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Identification of two Mycobacterium marinum loci that affect interactions with macrophages.

ABSTRACT: Mycobacterium marinum is closely related to Mycobacterium tuberculosis, the cause of tuberculosis in humans. M. marinum has become an important model system for the study of the molecular mechanisms involved in causing tuberculosis in humans. Through molecular genetic analysis of the differences between pathogenic and nonpathogenic mycobacteria, we identified two loci that affect the ability of M. marinum to infect macrophages, designated mel(1) and mel(2). In silico analyses of the 11 putative genes in these loci suggest that mel(1) encodes secreted proteins that include a putative membrane protein and two putative transglutaminases, whereas mel(2) is involved in secondary metabolism or biosynthesis of fatty acids. Interestingly, mel(2) is unique to M. marinum and the M. tuberculosis complex and not present in any other sequenced mycobacterial species. M. marinum mutants with mutations in mel(1) and mel(2), constructed by allelic exchange, are defective in the ability to infect both murine and fish macrophage cell lines. These data suggest that the genes in mel(1) and mel(2) are important for the ability of M. marinum to infect host cells.

SUBMITTER: El-Etr SH

PROVIDER: S-EPMC529147 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

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Identification of two Mycobacterium marinum loci that affect interactions with macrophages.

El-Etr Sahar H SH Subbian Selvakumar S Cirillo Suat L G SL Cirillo Jeffrey D JD

Infection and immunity 20041201 12

Mycobacterium marinum is closely related to Mycobacterium tuberculosis, the cause of tuberculosis in humans. M. marinum has become an important model system for the study of the molecular mechanisms involved in causing tuberculosis in humans. Through molecular genetic analysis of the differences between pathogenic and nonpathogenic mycobacteria, we identified two loci that affect the ability of M. marinum to infect macrophages, designated mel(1) and mel(2). In silico analyses of the 11 putative ...[more]

PMID: 15557611

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Project description:Multi-drug therapy is the standard-of-care treatment for tuberculosis. Despite this, virtually all studies of the pharmacodynamics (PD) of mycobacterial drugs employed for the design of treatment protocols are restricted to single agents. In this report, mathematical models and in vitro experiments with Mycobacterium marinum and five antimycobacterial drugs are used to quantitatively evaluate the pharmaco-, population and evolutionary dynamics of two-drug antimicrobial chemotherapy regimes. Time kill experiments with single and pairs of antibiotics are used to estimate the parameters and evaluate the fit of Hill-function-based PD models. While Hill functions provide excellent fits for the PD of each single antibiotic studied, rifampin, amikacin, clarithromycin, streptomycin and moxifloxacin, two-drug Hill functions with a unique interaction parameter cannot account for the PD of any of the 10 pairs of these drugs. If we assume two antibiotic-concentration dependent functions for the interaction parameter, one for sub-MIC and one for supra-MIC drug concentrations, the modified biphasic Hill function provides a reasonably good fit for the PD of all 10 pairs of antibiotics studied. Monte Carlo simulations of antibiotic treatment based on the experimentally-determined PD functions are used to evaluate the potential microbiological efficacy (rate of clearance) and evolutionary consequences (likelihood of generating multi-drug resistance) of these different drug combinations as well as their sensitivity to different forms of non-adherence to therapy. These two-drug treatment simulations predict varying outcomes for the different pairs of antibiotics with respect to the aforementioned measures of efficacy. In summary, Hill functions with biphasic drug-drug interaction terms provide accurate analogs for the PD of pairs of antibiotics and M. marinum. The models, experimental protocols and computer simulations used in this study can be applied to evaluate the potential microbiological and evolutionary efficacy of two-drug therapy for any bactericidal antibiotics and bacteria that can be cultured in vitro.

Project description:Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.

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Identification of two Mycobacterium marinum loci that affect interactions with macrophages.

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Identification of two Mycobacterium marinum loci that affect interactions with macrophages.

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