Molecular Detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in Soil

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 × 10³ to 3.6 × 10³ gene copies g of soil⁻¹, depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37°C with moist soil (−20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.     

Molecular detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in soil

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 x 10(3) to 3.6 x 10(3) gene copies g of soil(-1), depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37 degrees C with moist soil (-20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.     

The effect of oxygenated mycolic acid composition on cell wall function and macrophage growth in Mycobacterium tuberculosis

There are three major structural classes of mycolic acids in the cell envelope of Mycobacterium tuberculosis (MTB): alpha-, methoxy- and ketomycolate. The two oxygen-containing classes are biosynthetically related through a common α-methyl hydroxymycolate intermediate. BCG strains that fail to produce methoxymycolate and instead produce only keto- and alpha-mycolic acids show apparent defects in the O -methyltransferase MMAS-3. Overproduction of MMAS-3 from MTB resulted in a complete replacement of ketomycolate by methoxymycolate in both BCG and MTB. In vitro growth of these recombinant strains lacking ketomycolate was impaired at reduced temperatures but appeared to be normal at 37°C. Glucose uptake was significantly decreased in such strains, but uptake of chenodeoxycholate and glycine was unaffected. Although sensitivity to INH remained unchanged, these cells were found to be hypersensitive to ampicillin and rifampicin. Infectivity of BCG and H37Rv wild type or MMAS-3 overproducers in THP-1 cells was somewhat affected, but the ability of the strains lacking ketomycolate to grow within this macrophage-like cell line was severely compromised. In vivo labelling of mycolic acids during growth of H37Rv within THP-1 cells revealed a substantial increase in ketomycolate and alphamycolate synthesized by intracellularly grown mycobacteria. These results establish a critical role for mycolate composition in proper cell wall function during the growth of MTB in vivo .     

DNA restriction endonuclease analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG

DNA preparations from two reference (H37Ra and H37Rv) and two wild strains of Mycobacterium tuberculosis and one re-isolated strain of Mycobacterium bovis BCG were analysed using 17 restriction endonucleases. The enzyme BstEII revealed the greatest differences between strains. Electrophoretic DNA patterns from the wild M. tuberculosis strains differed from each other and from the reference strains at relatively few positions. At the highest resolution attained, patterns from the two reference strains remained indistinguishable from each other. The pattern of the M. bovis BCG strain was substantially different from, but had many bands in common with, the M. tuberculosis patterns.     

Catalase-peroxidase of Mycobacterium bovis BCG converts isoniazid to isonicotinamide, but not to isonicotinic acid: differentiation parameter between enzymes of Mycobacterium bovis BCG and Mycobacterium tuberculosis

Isonicotinic acid hydrazide (Isoniazid, INH) is one of the major drugs worldwide used in the chemotherapy of tuberculosis. Many investigators have emphasized that INH activation is associated with mycobacterial catalase-peroxidase (katG). However, INH activation mechanism is not completely understood. In this study, katG of M. bovis BCG was separated and purified into two katGs, katG I (named as relatively higher molecular weight than katG II) and katG II, indicating that there is some difference in protein structure between two katGs. The molecular weight of the enzymes of katG I and katG II was estimated to be approximately 150,000 Da by gel filtration, and its subunit was 75,000 Da as determined by SDS-PAGE, indicating that purified enzyme was composed of two identical subunits. The specific activity of the purified enzyme katG I was 991.1 (units/mg). The enzymes were then investigated in INH activation by using gas chromatography mass spectrometry (GC-MS). The analysis of GC-MS showed that the katG I from M. bovis BCG directly converted INH (Mr, 137) to isonicotinamide (Mr, 122), not to isonicotinic acid (Mr, 123), in the presence or absence of H2O2. Therefore, this is the first report that katG I, one of two katGs with almost same molecular weight existed in M. bovis BCG, converts INH to isonicotinamide and this study may give us important new light on the activation mechanism of INH by KatG between M. bovis BCG and M. tuberculosis.     

The composition of cell wall skeleton and outermost lipids of Mycobacterium vaccae is modified by ethambutol treatment

Ethambutol (EMB) is a first line drug in tuberculosis treatment inhibiting the biosynthesis of arabinogalactan, which is a component of the mycobacterial cell wall. The growth of Mycobacterium vaccae cells in the presence of EMB increases cell wall permeability, which was monitored by beta-sitosterol biotransformation. GC/MS and GLC/MS (gas chromatography/mass spectrometry) analysis revealed dramatic changes in the content of covalently bound mycolic acids and in molar ratio galactose (Gal) to arabinose (Ara) in the cell envelopes of EMB-treated cells. The detected variations in the compositions of fatty acids indicate that both the cell wall skeleton and outer layer (free lipids) are decomposed due to EMB treatment.     

Chemical and ultrastructural investigations of Mycobacterium bovis BCG: implications for the molecular structure of the mycobacterial cell envelope

Abstract The mycobacterial cell wall visualized by transmission electron microscopy (TEM) of thin sections of resin-embedded specimens is generally believed to consist of an electron-dense peptidoglycan, an electron-transparent arabinogalactan-mycolate layer and an electron-dense outer layer (OL). In addition, a pseudocapsule known as the ‘electron-transparent zone’ (ETZ) has been observed after phagocytosis of mycobacteria by macrophages. TEM of thin sections of Mycobacterium bovis BCG, Tice^® substrain, revealed an OL bilayer, each of which measured 2–4 nm in diameter. The intermediate electron-transparent layer varied from 1 to about 250 nm in diameter and appears to be a previously observed oxygen-dependent amorphous integument that consists of hot water-extractable neutral polysaccharides, especially a recently characterized α-glucan, comprising about 12% of the dry cell weight. This and other recent studies of BCG have revealed cell-surface features that may provide a better understanding of the outer mycobacterial cell envelope.     

Immune responses of white-tailed deer (Odocoileus virginianus) to Mycobacterium bovis BCG vaccination

The objective was to evaluate cellular immune response of captive white-tailed deer (Odocoileus virginianus) to live Mycobacterium bovis bacille Calmette Guerin (BCG) vaccination and to determine diagnostic implications of these responses. In vitro proliferative and interferon-gamma (IFN-gamma) responses to M. bovis purified protein derivative (PPD) were detected beginning 9 days postvaccination. Responses to Mycobacterium avium PPD, however, generally exceeded responses to M. bovis PPD. Interferon-gamma responses to M. avium PPD were not detected prior to vaccination nor in nonvaccinated deer, suggesting that vaccination with BCG boosted prior quiescent M. avium-sensitized cells. Both CD4+ and gammadelta T cells from vaccinated deer proliferated in response to M. bovis PPD stimulation. Intradermal administration of M. bovis PPD resulted in increases in skin thickness of vaccinated deer beginning 24 hr postinjection. Such early reactions were characterized by edema and minimal mononuclear cell infiltration, whereas later reactions (i.e., 72 hr postinjection) were more typical of delayed type hypersensitivity. Upon in vitro activation with pokeweed mitogen, CD44 expression increased and CD62L expression decreased on lymphocytes from deer regardless of vaccination status. Likewise, M. bovis PPD stimulation of lymphocytes from vaccinated deer resulted in increases in CD44 expression and decreases in CD62L expression. These findings demonstrate the potential of BCG vaccination to elicit strong cell-mediated immune responses and appropriate alterations in CD44 and CD62L expression with in vitro stimulation of white-tailed deer lymphocytes. In relation to M. bovis diagnosis, vaccination of white-tailed deer with BCG can induce skin test responses that classify the animal as a tuberculosis reactor. In contrast, BCG vaccination will likely not interfere with tuberculosis testing by the IFN-gamma assay.     

Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis

The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen’s persistence.     

Structural-functional relationship of pathogen-associated molecular patterns: lessons from BCG cell wall skeleton and mycoplasma lipoprotein M161Ag

The innate immune system senses microbial components by signaling receptors and induces phagocytosis by uptake receptors. The Toll-like receptor represents the signaling receptors that cause maturation of dendritic cells, while phagocytosis is supported by other receptor families. We identify the structural signatures of microbial components recognized by these receptors to establish the two-receptor hypothesis in innate immunity.     

Evidence for a small anion-selective channel in the cell wall of Mycobacterium bovis BCG besides a wide cation-selective pore.

Two channels were observed in extracts of whole Mycobacterium bovis BCG cells using organic solvents and detergents. The channels derived from organic solvent treatment had a single-channel conductance of about 4.0 nS in 1 M KCl in lipid bilayer membranes with properties similar to those of the channels discovered previously in Mycobacterium smegmatis and Mycobacterium chelonae. The channel was in its open configuration only at low transmembrane potentials. At higher voltages it switched to closed states that were almost impermeable for ions. Lipid bilayer experiments in the presence of detergent extracts of whole cells revealed another channel with a single-channel conductance of only 780 pS in 1 M KCl. Our results indicate that the mycolic acid layer of M. bovis BCG contains two channels, one is cation-selective and its permeability properties can be finely controlled by cell wall asymmetry or potentials. The other one is anion-selective, has a rather small single-channel conductance and is voltage-insensitive. The concentration of channel-forming proteins in the cell wall seems to be small, which is in agreement with the low cell wall permeability for hydrophilic solutes.     

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).     

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.     

Demonstration that fbiC is required by Mycobacterium bovis BCG for coenzyme F(420) and FO biosynthesis

Using the nitroimidazopyran-based antituberculosis drug PA-824 as a selective agent, transposon-generated Mycobacterium bovis strain BCG (M. bovis) mutants that could not make coenzyme F(420) were identified. Four independent mutants that could not make F(420) or the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in the M. bovis homologue of the Mycobacterium tuberculosis gene Rv1173, which we have named fbiC. Complementation of an M. bovis FbiC(-) mutant with fbiC restored the F(420) phenotype. These data demonstrate that fbiC is essential for F(420) production and that FbiC participates in a portion of the F(420) biosynthetic pathway between pyrimidinedione and FO. Homologues of fbiC were found in all 11 microorganisms that have been fully sequenced and that are known to make F(420). Four of these homologues (all from members of the aerobic actinomycetes) coded for proteins homologous over the entire length of the M. bovis FbiC, but in seven microorganisms two separate genes were found to code for proteins homologous with either the N-terminal or C-terminal portions of the M. bovis FbiC. Histidine-tagged FbiC overexpressed in Escherichia coli produced a fusion protein of the molecular mass predicted from the M. bovis BCG sequence (approximately 95,000 Da), as well as three other histidine-tagged proteins of significantly smaller size, which are thought to be proteolysis products of the FbiC fusion protein.     

Cloning and sequence analysis of the gene encoding an NADP-dependent alcohol dehydrogenase in Mycobacterium bovis BCG.

The nucleotide sequence of a 1619-bp fragment of Mycobacterium bovis BCG containing the gene that encodes an alcohol dehydrogenase (ADH) has been determined. The M(r) calculated from the deduced amino acid (aa) sequence, as well as the N terminus, are in good accordance with those determined for the ADH purified from M. bovis BCG extracts. The M. bovis BCG cloned adh gene was expressed in Escherichia coli by its own promoter and the synthesized product shows ADH activity in the butane-1-ol-NADP system. Based on comparison of the aa sequence, this enzyme belongs to the zinc-containing, long-chain alcohol/polyol dehydrogenase family, which has been primarily described in eukaryotes. Of the 22 strictly conserved residues in this group, 19 are also conserved in M. bovis BCG ADH (BCGADH).