Cholesterol oxidase ChoD is not a critical enzyme accounting for oxidation of sterols to 3-keto-4-ene steroids in fast-growing Mycobacterium sp. VKM Ac-1815D

Fast-growing strain of Mycobacterium sp. VKM Ac-1815D is capable of effective oxidizing of sterols (phytosterol, cholesterol, ergosterol) to androstenedione and other valuable 3-oxo-steroids. To elucidate the role of cholesterol oxidase in sterol catabolism by the strain, the choD gene has been cloned and sequenced. The deduced gene product (M(r) 63.5kDa) showed homologies over its entire length to a large number of proteins belonging to the InterPro-family EPR006076, which includes various FAD dependent oxidoreductases. The expression of choD in Escherichia coli was shown to result in the synthesis of membrane associated cholesterol oxidase. In addition to cholesterol, the enzyme oxidized β-sitosterol, dehydroepiandrosterone, ergosterol, pregnenolone, and lithocholic acid. Knock-out of choD in Mycobacterium sp. VKM Ac-1815D strain was obtained by the gene replacement technique. The mutant strain transformed sitosterol forming exclusively 3-keto-4-ene steroids with androstenedione as a major product, thus evidencing that choD knock out did not abrogate sterol A-ring oxidation. The results indicated that ChoD is not a critical enzyme responsible for modification of 3β-hydroxy-5-ene- to 3-keto-4-ene steroids in Mycobacterium sp. VKM Ac-1815D. Article from a special issue on steroids and microorganisms.     

Initial step in the catabolism of cholesterol by Mycobacterium smegmatis mc2 155

The first step in the catabolism of cholesterol, i.e. the transformation of cholesterol into cholestenone, has been investigated in Mycobacterium smegmatis. In silico analysis identified the MSMEG_1604 gene encoding a putative protein similar to the ChoD cholesterol oxidase of M. tuberculosis H37Rv (Rv3409c) and the MSMEG_5228 gene coding for a protein similar to the NAD(P)-dependent cholesterol dehydrogenase/isomerase of Nocardia sp. The expression of the MSMEG_5228 gene was inducible by cholesterol whereas the expression of MSMEG_1604 gene was constitutive. When both genes were expressed in Escherichia coli only the MSMEG_5228 protein was active on cholesterol. The function of ChoD-like MSMEG_1604 protein remains to be elucidated, but it does not appear to play a critical role in the mineralization of cholesterol as a MSMEG_1604(-) mutant was not affected in the production of cholestenone. However, a MSMEG_5228(-) mutant showed a drastic reduction in the synthesis of cholestenone. The finding that this mutant was still able to grow in cholesterol, allowed us to demonstrate that the cholesterol-inducible MSMEG_5233 gene encodes an additional cholesterol dehydrogenase/isomerase similar to the AcmA dehydrogenase of Sterolibacterium denitrificans. The observation that the double MSMEG_5228-5233(-) mutant was able to grow in cholesterol suggests that in addition to these enzymes other dehydrogenase/isomerases can also catalyse the first reaction of the cholesterol degradation pathway in M. smegmatis, which is not the limiting step of the process.     

Monitoring promoter activity and protein localization in Mycobacterium spp. using green fluorescent protein

Two green fluorescent protein (Gfp) fusion vectors were constructed for use in Mycobacterium spp. The first plasmid facilitates quantification of mycobacterial promoter activity. The second vector permits construction of translational fusions of mycobacterial proteins to Gfp in order to study subcellular localization including protein secretion. Using this translational fusion construct, we verify that a Gfp fusion to the putative secreted M. tuberculosis protein ChoD is translocated to the extracellular milieu when cloned and expressed in Mycobacterium smegmatis.     

Targeting of Mycobacterium tuberculosis heparin-binding hemagglutinin to mitochondria in macrophages

Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA), a virulence factor involved in extrapulmonary dissemination and a strong diagnostic antigen against tuberculosis, is both surface-associated and secreted. The role of HBHA in macrophages during M. tuberculosis infection, however, is less well known. Here, we show that recombinant HBHA produced by Mycobacterium smegmatis effectively induces apoptosis in murine macrophages. DNA fragmentation, nuclear condensation, caspase activation, and poly (ADP-ribose) polymerase cleavage were observed in apoptotic macrophages treated with HBHA. Enhanced reactive oxygen species (ROS) production and Bax activation were essential for HBHA-induced apoptosis, as evidenced by a restoration of the viability of macrophages pretreated with N-acetylcysteine, a potent ROS scavenger, or transfected with Bax siRNA. HBHA is targeted to the mitochondrial compartment of HBHA-treated and M. tuberculosis-infected macrophages. Dissipation of the mitochondrial transmembrane potential (ΔΨ(m)) and depletion of cytochrome c also occurred in both macrophages and isolated mitochondria treated with HBHA. Disruption of HBHA gene led to the restoration of ΔΨ(m) impairment in infected macrophages, resulting in reduced apoptosis. Taken together, our data suggest that HBHA may act as a strong pathogenic factor to cause apoptosis of professional phagocytes infected with M. tuberculosis.     

Host defense responses to infection by Mycobacterium tuberculosis. Induction of IRF-1 and a serine protease inhibitor

Alveolar macrophages and newly recruited monocytes are targets of infection by Mycobacterium tuberculosis. Therefore, we examined the expression of interferon regulatory factor 1 (IRF-1), which plays an important role in host defense against M. tuberculosis, in undifferentiated and differentiated cells. Infection induced IRF-1 in both. IRF-1 from undifferentiated, uninfected monocytic cell lines was modified during extraction to produce specific species that were apparently smaller than intact IRF-1. After infection by M. tuberculosis or differentiation, intact IRF-1 was recovered. Subcellular fractions were assayed for the ability to modify IRF-1 or inhibit its modification. A serine protease on the cytoplasmic surface of an organelle or vesicle in the "lysosomal/mitochondrial" fraction from undifferentiated cells was responsible for the modification of IRF-1. Thus, the simplest explanation of the modification is cleavage of IRF-1 by the serine protease. Recovery of intact IRF-1 correlated with induction of a serine protease inhibitor that was able to significantly reduce the modification of IRF-1. The inhibitor was present in the cytoplasm of M. tuberculosis-infected or -differentiated cells. It is likely that induction of both IRF-1 and the serine protease inhibitor in response to infection by M. tuberculosis represent host defense mechanisms.     

Loss of a conserved 7-methylguanosine modification in 16S rRNA confers low-level streptomycin resistance in bacteria

Streptomycin has been an important drug for the treatment of tuberculosis since its discovery in 1944. But numerous strains of Mycobacterium tuberculosis, the bacterial pathogen that causes tuberculosis, are now streptomycin resistant. Although such resistance is often mediated by mutations within rrs, a 16S rRNA gene or rpsL, which encodes the ribosomal protein S12, these mutations are found in a limited proportion of clinically isolated streptomycin-resistant M. tuberculosis strains. Here we have succeeded in identifying a mutation that confers low-level streptomycin resistance to bacteria, including M. tuberculosis. We found that mutations within the gene gidB confer low-level streptomycin resistance and are an important cause of resistance found in 33% of resistant M. tuberculosis isolates. We further clarified that the gidB gene encodes a conserved 7-methylguanosine (m(7)G) methyltransferase specific for the 16S rRNA, apparently at position G527 located in the so-called 530 loop. Thus, we have identified gidB as a new streptomycin-resistance locus and uncovered a resistance mechanism that is mediated by loss of a conserved m(7)G modification in 16S rRNA. The clinical significance of M. tuberculosis gidB mutation also is noteworthy, as gidB mutations emerge spontaneously at a high frequency of 10(-6) and, once emerged, result in vigorous emergence of high-level streptomycin-resistant mutants at a frequency more than 2000 times greater than that seen in wild-type strains. Further studies on the precise function of GidB may provide a basis for developing strategies to suppress pathogenic bacteria, including M. tuberculosis.     

A parallel intraphagosomal survival strategy shared by mycobacterium tuberculosis and Salmonella enterica

Mycobacterium tuberculosis and Salmonella enterica cause very different diseases and are only distantly related. However, growth within macrophages is crucial for virulence in both of these intracellular pathogens. Here, we demonstrate that in spite of the phylogenetic distance, M. tuberculosis and Salmonella employ a parallel survival strategy for growth within macrophage phagosomes. Previous studies established that the Salmonella mgtC gene is required for growth within macrophages and for virulence in vivo. M. tuberculosis contains an open reading frame exhibiting 38% amino acid identity with the Salmonella MgtC protein. Upon inactivation of mgtC, the resulting M. tuberculosis mutant was attenuated for virulence in cultured human macrophages and impaired for growth in the lungs and spleens of mice. Replication of the mgtC mutant was inhibited in vitro by a combination of low magnesium and mildly acidic pH suggesting that the M. tuberculosis-containing phagosome has these characteristics. The similar phenotypes displayed by the mgtC mutants of M. tuberculosis and Salmonella suggest that the ability to acquire magnesium is essential for virulence in intracellular pathogens that proliferate within macrophage phagosomes.     

Generation of nitric oxide by the inducible nitric oxide synthase protects gamma delta T cells from Mycobacterium tuberculosis-induced apoptosis.

Gamma delta T cells are early recruited into mycobacterial lesions. Upon microbial Ag recognition, gamma delta cells secrete cytokines and chemokines and undergo apoptosis via CD95/CD95 ligand (CD95L) interaction, possibly influencing the outcome of infection and the characteristics of the disease. In this paper we show that activated phagocytes acquire, upon challenge with Mycobacterium tuberculosis, the ability to inhibit M. tuberculosis-induced gamma delta cell apoptosis. Apoptosis protection was due to NO because it correlated with NO synthase (NOS)-2 induction and activity in scavenger cells and was abrogated by NOS inhibitors. Furthermore, the NO donor S-nitrosoacetylpenicillamine mimicked the effect of enzyme induction. NO left unaffected the expression of CD95 and CD95L, suggesting interference with an event ensuing CD95/CD95L interaction. NO was found to interfere with the intracellular accumulation of ceramide and the activation of caspases, which were involved in gamma delta T cells apoptosis after M. tuberculosis recognition. We propose that NO generated by infected macrophages determines the life span and therefore the function of lymphocytes at the infection site, thus linking innate and adaptive immunity.     

Inactivation of the Mycobacterium tuberculosis fadB4 gene results in increased virulence in host cell and mice

The genome of Mycobacterium tuberculosis encodes many proteins involved in fatty acid metabolism, a subset of which are required for virulence. The Mycobacterium tuberculosis fadB4 gene, which shares strong similarity with oxidoreductases and fatty acid synthases, is up-regulated during infection of macrophages and is predicted to protect the bacterium from the hostile environment of the host cell. In order to determine if fadB4 plays a role in the virulence of M. tuberculosis, we constructed a M. tuberculosis mutant in which the fadB4 had been disrupted (DeltafadB4). Surprisingly, DeltafadB4, grew more rapidly in host cells compared to wild-type M. tuberculosis or the DeltafadB4 or the gene-disrupted strain complemented with fadB4. In addition, macrophages infected with DeltafadB4 displayed reduced secretion of the cytokine TNF-alpha, suggesting a role for the FadB4 protein in influencing the pro-inflammatory host response to M. tuberculosis. After infection of mice, DeltafadB4 demonstrated an increased replication at early time-points post-infection compared to the growth of wild-type M. tuberculosis. This increased capacity of DeltafadB4 to replicate in vivo was reflected in the decreased time to death of immuno-deficient RAG-1(-/-) mice infected with M. tuberculosis lacking the fadB4 gene. Therefore fadB4 is part of the family of genes whose expression serves to regulate the virulence of M. tuberculosis within the host.     

Mycobacterium tuberculosis triggers host type I IFN signaling to regulate IL-1β production in human macrophages

Mycobacterium tuberculosis is a virulent intracellular pathogen that survives in macrophages even in the presence of an intact adaptive immune response. Type I IFNs have been shown to exacerbate tuberculosis in mice and to be associated with disease progression in infected humans. Nevertheless, the mechanisms by which type I IFNs regulate the host response to M. tuberculosis infection are poorly understood. In this study, we show that M. tuberculosis induces an IFN-related gene expression signature in infected primary human macrophages, which is dependent on host type I IFN signaling as well as the mycobacterial virulence factor, region of difference-1. We further demonstrate that type I IFNs selectively limit the production of IL-1β, a critical mediator of immunity to M. tuberculosis. This regulation occurs at the level of IL1B mRNA expression, rather than caspase-1 activation or autocrine IL-1 amplification and appears to be preferentially used by virulent mycobacteria since avirulent M. bovis bacillus Calmette-Guérin (BCG) fails to trigger significant expression of type I IFNs or release of mature IL-1β protein. The latter property is associated with decreased caspase-1-dependent IL-1β maturation in the BCG-infected macrophages. Interestingly, human monocytes in contrast to macrophages produce comparable levels of IL-1β in response to either M. tuberculosis or BCG. Taken together, these findings demonstrate that virulent and avirulent mycobacteria employ distinct pathways for regulating IL-1β production in human macrophages and reveal that in the case of M. tuberculosis infection the induction of type I IFNs is a major mechanism used for this purpose.     

Coronin-1a inhibits autophagosome formation around Mycobacterium tuberculosis-containing phagosomes and assists mycobacterial survival in macrophages

Mycobacterium tuberculosis is an intracellular bacterium that can survive within macrophages. Such survival is potentially associated with Coronin-1a (Coro1a). We investigated the mechanism by which Coro1a promotes the survival of M. tuberculosis in macrophages and found that autophagy was involved in the inhibition of mycobacterial survival in Coro1a knock-down (KD) macrophages. Fluorescence microscopy and immunoblot analyses revealed that LC3, a representative autophagic protein, was recruited to M. tuberculosis-containing phagosomes in Coro1a KD macrophages. Thin-section electron microscopy demonstrated that bacilli were surrounded by the multiple membrane structures in Coro1a KD macrophages. The proportion of LC3-positive mycobacterial phagosomes colocalized with p62/SQSTM1, ubiquitin or LAMP1 increased in Coro1a KD macrophages during infection. These results demonstrate the formation of autophagosomes around M. tuberculosis in Coro1a KD macrophages. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) was induced in response to M. tuberculosis infection in Coro1a KD macrophages, suggesting that Coro1a blocks the activation of the p38 MAPK pathway involved in autophagosome formation. LC3 recruitment to M. tuberculosis-containing phagosomes was also observed in Coro1a KD alveolar or bone marrow-derived macrophages. These results suggest that Coro1a inhibits autophagosome formation in alveolar macrophages, thereby facilitating M. tuberculosis survival within the lung.     

The central helices of ApoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-type ApoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo

We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by approximately 80-92% by apoA-I[delta(185-243)], only by 15% by apoA-I[delta(1-41)] or apoA-I[delta(1-59)], and was restored to 75-80% of the wild-type apoA-I control value by double deletion mutants apoA-I[delta(1-41)delta(185-243)] and apoA-I[delta(1-59)delta(185-243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[delta(232-243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30-40% and 50-65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[delta(1-41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[delta(1-41)delta(185-243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220-231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.     

Stimulation of a major subset of lymphocytes expressing T cell receptor gamma delta by an antigen derived from Mycobacterium tuberculosis

To investigate the possible function(s) of T cell receptor (TcR) gamma delta expressing lymphocytes, we generated a series of gamma delta TcR surface positive hybridomas. Spontaneous producers of IL-2 were quite common among these hybridomas, particularly those expressing a certain V delta gene or gene family (V delta M23). Several other experiments indicated that IL-2 production in these hybridomas is triggered via TcR gamma delta. Surprisingly, every spontaneously reactive gamma delta+ hybridoma was further stimulated by purified protein derivative (PPD) of Mycobacterium tuberculosis, perhaps due to crossreaction with a bacterial antigen homologous to certain eukaryotic heat shock proteins. The finding of an antigen recognized by a gamma delta TcR could aid in understanding the functional role of the gamma delta TcR+ lymphocytes.