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The innate immune response plays an important but unknown role in host defense against Mycobacterium tuberculosis . To define the function of innate immunity during tuberculosis, we evaluated M. tuberculosis replication dynamics during murine infection. Our data show that the early pulmonary innate immune response limits M. tuberculosis replication in a MyD88-dependent manner. Strikingly, we found that little M. tuberculosis cell death occurs during the first 2 weeks of infection. In contrast, M. tuberculosis cells deficient in the surface lipid phthiocerol dimycocerosate (PDIM) exhibited significant death rates, and consequently, total bacterial numbers were reduced. Host restriction of PDIM-deficient M. tuberculosis was not alleviated by the absence of interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), or the phagocyte oxidase subunit p47. Taken together, these data indicate that PDIM protects M. tuberculosis from an early innate host response that is independent of IFN-γ, reactive nitrogen intermediates, and reactive oxygen species. By employing a pathogen replication tracking tool to evaluate M. tuberculosis replication and death during infection, we identify both host and pathogen factors affecting the outcome of infection.

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.

The RD1 genomic region is present in virulent strains of Mycobacterium tuberculosis (MTB), missing from the vaccine strain M. bovis BCG, and its importance to virulence has been established experimentally. Based on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this region affects virulence is unknown. Here we examined mutants disrupted in five individual RD1 genes. Both in vitro and in vivo, each mutant displayed an attenuated phenotype very similar to a mutant missing the entire RD1 region. Genetic complementation of individual genes restored virulence. Attenuated mutants could multiply within THP-1 cells, but they were unable to spread to uninfected macrophages. We also examined export of two immunodominant RD1 proteins, CFP-10 and ESAT-6. Export of these proteins was greatly reduced or abolished in each attenuated mutant. Again, genetic complementation restored a wild-type phenotype. Our results indicate that RD1 genes work together to form a single virulence determinant, and argue that RD1 encodes a novel specialized secretion system that is required for pathogenesis of MTB.

Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20–30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 ‘dormant’ DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing global models of coordinated gene expression., Adaptation of Mycobacterium tuberculosis to the host environment is principally mediated through its transcription factors. Here, the authors report the DNA binding and transcriptional profile of ~80% of all predicted M. tuberculosis transcription factors, and find wide-spread dormant DNA binding.

Background Mycobacterium tuberculosis senses and responds to the shifting and hostile landscape of the host. To characterize the underlying intertwined gene regulatory network governed by approximately 200 transcription factors of M. tuberculosis, we have assayed the global transcriptional consequences of overexpressing each transcription factor from an inducible promoter. Results We cloned and overexpressed 206 transcription factors in M. tuberculosis to identify the regulatory signature of each. We identified 9,335 regulatory consequences of overexpressing each of 183 transcription factors, providing evidence of regulation for 70% of the M. tuberculosis genome. These transcriptional signatures agree well with previously described M. tuberculosis regulons. The number of genes differentially regulated by transcription factor overexpression varied from hundreds of genes to none, with the majority of expression changes repressing basal transcription. Exploring the global transcriptional maps of transcription factor overexpressing (TFOE) strains, we predicted and validated the phenotype of a regulator that reduces susceptibility to a first line anti-tubercular drug, isoniazid. We also combined the TFOE data with an existing model of M. tuberculosis metabolism to predict the growth rates of individual TFOE strains with high fidelity. Conclusion This work has led to a systems-level framework describing the transcriptome of a devastating bacterial pathogen, characterized the transcriptional influence of nearly all individual transcription factors in M. tuberculosis, and demonstrated the utility of this resource. These results will stimulate additional systems-level and hypothesis-driven efforts to understand M. tuberculosis adaptations that promote disease. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0502-3) contains supplementary material, which is available to authorized users.

As a strategy to increase the penetration of antibiotic drugs through the outer membrane of gram-negative pathogens, facilitated transport through siderophore receptors has been frequently exploited. Hydroxamic acids, catechols, or very close isosteres of catechols, which are mimics of naturally occurring siderophores, have been used successfully as covalently linked escorting moieties, but a much wider diversity of iron binding motifs exists. This observation, coupled to the relative lack of specificity of siderophore receptors, prompted us to initiate a program to identify novel, noncatechol siderophoric structures. We screened over 300 compounds for their ability to (1) support growth in low iron medium of a Pseudomonas aeruginosa siderophore biosynthesis deletion mutant, or (2) compete with a bactericidal siderophore-antibiotic conjugate for siderophore receptor access. From these assays we identified a set of small molecules that fulfilled one or both of these criteria. We then synthesized these compounds with functional groups suitable for attachment to both monobactam and cephalosporin core structures. Siderophore-beta-lactam conjugates then were tested against a panel of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus strains. Although several of the resultant chimeric compounds had antimicrobial activity approaching that of ceftazidime, and most compounds demonstrated very potent activity against their cellular targets, only a single compound was obtained that had enhanced, siderophore-mediated antibacterial activity. Results with tonB mutants frequently showed increased rather than decreased susceptibilities. suggesting that multiple factors influenced the intracellular concentration of the drugs.

Region of difference (RD1) genes are present in virulent Mycobacterium tuberculosis but not the vaccine strain M. bovis bacille Calmette-Guerin (BCG). The deletion of RD1 from M. tuberculosis produces an attenuation strikingly like that of BCG, which suggests the use of RD1 mutant strains for improvement of the tuberculosis (TB) vaccine. We performed long-term murine infection with M. tuberculosis H37Rv: Delta RD1 and BCG. Mice infected with H37Rv: Delta RD1 gained less weight than did BCG-infected control mice, and, after >1 year, their lungs harbored many more bacteria and displayed significant levels of inflammation. This difference in virulence has important implications for the pursuit of strains lacking RD1 in the development of the TB vaccine.

The tuberculosis (TB) vaccine bacille Calmette-Guerin (BCG) is a live attenuated organism, but the mutation responsible for its attenuation has never been defined. Recent genetic studies identified a single DNA region of difference, RD1, which is absent in all BCG strains and present in all Mycobacterium tuberculosis (MTB) strains. The 9 open-reading frames predicted within this 9.5-kb region are of unknown function, although they include the TB-specific immunodominant antigens ESAT-6 and CFP-10. In this study, RD1 was deleted from MTB strain H37Rv, and virulence of H37Rv:DeltaRD1 was assessed after infections of the human macrophage-like cell line THP-1, human peripheral blood monocyte-derived macrophages, and C57BL/6 mice. In each of these systems, the H37Rv:DeltaRD1 strain was strikingly less virulent than MTB and was very similar to BCG controls. Therefore, it was concluded that genes within or controlled by RD1 are essential for MTB virulence and that loss of RD1 was important in BCG attenuation.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidant proteins with alkylhydroperoxidase (Ahp) and thioredoxin-dependent peroxidase (TPx) activities. AhpC expression is associated with resistance to the front-line antitubercular drug isoniazid in the naturally resistant organisms E. coli and M. smegmatis. We identified several isoniazid-resistant M. tuberculosis isolates with ahpC promoter mutations resulting in AhpC overexpression. These strains were more resistant to cumene hydroperoxide than were wild-type strains. However, these strains were unchanged in their sensitivity to isoniazid, refuting a role for AhpC in detoxification of this drug. All the isoniazid-resistant, AhpC-overexpressing strains were also deficient in activity of the mycobacterial catalase-peroxidase KatG. KatG, the only known catalase in M. tuberculosis, is required for activation of isoniazid. We propose that compensatory ahpC promoter mutations are selected from KatG-deficient, isoniazid-resistant M. tuberculosis during infections, to mitigate the added burden imposed by organic peroxides on these strains.

Pseudomonas aeruginosa can employ many distinct mechanisms of resistance to aminoglycoside antibiotics; however, in cystic fibrosis patients, more than 90% of aminoglycoside-resistant P. aeruginosa isolates are of the impermeability phenotype. The precise molecular mechanisms that produce aminoglycoside impermeability-type resistance are yet to be elucidated. A subtractive hybridization technique was used to reveal gene expression differences between PAO1 and isogenic, spontaneous aminoglycoside-resistant mutants of the impermeability phenotype. Among the many genes found to be up-regulated in these laboratory mutants were the amrAB genes encoding a recently discovered efflux system. The amrAB genes appear to be the same as the recently described mexXY genes; however, the resistance profile that we see in P. aeruginosa is very different from that described for Escherichia coli with mexXY . Direct evidence for AmrAB involvement in aminoglycoside resistance was provided by the deletion of amrB in the PAO1-derived laboratory mutant, which resulted in the restoration of aminoglycoside sensitivity to a level nearly identical to that of the parent strain. Furthermore, transcription of the amrAB genes was shown to be up-regulated in P. aeruginosa clinical isolates displaying the impermeability phenotype compared to a genotypically matched sensitive clinical isolate from the same patient. This suggests the possibility that AmrAB-mediated efflux is a clinically relevant mechanism of aminoglycoside resistance. Although it is unlikely that hyperexpression of AmrAB is the sole mechanism conferring the impermeability phenotype, we believe that the Amr efflux system can contribute to a complex interaction of molecular events resulting in the aminoglycoside impermeability-type resistance phenotype.

Although the 16-kDa α-crystallin homologue ofMycobacterium tuberculosis(MTB) is the dominant protein produced by stationary phase culturesin vitro, it is undetectable in logarithmically growing cultures. By growing bacilli at defined oxygen concentrations,acrtranscription was shown to be strongly induced by mildly hypoxic conditions. Acr expression also was found to be induced during the course ofin vitroinfection of macrophages. Theacrgene was replaced with a hygromycin resistance cassette by allelic exchange in MTB H37Rv. The resulting Δacr∷hptstrain was shown to be equivalent to wild-type H37Rv inin vitrogrowth rate and infectivity but was significantly impaired for growth in both mouse bone marrow derived macrophages and THP-1 cells. In addition to its proposed role in maintenance of long-term viability during latent, asymptomatic infections, these results establish a role for the Acr protein in replication during initial MTB infection.

The object of this study was to investigate the ability of a rapid luciferase assay to detect antimycobacterial activity in plant extracts. Recombinant strains of Mycobacterium bovis BCG (rBCG) and Mycobacterium intracellulare expressing firefly luciferase were used as the test organisms. Assays were conducted in a 96-well minitube format under biosafety level 2 conditions. Control and test wells were sampled immediately after inoculation and after 3 (recombinant M. intracellulare) and 5 (rBCG) days of incubation to measure luminescence with a microplate luminometer, and the relative change in luminescence was calculated as a percentage of control values. As an alternative test method, Alamar blue was added after 12 days of incubation, and changes in color were read visually. A total of 480 extracts were tested. Sixteen extracts were active against rBCG, and of those, seven were also active against recombinant M. intracellulare. With activity defined as a relative change in luminescence of < or = 1% (i.e., > or = 99% inhibition) and a persistence of blue color after addition of Alamar blue, there was 99.0% agreement between the two methods. Our results suggest that the luciferase assay is rapid and accurate and has the potential to greatly accelerate the evaluation of antimycobacterial activity in plant extracts in vitro. With this method, it is possible to screen a large number of samples in a short period of time.

Mutations that eliminate KatG catalase-peroxidase activity prevent activation of isoniazid and are a major mechanism of resistance to this principal drug for the treatment of Mycobacterium tuberculosis infections. However, the loss of KatG activity in clinical isolates seemed paradoxical because KatG is considered an important factor for the survival of the organism. Expression of either KatG or the recently identified alkyl hydroperoxidase AhpC was sufficient to protect bacilli against the toxic effects of organic peroxides. To survive during infection, isoniazid-resistant KatG mutants have apparently compensated for the loss of KatG catalase-peroxidase activity by a second mutation, resulting in hyperexpression of AhpC.

Bioluminescence-based assays to indicate antimicrobial susceptibility have been developed and validated for recombinant strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium avium, and Mycobacterium intracellulare expressing an integrated eukaryotic luciferase gene. MICs determined with these bioluminescence assays for several antimycobacterial agents, including isoniazid, ethambutol, rifampin, amikacin, streptomycin, ciprofloxacin, and clarithromycin, compared favorably with traditional BACTEC methods and visual estimations of the inhibitory end point. Assay methodology has been optimized for the analysis of large numbers of novel compounds and is simple, inexpensive, and labor efficient. The availability of these four recombinant mycobacteria has permitted a strategy for drug discovery employing the nonpathogenic BCG strain for mass screening purposes with subsequent confirmation of activity against the pathogenic mycobacteria. Furthermore, evidence suggests that the BCG-based screen may allow the direct identification of bactericidal agents.

To persist in macrophages and in granulomatous caseous lesions, pathogenic mycobacteria must be equipped to withstand the action of toxic oxygen metabolites. In Gram-negative bacteria, the OxyR protein is a critical component of the oxidative stress response. OxyR is both a sensor of reactive oxygen species and a transcriptional activator, inducing expression of detoxifying enzymes such as catalase/hydroperoxidase and alkyl hydroperoxidase. We have characterized the responses of various mycobacteria to hydrogen peroxide both phenotypically and at the levels of gene and protein expression. Only the saprophytic Mycobacterium smegmatis induced a protective oxidative stress response analogous to the OxyR response of Gram-negative bacteria. Under similar conditions, the pathogenic mycobacteria exhibited a limited, nonprotective response, which in the case of Mycobacterium tuberculosis was restricted to induction of a single protein, KatG. We have also isolated DNA sequences homologous to oxyR and ahpC from M. tuberculosis and Mycobacterium avium. While the M. avium oxyR appears intact, the oxyR homologue of M. tuberculosis contains numerous deletions and frameshifts and is probably nonfunctional. Apparently the response of pathogenic mycobacteria to oxidative stress differs significantly from the inducible OxyR response of other bacteria.

The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels. GPIX is a relatively small (Mr, 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex, GPIb (Mr, 165,000). Using antibody screening, we cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage lambda gt11. Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end. By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A)+ RNA. Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acid followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX. The predicted amino acid sequence of mature GPIX includes an NH2-terminal extracytoplasmic domain of 134 residues, a transmembrane domain of 20 residues, 6 intracytoplasmic residues, and 1 N-linked glycosylation site. GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast. "Flanking" sequences of approximately 22 amino acids are present at the NH2 and/or COOH sides of the "central" LRG sequence(s) in GPIX, GPIb, and the other human and Drosophila members of the LRG family. The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined.

The receptor on human neutrophils (polymorphonuclear leukocytes) that mediates cellular adherence consists of two noncovalently associated subunits, designated alpha M (Mac-1 alpha, Mol alpha, or CD11b; Mr, 170,000) and beta (Mac-1 beta, Mol beta, or CD18; Mr, 100,000). We isolated a cDNA clone for the human neutrophil alpha M subunit by screening a lambda gt 11 cDNA library made from chronic myelogenous leukemia neutrophils by using an affinity-purified rabbit polyclonal antibody directed against the alpha M subunit. We used this cDNA clone to obtain additional clones from cDNA libraries made from differentiated HL-60 promyelocytic leukemia cells. Together these cDNAs constitute the complete 1137-amino acid sequence for the mature human alpha M subunit protein. The deduced amino acid sequence indicates the presence of an extensive extracellular domain with three putative metal-binding regions, (i) an amino acid region that is homologous to the A domain of von Willebrand factor, (ii) a 26-amino acid hydrophobic sequence that is a potential transmembrane domain, and (iii) a 19-amino acid cytoplasmic region. The amino acid sequence for the human neutrophil alpha M subunit contains regions that are closely related to amino acid sequences of adhesion receptors belonging to the integrin family.

We characterized the surface antigen and mRNA expression for the CD11c (αX, p150) subunit of the human leukocyte adherence receptor family during hematopoietic cell differentiation. The CD11c subunit antigen and mRNA are constituitively expressed in undifferentiated HL-60 promyelocytic leukemia cells, and levels increase markedly with differentiation along the monocyte/macrophage pathway using phorbol myristate acetate. Human monocyte-derived macrophages and human alveolar macrophages express elevated levels of the CD11c subunit antigen and mRNA, indicating that the changes observed in vitro are present in vivo . Dot blot analysis of immature and mature lymphoid and myeloid cells and cell lines demonstrate equivalent levels of CD11c mRNA expression. We conclude that CD11c gene expression is selectively increased during hematopoietic cell differentiation along the monocyte/macrophage pathway.

Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III collagen in a saturable (KD = 1 X 10(-8) M), specific, and rapid manner with a stoichiometry of approximately 1:15 [vWf subunit (Mr 270,000):collagen trimer (Mr 300,000)]. Two reduced and alkylated CNBr peptides of vWf, termed M11 residues 542-622 and M20 residues 948-998 [Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., & Fujikawa, K. (1986) Biochemistry 25, 3171-3184], inhibited vWf binding to collagen. With 125I-vWf (2 X 10(-9) M) as ligand, M11, M20, fragment III (a dimeric, V8 protease, NH2-terminal fragment, Mr 320,000 referenced above), and unlabeled vWf inhibited binding to collagen with EC50 values of 4.8 X 10(-7), 9.4 X 10(-7), 1.1 X 10(-7), and 0.2 X 10(-7) M, respectively. M11 and M20 bind to collagen directly when 125I-labeled peptides are used as ligands. Other CNBr fragments of vWf were less effective as inhibitors (5-fold or less) and bound less avidly to collagen (5-fold or less) compared to M11 and M20. A murine anti-human vWf monoclonal antibody (MR5), which blocks the binding of vWf to collagen, bound selectively to both M11 and M20 when tested in an enzyme-linked immunoadsorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to an apparent lack of messenger RNA, precluding the construction of platelet cDNA libraries and hindering the cloning of authentic platelet cDNA's. We reasoned that vestigial amounts of messenger RNA may remain in platelets when they first separate from the megakaryocyte and circulate in the peripheral blood. We isolated poly (A)+ RNA from platelets obtained by pheresis of individuals with elevated blood platelet counts due to a myeloproliferative syndrome termed essential thrombocythemia. Northern blots using probes for platelet glycoprotein Ib indicate that the poly (A)+ RNA obtained from the platelets of these donors is, in fact, derived from platelets. Cell free translation studies using the platelet poly (A)+ RNA indicate that the material is translationally active. We conclude that, contrary to prevailing information, circulating human blood platelets retain appreciable amounts of poly (A)+ RNA and that this RNA can be harvested by the described approach. The poly (A)+ RNA provides templates for the synthesis of cDNA's that code for platelet proteins.

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.

The development of new drugs and vaccines directed against Mycobacterium tuberculosis is severely impeded by the slow growth of this organism and the need to work under stringent biosafety conditions. These difficulties pose considerable obstacles when animal studies with M. tuberculosis are performed. We investigated whether a novel approach termed luciferase in vivo expression, using an enhanced luciferase-expressing mycobacterial strain, could be used to evaluate antimycobacterial activity in mice. Vectors that expressed firefly luciferase (lux gene) at high levels in the bacillus Calmette-Gu-erin (BCG) strain of Mycobacterium bovis were constructed for use in vivo. One recombinant BCG reporter strain (rBCG-lux) was selected for high-level expression of the lux gene product and for its ability to replicate in mice. Methodology to monitor in vivo growth of the rBCG-lux reporter strain in mice by direct assay of luciferase luminescence in organ homogenates was developed. The utility of this approach for assessing the in vivo efficacies of antimycobacterial compounds was evaluated. The activities of standard antimycobacterial drugs were directly apparent in mice infected with the rBCG-lux reporter strain by statistically significant reductions in spleen luminescence. In addition, antimycobacterial immunity was also evident in BCG-immunized mice, in which suppression of rBCG-lux growth in comparison with that in naive mice was clearly observed. The use of luciferase in vivo expression for the in vivo evaluation of antimycobacterial activity compared favorably with standard CFU determinations in terms of time, labor, expense, and statistical significance but permitted the evaluation of antimycobacterial drugs and immunity in mice in 7 days or less. Thus, the use of this technology can greatly accelerate the process of evaluation of antibiotics and immunogens in animal models for the slowly growing pathogenic mycobacteria.

An examination of the pattern of lipid biosynthetic responses to isoniazid (INH) treatment of Mycobacterium tuberculosis and Mycobacterium smegmatis suggests that the mode of action of activated INH differs between these 2 organisms. Transformation of M. smegmatis with inhA on a plasmid construct conferred high-level resistance to INH, while the same construct failed to confer resistance upon M. tuberculosis. The inhA region from 2 clinical isolates whose resistance has been attributed to changes in the upstream promoter region has been cloned and was not sufficient to impart INH resistance to the level of the parent strain on sensitive M. tuberculosis. These putative mutant promoter elements appear to elevate expression levels of gene fusion reporter constructs, suggesting some noncausal connection between the observed mutations and the lipid metabolism of drug-resistant organisms. These results suggest that InhA is not the major target for activated INH in M. tuberculosis.