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.     

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.     

A Point Mutation in cycA Partially Contributes to the D-cycloserine Resistance Trait of Mycobacterium bovis BCG Vaccine Strains

In mycobacteria, CycA a D-serine, L- and D-alanine, and glycine transporter also functions in the uptake of D-cycloserine, an important second-line anti-tubercular drug. A single nucleotide polymorphism identified in the cycA gene of BCG was hypothesized to contribute to the increased resistance of Mycobacterium bovis bacillus Calmette-Guérin (BCG) to D-cycloserine compared to wild-type Mycobacterium tuberculosis or Mycobacterium bovis. Working along these lines, a merodiploid strain of BCG expressing Mycobacterium tuberculosis CycA was generated and found to exhibit increased susceptibility to D-cycloserine albeit not to the same extent as wild-type Mycobacterium tuberculosis or Mycobacterium bovis. In addition, recombinant Mycobacterium smegmatis strains expressing either Mycobacterium tuberculosis or Mycobacterium bovis CycA but not BCG CycA were rendered more susceptible to D-cycloserine. These findings support the notion that CycA-mediated uptake in BCG is impaired as a result of a single nucleotide polymorphism; however, the partial contribution of this impairment to D-cycloserine resistance suggests the involvement of additional genetic lesions in this phenotype.     

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.     

Identification of cyclic AMP-regulated genes in Mycobacterium tuberculosis complex bacteria under low-oxygen conditions

Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), which kills approximately 2 million people a year despite current treatment options. A greater understanding of the biology of this bacterium is needed to better combat TB disease. The M. tuberculosis genome encodes as many as 15 adenylate cyclases, suggesting that cyclic AMP (cAMP) has an important, yet overlooked, role in mycobacteria. This study examined the effect of exogenous cAMP on protein expression in Mycobacterium bovis BCG grown under hypoxic versus ambient conditions. Both shaking and shallow standing cultures were examined for each atmospheric condition. Different cAMP-dependent changes in protein expression were observed in each condition by two-dimensional gel electrophoresis. Shaking low-oxygen cultures produced the most changes (12), while standing ambient conditions showed the fewest (2). Five upregulated proteins, Rv1265, Rv2971, GroEL2, PE_PGRS6a, and malate dehydrogenase, were identified from BCG by mass spectrometry and were shown to also be regulated by cAMP at the mRNA level in both M. tuberculosis H37Rv and BCG. To our knowledge, these data provide the first direct evidence for cAMP-mediated gene regulation in TB complex mycobacteria.     

In vivo efficiency of targeted norfloxacin against persistent, isoniazid-insensitive, Mycobacterium bovis BCG present in the physiologically hypoxic mouse liver

Persistence of Mycobacterium tuberculosis is a hypoxia-inducible state in which the bacteria are phenotypically insensitive to currently available antituberculous drugs. In humans, persistent M. tuberculosis is found in granulomatous lesions, either inside macrophages or in necrotic tissue, where the partial oxygen pressure (pO(2)) is very low. Persistent bacteria can remain silent for decades before overt tuberculosis develops. Due to insensitivity to classical drugs, M. tuberculosis persistence prevents rapid and definitive clearance of bacteria. Consequently, therapeutic molecules are required that are both active against persistent bacilli and able to reach their intramacrophagic location. In contrast to its native form, norfloxacin is active in vivo against Mycobacterium bovis BCG present in the lungs when temporarily linked to a macromolecular carrier targeted to macrophages. To study the efficiency of this macromolecular prodrug targeted to persistent mycobacteria confined inside macrophages, we established a short-term in vivo model based on the physiological pO(2) differences between lungs, spleen and liver. Whereas lungs and spleen are well oxygenated, the liver has a low pO(2) due to its portal irrigation. Therefore, studying mycobacteria in the liver yields information about in vivo persistent bacilli exposed to low pO(2). To our knowledge, no similar short-term in vivo model has been published to date. Using this model, we demonstrated the insensitivity to isoniazid of M. bovis BCG present in hypoxic sites, and showed that norfloxacin given as a mannosylated macrophage-targeted prodrug was able to kill these isoniazid-insensitive mycobacteria. This demonstrates that intracellular persistent mycobacteria are amenable to antibiotic treatment.     

A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.     

Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis Bacille Calmette-Guerin. Structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains

The attenuated strain of Mycobacterium bovis Bacille Calmette-Guérin (BCG), used worldwide to prevent tuberculosis and leprosy, is also clinically used as an immunotherapeutic agent against superficial bladder cancer. An anti-tumor polysaccharide has been isolated from the boiling water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues. Mycobacterium tuberculosis and other mycobacterial species produce a glycogen-like alpha-glucan composed of repeating units of 4-linked glucosyl residues substituted at some 6 positions by short oligoglucosyl units that also exhibits an anti-tumor activity. Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property. The present study addresses this question through the comparative analysis of alpha-glucans purified from the extracellular materials and boiling water extracts of three vaccine substrains. The polysaccharides were purified, and their structural features were established by mono- and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds. The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like alpha-glucan of M. tuberculosis and other mycobacteria. Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted alpha-glucans in mycobacteria.     

Identification of arylamine N -acetyltransferase inhibitors as an approach towards novel anti-tuberculars

New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules— triazoles—in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.     

Mycobacterial growth in human macrophages: variation according to donor, inoculum and bacterial strain

The microbicidal capacity of the macrophage is frequently evaded by mycobacteria, leading to tuberculosis (TB). We investigated a number of parameters affecting the rate of growth of mycobacteria in human monocyte-derived macrophages (MDM). The results show a great deal of variation in the growth of both Mycobacterium bovis BCG and M. tuberculosis H37Rv, using a large number of human macrophage donors, (132 and 40, respectively), but no correlation was seen with the TB status of the MDM donor. Clumping of the mycobacteria resulted in more vigorous growth in MDM, suggesting that inoculum size could affect disease progression. The growth rates of 17 clinical isolates of M. tuberculosis were measured in macrophages derived from three donors and no consistent or marked differences between isolates were observed over the 5-day period of growth measurement. However, all 17 clinical strains grew consistently faster than H37Rv in the same experiments.     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum , like Mycobacterium tuberculosis , is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200–1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2–20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis , Mycobacterium bovis BCG and Mycobacterium tuberculosis . These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.     

Molecular cloning and sequencing of the merozoite surface antigen 2 gene from Plasmodium falciparum strain FCC-1/HN and expression of the gene in mycobacteria

Strain bacillus Calmette-Guerin (BCG) of Mycobacterium bovis has been used as a live bacterial vaccine to immunize more than 3 billion people against tuberculosis. In an attempt to use this vaccine strain as a vehicle for protective antigens, the gene encoding merozoite surface antigen 2 (MSA2) was amplified from strain FCC-1/HN Plasmodium falciparum genome, sequenced, and expressed in M. bovis BCG under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) from Mycobacterium tuberculosis. The recombinant shuttle plasmid pBCG/MSA2 was introduced into mycobacteria by electroporation, and the recombinant mycobacteria harboring pBCG/MSA2 could be induced by heating to express MSA2; the molecular mass of recombinant MSA2 was about 31 kDa. This first report of expression of the full-length P. falciparum MSA2 gene in BCG provides evidence for use of the HSP70 promoter in expressing a foreign gene in BCG and in development of BCG as a multivalent vectoral vaccine for malaria.     

A family of autocrine growth factors in Mycobacterium tuberculosis

Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross-species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinity-purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo. The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.     

Bacterial luciferase is naturally destabilized in Mycobacterium tuberculosis and can be used to monitor changes in gene expression

Reporter systems efficient at monitoring temporal gene expression in slow-growing mycobacteria would significantly aid the characterization of gene expression in specific environments. Bacterial luciferase is a reporter that has not been widely used to study gene expression in mycobacteria. This report describes the determination of the degradation of bacterial luciferase in Mycobacterium tuberculosis H37Ra and its utility as a reporter of temporal gene expression in this slow-growing mycobacterium. The inducible/repressible alanine dehydrogenase promoter of M. tuberculosis H37Rv was used to track the decay kinetics of Vibrio harveyi luciferase in both mid-log phase and stationary phase grown M. tuberculosis H37Ra, which proved to be highly similar during both phases of growth. The luciferase reporter was then used to detect changes in expression from the heat-shock promoter, phsp60, of M. bovis BCG during M. tuberculosis H37Ra growth in culture. Quantitative real-time PCR analysis of groEL2, the hsp60 homologue in M. tuberculosis, displayed a similar pattern of expression to phsp60-driven luciferase. These results strongly suggest that the luciferase reporter can be used to monitor temporal changes in gene expression in M. tuberculosis and may serve as a novel system to examine gene expression under specific conditions.     

应用多重PCR方法检测石蜡包埋组织标本中的分枝杆菌DNA

应用多重PCR方法检测并鉴别石蜡包埋组织中的结核分枝杆菌复合体与非结核分枝杆菌DNA扩增片段类型 ,为结核分枝杆菌复合体感染与非结核分枝杆菌感染的病理学诊断提供一种补充的鉴别诊断方法。应用三对具有特异性的寡核苷酸引物 ,进行多重PCR扩增。这三对引物分别对应于分枝杆菌 6 5kD表面抗原、结核分枝杆菌插入序列IS6 1 1 0及人类β 珠蛋白基因的部分序列 ,其扩增产物分别为 3 83bp、1 2 3bp和 2 6 8bp。此种多重PCR方法检测的灵敏度为 0 6pg。经多重PCR扩增后进行凝胶电泳 ,结核分枝杆菌复合体 (结核分枝杆菌、牛型结核分枝杆菌、BCG)均可见 3 83bp、1 2 3bp片段 ,而非结核分枝杆菌 (鸟、龟、瘰疬、蟾蜍、堪萨斯、胞内、耻垢分枝杆菌 )仅见 3 83bp片段 (猿猴分枝杆菌与结核分枝杆菌复合体相同 )。与上述相比 ,分枝杆菌感染的临床标本分别增加了一条 2 6 8bp片段。对 2 0 9例临床初步诊断为淋巴结结核病人的石蜡包埋组织标本进行了多重PCR检测 ,1 93例病理诊断为淋巴结结核、结核性肉芽组织、结核性肉芽肿性炎症病人的标本 ,检测结果符合结核分枝杆菌复合体感…     

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.     

Low molecular mass protein patterns in mycobacterial culture filtrates and purified protein derivatives

Mycobacterial polypeptides from 2 kDa to 14 kDa may be involved in host response to infection and be useful for new diagnostics and vaccines. Tris-tricine SDS-polyacrylamide gel electrophoresis separation of proteins in tuberculin purified protein derivative (PPD) and in 6-8-week culture filtrates of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and other Mycobacterium species demonstrated as many as 10 low molecular mass bands. Common and distinct bands were observed among different species and PPD. These low molecular mass culture filtrate proteins may represent potential diagnostic reagents and vaccines for Mycobacterium tuberculosis.