William R. Jacobs

Genetic systems for mycobacteria

Publisher Summary This chapter discusses genetic systems for mycobacteria. The ability to perform genetic analyses on bacteria has provided powerful tools and experimental systems to unravel fundamental biological processes. The advances of recombinant DNA technologies have ignited the development of genetic systems for bacteria that are difficult to work with. The genus Mycobacterium contains a set of the most difficult bacterial species to manipulate experimentally. The tuberculosis vaccine strain, bacille Calmette Guerin (BCG) has been used to vaccinate more individuals than any other live bacterial vaccine, yet little is known about mycobacterial gene structure and expression. The recent development of phage, plasmid, and gene replacement systems for the introduction of recombinant DNA into mycobacteria has opened up a new era of research on members of the genus Mycobacterium .

Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis

The inhibition of apoptosis of infected host cells is a well-known but poorly understood function of pathogenic mycobacteria. We show that inactivation of the secA2 gene in Mycobacterium tuberculosis, which encodes a component of a virulence-associated protein secretion system, enhanced the apoptosis of infected macrophages by diminishing secretion of mycobacterial superoxide dismutase. Deletion of secA2 markedly increased priming of antigen-specific CD8(+) T cells in vivo, and vaccination of mice and guinea pigs with a secA2 mutant significantly increased resistance to M. tuberculosis challenge compared with standard M. bovis bacille Calmette-Guérin vaccination. Our results define a mechanism for a key immune evasion strategy of M. tuberculosis and provide what we believe to be a novel approach for improving mycobacterial vaccines.

Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis

Tuberculosis treatment is shortened to six months by the indispensable addition of pyrazinamide (PZA) to the drug regimen that includes isoniazid and rifampin. PZA is a pro-drug of pyrazinoic acid (POA) (ref. 3), whose target of action has never been identified. Although PZA is active only against Mycobacterium tuberculosis, the PZA analog 5-chloro-pyrazinamide (5-Cl-PZA) displays a broader range of anti-mycobacterial activity. We have found that the eukaryotic-like fas1 gene (encoding fatty acid synthetase I, FASI) from M. avium, M. bovis BCG or M. tuberculosis confers resistance to 5-Cl-PZA when present on multi-copy vectors in M. smegmatis. 5-Cl-PZA and PZA markedly inhibited the activity of M. tuberculosis FASI, the biosynthesis of C16 to C24/C26 fatty acids from acetyl-CoA (ref. 6). Importantly, PZA inhibited FASI in M. tuberculosis in correlation with PZA susceptibility. These results indicate that FASI is a primary target of action for PZA in M. tuberculosis. Further characterization of FASI as a drug target for PZA may allow the development of new drugs to shorten the therapy against M. tuberculosis and may provide more options for treatment against M. bovis, M. avium and drug resistant M. tuberculosis.

Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice

Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.

Trichoderins, novel aminolipopeptides from a marine sponge-derived Trichoderma sp., are active against dormant mycobacteria

Three new aminolipopeptides, designated trichoderins A (1), A1 (2), and B (3), were isolated from a culture of marine sponge-derived fungus of Trichoderma sp. as anti-mycobacterial substances with activity against active and dormant bacilli. The chemical structures of trichoderins were determined on the basis of spectroscopic study. Trichoderins showed potent anti-mycobacterial activity against Mycobacterium smegmatis, Mycobacterium bovis BCG, and Mycobacterium tuberculosis H37Rv under standard aerobic growth conditions as well as dormancy-inducing hypoxic conditions, with MIC values in the range of 0.02-2.0 microg/mL.

Genome organization and characterization of mycobacteriophage Bxb1

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis. The morphology of Bxb1 particles is similar to that of mycobacteriophages L5 and D29, although Bxb1 differs from these phages in other respects. First, it is heteroimmune with L5 and efficiently forms plaques on an L5 lysogen. Secondly, it has a different host range and fails to infect slow‐growing mycobacteria, using a receptor system that is apparently different from that of L5 and D29. Thirdly, it is the first mycobacteriophage to be described that forms a large prominent halo around plaques on a lawn of M. smegmatis. The sequence of the Bxb1 genome shows that it possesses a similar overall organization to the genomes of L5 and D29 and shares weak but detectable DNA sequence similarity to these phages within the structural genes. However, Bxb1 uses a different system of integration and excision, a repressor with different specificity to that of L5 and encodes a large number of novel gene products including several with enzymatic functions that could degrade or modify the mycobacterial cell wall.

CONSTRUCTION OF D29 SHUTTLE PHASMIDS AND LUCIFERASE REPORTER PHAGES FOR DETECTION OF MYCOBACTERIA

Diseases caused by Mycobacterium tuberculosis, M. leprae and M. avium, cause significant morbidity and mortality worldwide. Effective treatments require that the organisms be speciated and that drug susceptibilities for the causative organisms be characterized. Reporter phage technology has been developed as a rapid and convenient method for identifying mycobacterial species and evaluating drug resistance. In this report we describe the construction of luciferase reporter phages from mycobacteriophage D29 DNA. Shuttle phasmids were first constructed with D29 in order to identify non-essential regions of the D29 genomes and to introduce unique cloning sites within that region. Using this approach, we observed that all of the D29 shuttle phasmids had the cosmid vector localized to one area of the phage genome near one cohesive end. These shuttle phasmids had been constructed with a cosmid that could be readily excised from the D29 genome with different sets of restriction enzymes. Luciferase reporter phages were made by substituting the luciferase cassette for the cosmid vector. Recombinant phages with the luciferase cassette fall into two groups. One group produced light and had the expression cassette oriented with the promoter directing transcription away from the cohesive end. In contrast, the other group had the expression cassette in the opposite orientation and failed to produce light during lytic infection, but did produce light in L5 lysogens which are known to repress D29 promoters. These results suggest that a phage promoter of the D29 phage can occlude the expression of a promoter introduced into this region. D29 luciferase reporter phages are capable of detecting low numbers of L5 lysogens like L5 luciferase phages. However, unlike L5 luciferase phages, D29 luciferase phages can readily infect M. tuberculosis and M. bovis BCG, demonstrating that these phages can be used to evaluate drug susceptibilities of many types of mycobacteria.

3 Persisting problems in tuberculosis

Publisher Summary This chapter discusses the persisting problems in tuberculosis. The evolutionary origins of Mycobacterium tuberculosis , the bacterium that causes human tuberculosis, are uncertain. Tuberculosis may have originated in prehistoric humans as a zoonotic infection transmitted from tuberculous animals. A likely candidate for the most recent ancestor of M. tuberculosis is the closely related species, Mycobacterium bovis , which is the cause of bovine tuberculosis. Alternatively, these two species may have diverged independently from a common precursor. Acquired resistance to tuberculosis is thought to be mediated by M. tuberculosis-specific T lymphocytes displaying the αβT-cell receptor and either the CD4 or CD8 coreceptor. T cells expressing CD8 recognize antigens presented by major histocompatibility complex (MHC) Class I molecules, which are widely distributed on virtually every cell type in the human body. T cells expressing CD4 recognize antigens presented by MHC Class II, present only on specialized antigen-presenting cells, including macrophages. Both classes of T cells can produce IFNγ to activate macrophages.

Advances in Mycobacterial Genetics: New Promises for Old Diseases

Introduction: Mycobacterial infections and a mycobacterial vaccine Historically, members of the genus Mycobacterium have been some of the most important players in the drama of infectious disease and immunology of man.Mycobacterium leprae causes leprosy, a disease that dates back to biblical times and continues to afflict millions around the world today.M.leprae was discovered by G.A.HANSEN in 1873 and found to be the first bacteria associated with human disease.Despite this early discovery, this bacillus has yet to be cultivated on artificial media and can only be propagated in the laboratory in nine-banded armadillos or footpads of mice.Twenty years ago, tuberculosis was thought to be a disease that was no longer to be a menace in the world with the presence of a vaccine and the development of effective chemotherapies to control this infection.However, in the last five years the world has seen alarming resurgence of tuberculosis.The World Health Organization estimates that there are 8 million new cases of tuberculosis and 3 million deaths resulting from this dreaded illness every year.An increasing number of reports of infections with drug-resistant Mycobacterium tuberculosis cells makes this resurgence so much more frightening.Since the introduction of isoniazid in 1954,.the United States had thirty-two years of steady decline of the number of new cases of tuberculosis.This trend ended in 1986 and we have seen steady increases with a 5 % increase in 1989 alone.This increase is most likely a result of the epidemic of the Acquired Immunodeficiency Syndrome (AIDS), where M.tuberculosis infection appears to be one of the first signs of a loss of T cell function.AIDS patients also have a number of opportunistic infections, including infections by another mycobacterium, Mycobacterium avium.This bacterium rarely causes disease in immunocompetent individuals and yet is being found in greater than 40 of AIDS patients in the US.Infections caused by M.avium are difficult to treat as the bacterium appears to be refractory to existing chemotherapies.It is quite clear that all of these mycobacteria continue to be significant cause of morbidity and mortality in man.

Pyrazinamide, but not pyrazinoic acid, is a competitive inhibitor of NADPH binding to Mycobacterium tuberculosis fatty acid synthase I

Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty acid synthase I (FAS I). Both PZA and pyrazinoic acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity.