Vaccination of brushtail possums, Trichosurus vulpecula, with Bacille Calmette-Guerin induces T lymphocytes that reduce Mycobacterium bovis replication in alveolar macrophages via a contact-dependent/nitric oxide-independent mechanism

The permissiveness of alveolar macrophages from brushtail possums for the replication of Mycobacterium bovis was examined. Mycobacterium bovis replication was indirectly measured by assessing bacterial metabolism via the incorporation of [3-H]-uracil by bacilli released from lysed macrophages previously infected with mycobacteria. Alveolar macrophages allowed substantial replication of virulent M. bovis, in contrast to Bacille Calmette-Guerin (BCG) Pasteur, which replicated poorly. The addition of crude lymphokines enhanced the metabolic activity of phagocytosed M. bovis in possum macrophages. Possum lymphokines enhanced the ability of possum macrophages to generate reactive oxygen intermediates, measured by the reduction of nitroblue tetrazolium, which is indicative of an activation process. Similarly, the addition of recombinant possum TNF-alpha enhanced the permissiveness of alveolar macrophages for M. bovis. In contrast to mouse peritoneal macrophages, possum alveolar macrophages did not release significant levels of nitric oxide (NO) after stimulation with M. bovis and/or lymphokines. However, the uptake of virulent M. bovis by possum macrophages was associated with an enhanced ability of cells to release TNF-alpha, whereas very low levels of TNF-alpha were released after infection with BCG. The addition of a selective inhibitor of inducible NO synthase had no impact on the replication of M. bovis or BCG in possum macrophages in the presence or absence of lymphokines. Co-culturing infected possum alveolar macrophages with autologous blood mononuclear cells from BCG-vaccinated possums led to a significant decrease in the metabolic activity of intracellular M. bovis. This effect was contact dependent and NO independent and was mediated by a population of CD3+ cells. In addition, adding scavengers of reactive oxygen intermediates did not abrogate this phenomenon.     

The role of histamine in the intracellular survival of Mycobacterium bovis BCG

The course and outcome of infection with mycobacteria are determined by a complex interplay between the immune system of the host and the survival mechanisms developed by the bacilli. Histamine plays an important role in various processes, including cell division, metabolism, and apoptosis, and it modulates innate and adaptive immune responses. In the present study we investigated the intracellular survival of Mycobacterium bovis BCG in murine bone-marrow macrophages isolated from wild-type (WT) and histidine-decarboxylase knock-out [HDC (-/-)] mice. Mycobacterial titers were significantly higher in the HDC (-/-) macrophages as compared with the WT cells. M. bovis BCG growth in WT macrophages could be enhanced by pyrilamine and cimetidine. Exogenously added histamine decreased the intracellular counts of M. bovis BCG in HDC (-/-) macrophages. Infection of activated macrophages with M. bovis BCG elicited apoptosis, but there was no significant difference between the WT and the HDC (-/-) cells. These bacilli induced comparable levels of tumor necrosis factor-alpha production in the WT and the HDC (-/-) macrophages. M. bovis BCG stimulated interleukin-18 (IL-18) production in the macrophages from WT mice, but not in the HDC (-/-) cells. Exogenously added IL-18 decreased the titers of intracellular mycobacteria in HDC (-/-) cells. In conclusion, these data implicate histamine in the intracellular survival of M. bovis BCG. The cellular control mechanisms restricting the growth of M. bovis BCG are complex and involve H1 and H2 receptor-mediated events. Histamine might be an important mediator of M. bovis BCG-induced IL-18 production, which in turn contributes to immune protection.     

Anaerobic nitrate reductase (narGHJI) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice

Mycobacterium tuberculosis and Mycobacterium bovis cause tuberculosis, which is responsible for the deaths of more people each year than any other bacterial infectious disease. Disseminated disease with Mycobacterium bovis BCG, the only currently available vaccine against tuberculosis, occurs in immunocompetent and immunodeficient individuals. Although mycobacteria are obligate aerobes, they are thought to face an anaerobic environment during infection, notably inside abscesses and granulomas. The purpose of this study was to define a metabolic pathway that could allow mycobacteria to exist under these conditions. Recently, the complete genome of M. tuberculosis has been sequenced, and genes homologous to an anaerobic nitrate reductase (narGHJI), an enzyme allowing nitrate respiration when oxygen is absent, were found. Here, we show that the narGHJI cluster of M. tuberculosis is functional as it conferred anaerobic nitrate reductase activity to Mycobacterium smegmatis. A narG mutant of M. bovis BCG was generated by targeted gene deletion. The mutant lacked the ability to reduce nitrate under anaerobic conditions. Both mutant and M. bovis BCG wild type grew equally well under aerobic conditions in vitro. Histology of immunodeficient mice (SCID) infected with M. bovis BCG wild type revealed large granulomas teeming with acid-fast bacilli; all mice showed signs of clinical disease after 50 days and succumbed after 80 days. In contrast, mice infected with the mutant had smaller granulomas containing fewer bacteria; these mice showed no signs of clinical disease after more than 200 days. Thus, it seems that nitrate respiration contributes significantly to virulence of M. bovis BCG in immunodeficient SCID mice.     

Recombinant BCG approach for development of vaccines: cloning and expression of immunodominant antigens of M. tuberculosis

In spite of major advances in our understanding of the biology and immunology of tuberculosis, the incidence of the disease has not reduced in most parts of the world. In an attempt to improve the protective efficacy of Mycobacterium bovis bacille Calmette-Guérin (BCG), we have developed a generic vector system, pSD5, for expression of genes at varying levels in mycobacteria. In this study, we have cloned and overexpressed three immunodominant secretory antigens of M. tuberculosis, 85A, 85B and 85C, belonging to the antigen 85 complex. All the genes were cloned under the control of a battery of mycobacterial promoters of varying strength. The expression was analysed in the fast-growing strain M. smegmatis and the slow-growing vaccine strain M. bovis BCG. The recombinant BCG constructs were able to express the antigens at high levels and the majority of the expressed antigens was secreted into the medium. These results show that by using this strategy the recombinant BCG approach can be successfully used for the development of candidate vaccines against infections associated with mycobacteria as well as other pathogens.     

Impaired generation of reactive oxygen species during differentiation of dendritic cells (DCs) by Mycobacterium tuberculosis secretory antigen (MTSA) and subsequent activation of MTSA-DCs by mycobacteria results in increased intracellular survival

We investigated the role of reactive oxygen species (ROS) in dendritic cell (DC) differentiation by 10-kDa Mycobacterium tuberculosis secretory Ag (MTSA) and survival of mycobacteria therein. Compared with GM-CSF, MTSA induced lower ROS production during DC differentiation from precursors. This result correlated with higher superoxide dismutase 1 expression in MTSA stimulated precursors as compared with GM-CSF stimulation. Furthermore, a negative regulation of protein kinase C (PKC) activation by ROS was observed during DC differentiation. ROS inhibited the rapid and increased phosphorylation of PKCalpha observed during DC differentiation by MTSA. In contrast, ROS inhibition increased the weak and delayed PKCalpha phosphorylation by GM-CSF. Similar to DC differentiation, upon activation with either M. tuberculosis cell extract (CE) or live Mycobacterium bovis bacillus Calmette-Guérin (BCG), DCs differentiated with MTSA (MTSA-DCs) generated lower ROS levels when compared with DCs differentiated with GM-CSF (GM-CSF-DCs). Likewise, a negative regulation of PKCalpha phosphorylation by ROS was once again observed in DCs activated with either M. tuberculosis CE or live M. bovis BCG. However, a reciprocal positive regulation between ROS and calcium was observed. Compared with MTSA-DCs, stimulation of GM-CSF-DCs with M. tuberculosis CE induced a 2-fold higher ROS-dependent calcium influx. However, pretreatment of MTSA-DCs with H(2)O(2) increased calcium mobilization. Finally, lower ROS levels in MTSA-DCs correlated with increased intracellular survival of M. bovis BCG when compared with survival in GM-CSF-DCs. Although inhibiting ROS in GM-CSF-DCs increased M. bovis BCG survival, H(2)O(2) treatment of MTSA-DCs decreased survival of M. bovis BCG. Overall our results suggest that DCs differentiated with Ags such as MTSA may provide a niche for survival and/or growth of mycobacteria following sequestration of ROS.     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

Adenylylation and catalytic properties of Mycobacterium tuberculosis glutamine synthetase expressed in Escherichia coli versus mycobacteria

Bacterial glutamine synthetases (GSs) are complex dodecameric oligomers that play a critical role in nitrogen metabolism, converting ammonia and glutamate to glutamine. Recently published reports suggest that GS from Mycobacterium tuberculosis (MTb) may be a therapeutic target (Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456-464). In some bacteria, GS is regulated via adenylylation of some or all of the subunits within the aggregate; catalytic activity is inversely proportional to the extent of adenylylation. The adenylylation and deadenylylation of GS are catalyzed by adenylyl transferase (ATase). Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially ( approximately 25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated ( approximately 30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.     

Role of apoptosis-regulating signal kinase 1 in innate immune responses by Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces innate immune responses through Toll-like receptor (TLR) 2 and TLR4. We investigated the role of apoptosis-regulating signal kinase (ASK) 1 in reactive oxygen species (ROS)-mediated innate immune responses induced by BCG mycobacterial infection. In macrophages, M. bovis BCG stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs), secretion of inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and ROS generation in a TLR2- and TLR4-dependent manner. M. bovis BCG-induced ROS production led to robust activation of ASK1 upstream of the c-jun-N-terminal kinase and p38 MAPK, but not extracellular-regulated kinase 1/2. Blocking ASK1 activity markedly attenuated M. bovis BCG-induced TNF-alpha and IL-6 production by macrophages. Both TLR2 and TLR4 were required for optimal activation of ASK1 in response to M. bovis BCG. Furthermore, we present evidence that TNF receptor-associated factor (TRAF) 6 activities were essential for ROS-mediated ASK1 activation by M. bovis BCG. Finally, ASK1 activities were required for effective control of intracellular mycobacterial survival. Thus, the results of this study suggest a novel role of the TLR-ROS-TRAF6-ASK1 axis in the innate immune response to mycobacteria as a signaling intermediate.     

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. II. Comparative skin testing on guinea-pigs

As part of a cooperative inter-laboratory WHO supported project for the fractionation of Mycobacterium tuberculosis skin test preparations, four fractions (designated 15, 7, 4.75 and 3.5%) were evaluated by comparative skin tests on sensitized guinea-pigs. The 7% fraction was the most potent in both homologously and heterologously sensitized animals, and the 4.75% and 3.5% gel fractions showed the lowest activity. Significant levels of cross-reactivity in guinea-pigs immunized with M. bovis BCG, M. kansasii, M. avium and M. intracellulare were demonstrated for all fractions examined, thus reflecting the antigenic relationships among these mycobacteria. These four fractions may qualify as starting material for further studies aiming at a reduction of skin test cross-reactivity.     

Cytolytic T-cell responses to human dendritic cells and macrophages infected with Mycobacterium bovis BCG and recombinant BCG secreting listeriolysin

Cytolytic T-cell responses from 63 normal blood donors were monitored in a Mycobacterium bovis BCG infection system in vitro. We wanted to know whether cultured dendritic cells were capable of potentiating the cytolytic T-cell responses to M. bovis BCG. Infected cultured dendritic cells were up to ten times more effective antigen-presenting cells than macrophages in proliferative assays, while cytolytic T-cell induction did not differ significantly between dendritic cells and macrophages. Separated CD4+ and CD8+ T-cell subsets contributed equally to lysis of infected targets. Experiments comparing wild-type M. bovis BCG strain with two new recombinant M. bovis BCG strains secreting listeriolysin revealed statistically significant higher maximal lysis values for recombinant M. bovis BCG. We conclude from our in vitro infection system with mycobacteria that dendritic cells are superior to macrophages in proliferative assays but equal to macrophages in their ability to induce cytolytic T-cell responses. Moreover, our data suggest that recombinant M. bovis BCG vaccine strains secreting listeriolysin improve cytolytic T-cell responses.     

Specificity of antigenic fractions of tuberculin.

Purified protein derivative from Mycobacterium bovis was separated into 6 fractions by electrophoresis at 1500 V/40 mA in pH 4.2 acetic acid-pyridine buffer. Further purification of one of the fractions on Sephadex G 25 column and by acid hydrolysis yielded antigen "PS" eliciting tuberculin reaction only in animals vaccinated with M. bovis BCG but not in those sensitized with M. avium and some fast-growing mycobacteria. The specificity of antigen "PS" was confirmed in vitro: the antigen induced blastic transformation only in lymphocytes of guinea pigs sensitized with M. bovis BCG.     

Production of volatile organic compounds by mycobacteria

The need for improved rapid diagnostic tests for tuberculosis disease has prompted interest in the volatile organic compounds (VOCs) emitted by Mycobacterium tuberculosis complex bacteria. We have investigated VOCs emitted by Mycobacterium bovis BCG grown on Lowenstein-Jensen media using selected ion flow tube mass spectrometry and thermal desorption-gas chromatography-mass spectrometry. Compounds observed included dimethyl sulphide, 3-methyl-1-butanol, 2-methyl-1-propanol, butanone, 2-methyl-1-butanol, methyl 2-methylbutanoate, 2-phenylethanol and hydrogen sulphide. Changes in levels of acetaldehyde, methanol and ammonia were also observed. The compounds identified are not unique to M.?bovis BCG, and further studies are needed to validate their diagnostic value. Investigations using an ultra-rapid gas chromatograph with a surface acoustic wave sensor (zNose) demonstrated the presence of 2-phenylethanol (PEA) in the headspace of cultures of M.?bovis BCG and Mycobacterium smegmatis, when grown on Lowenstein-Jensen supplemented with glycerol. PEA is a reversible inhibitor of DNA synthesis. It is used during selective isolation of gram-positive bacteria and may also be used to inhibit mycobacterial growth. PEA production was observed to be dependent on growth of mycobacteria. Further study is required to elucidate the metabolic pathways involved and assess whether this compound is produced during in vivo growth of mycobacteria.     

Nisin depletes ATP and proton motive force in mycobacteria

This study examined the inhibitory effect of nisin and its mode of action against Mycobacterium smegmatis, a non-pathogenic species of mycobacteria, and M. bovis-Bacill Carmette Guerin (BCG), a vaccine strain of pathogenic M. bovis. In agar diffusion assays, 2.5 mg ml(-1) nisin was required to inhibit M. bovis-BCG. Nisin caused a slow, gradual, time- and concentration-dependent decrease in internal ATP levels in M. bovis-BCG, but no ATP efflux was detected. In mycobacteria, nisin decreased both components of proton motive force (membrane potential, Delta Psi and Delta pH) in a time- and concentration-dependent manner. However, mycobacteria maintained their intracellular ATP levels during the initial time period of Delta Psi and Delta pH dissipation. These data suggest that the mechanism of nisin in mycobacteria is similar to that in food-borne pathogens.     

The formation of delayed hypersensitivity to mycobacterial antigens

In experiments on guinea pigs and BALB/c mice delayed hypersensitivity to mycobacterial antigens was induced by the sensitization of the animals with live BCG or killed Mycobacterium bovis or M. avium in incomplete Freund's adjuvant. In the study of the dynamics of the development of skin reactivity to tuberculin some advantages of the sensitization of guinea pigs with live mycobacteria were revealed, while after the revaccination of the animals no development of secondary cell-mediated immune response was observed. The immunization of guinea pigs with atypical mycobacteria prior to their sensitization with BCG was found to lead to the development of higher skin reactivity to allergen prepared from atypical mycobacteria than skin reactivity to tuberculin.     

TNF-α-mediated activation of HIV-1 LTR in monocytoid cells by mycobacteria

Mycobacterial infection occurs commonly in patients with acquired immune deficiency syndrome. Incubation of monocytoid cell line U937 cells, which was cotransfected HIV-1 long terminal repeat sequence (LTR) chloramphenicol acetyltransferase (CAT) plasmid and Tat expression plasmid, with Mycobacterium smegmatis, Mycobacterium avium, Mycobacterium bovis BCG and Mycobacterium tuberculosis resulted in enhancement of CAT production, indicating that these mycobacteria could activate LTR in this cell line. The amount of CAT in the cells coexisting with M. smegmatis was higher than that infected with other mycobacteria. The amounts of CAT production in the cells coculturing with M. avium and M. bovis BCG were intermediate. M. tuberculosis slightly stimulated CAT production. The amount of tumor necrosis factor (TNF)-alpha produced by transfected U937 cells was correlated with the amount of CAT production. The interleukin (IL)-1beta and IL-6 levels in the supernatant from coculturing with all species were similar. The antibody to TNF-alpha inhibited CAT production induced by mycobacterial infections. The anti-IL-1beta and anti-IL-6 antibodies, however, scarcely influenced stimulation of LTR by mycobacteria. In addition, U937 cells transfected with full length LTR CAT plasmid showed increased CAT production by activation with mycobacteria, but the cells transfected with mutant LTR CAT constructs from which the nuclear factor (NF)-kappaB binding site was deleted did not show activation. These findings indicated that activation of Mycobacterium-induced LTR CAT is NF-kappaB dependent. These findings suggested that activation of HIV-1 LTR by mycobacteria was mainly mediated by NF-kappaB-induced secondary release of cytokine TNF-alpha.     

The uraA locus and homologous recombination in Mycobacterium bovis BCG.

Molecular genetic manipulation of mycobacteria would benefit from the isolation of mycobacterial genes that could serve both as genetic markers and as sequences used to target homologous integration of recombinant DNA into the genome. We isolated the Mycobacterium bovis BCG gene encoding orotidine-5'-monophosphate decarboxylase (OMP-DCase) by complementing an Escherichia coli mutant defective in this activity. The BCG OMP-DCase gene (uraA) and the flanking DNA were sequenced. The predicted BCG OMP-DCase protein sequence is closely related to the Myxococcus xanthus OMP-DCase and more distantly related to the other known prokaryotic and eukaryotic OMP-DCases. To investigate whether homologous integration can occur in M. bovis BCG, an improved protocol for transformation of BCG was developed and a linear fragment of mycobacterial DNA containing the uraA locus, marked with a kanamycin resistance gene, was introduced into BCG cells by electroporation. The kanamycin-resistant BCG transformants all contained vector DNA integrated into the genome. The marked DNA had integrated into the homologous uraA locus in approximately 20% of the transformants. These results have implications for understanding the role of mycobacterial genes in disease pathogenesis and for the genetic engineering of improved mycobacterial vaccines.