv. ST of CBA/N, differing from CBA by xid mutation, was not prolonged by vaccination. Mouse strains with alternative alleles of BCG gene (s and r) and fzy gene as a genetic marker for Bcg5 were used for segregation analysis. It was shown that ST, the level of DTH reaction of mice infected with M. tuberculosis H37Rv, and protective effect of BCG vaccination did not depend on Bcg gene. However, Bcg gene, apparently, regulate the DTH response to PPD in mice only vaccinated with M. bovis (BCG).     

多重PCR方法特异性鉴定卡介苗菌株多糖核酸的初探

与结核分枝杆菌H37Rv菌株进行比较,BCG菌株可找到一个特殊的缺失片段RD1,它存在于所有有毒分枝杆菌中,而在所有的卡介苗菌株中均缺失。应用多重PCR方法检测RD1区的存在与否,可以区别BCG和其它有毒的分枝杆菌。卡介菌多糖核酸来源于卡介菌,检测成品中DNA是否含有RD1区,能特异性地鉴别该制品。结果显示牛分枝杆菌标准株和结核分枝杆菌H37Rv存在RD1区;而卡介菌多糖核酸注射液和国内皮内注射用BCG疫苗生产用菌株扩增产物一致,提示均缺失RD1区。因此,这种多重PCR方法适用于卡介菌多糖核酸注射液的特异性鉴别试验。     

Mutations in Mycobacterium tuberculosis Rv0444c, the gene encoding anti-SigK, explain high level expression of MPB70 and MPB83 in Mycobacterium bovis

It has recently been advanced that Mycobacterium tuberculosis sigma factor K (SigK) positively regulates expression of the antigenic proteins MPB70 and MPB83. As expression of these proteins differs between M. tuberculosis (low) and Mycobacterium bovis (high), this study set out to determine whether M. bovis lacks a functional SigK repressor (anti-SigK). By comparing genes near sigK in M. tuberculosis H37Rv and M. bovis AF2122/97, we observed that Rv0444c, annotated as unknown function, had variable sequence in M. bovis. Analysis of in vitro mpt70/mpt83 expression and Rv0444c sequencing across M. tuberculosis complex (MTC) members revealed that high-level expression was associated with a mutated Rv0444c. Complementation of M. bovis bacillus Calmette-Guerin Russia, a high producer of MPB70/MPB83, with wild-type Rv0444c resulted in a significant decrease in mpb70/mpb83 expression. Conversely, a M. tuberculosis H37Rv mutant which expressed sigK but not Rv0444c manifested the M. bovis phenotype of high-level MPB70/MPB83 expression. Further support that Rv0444c encodes the anti-SigK was obtained by yeast two-hybrid studies, where the N-terminal region of Rv0444c-encoded protein interacted with SigK. Together these findings indicate that Rv0444c encodes the regulator of SigK (RskA) and mutations in this gene explain high-level MPT70/MPT83 expression by certain MTC members.     

Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays

Whole-genome comparisons of the tubercle bacilli were undertaken using ordered bacterial artificial chromosome (BAC) libraries of Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG-Pasteur, together with the complete genome sequence of M. tuberculosis H37Rv. Restriction-digested BAC arrays of M. tuberculosis H37Rv were used in hybridization experiments with radiolabelled M. bovis BCG genomic DNA to reveal the presence of 10 deletions (RD1-RD10) relative to M. tuberculosis. Seven of these regions, RD4-RD10, were also found to be deleted from M. bovis, with the three M. bovis BCG-specific deletions being identical to the RD1-RD3 loci described previously. The distribution of RD4-RD10 in Mycobacterium africanum resembles that of M. tuberculosis more closely than that of M. bovis, whereas an intermediate arrangement was found in Mycobacterium microti, suggesting that the corresponding genes may affect host range and virulence of the various tubercle bacilli. Among the known products encoded by these loci are a copy of the proposed mycobacterial invasin Mce, three phospholipases, several PE, PPE and ESAT-6 proteins, epoxide hydrolase and an insertion sequence. In a complementary approach, direct comparison of BACs uncovered a third class of deletions consisting of two M. tuberculosis H37Rv loci, RvD1 and RvD2, deleted from the genome relative to M. bovis BCG and M. bovis. These deletions affect a further seven genes, including a fourth phospholipase, plcD. In summary, the insertions and deletions described here have important implications for our understanding of the evolution of the tubercle complex.