Infection of “nonprofessional phagocytes” with Mycobacterium avium complex

doi:10.1016/S0090-1229(05)80026-1

Clinical Immunology and Immunopathology

Volume 61, Issue 2, Part 1, November 1991, Pages 225-235

https://doi.org/10.1016/S0090-1229(05)80026-1 Get rights and content

Organisms belonging to the Mycobacterium avium complex are the most common bacteria isolated from patients with AIDS. In these patients, M. avium is associated with disseminated disease, and bacteria are found within macrophages in the liver and spleen. To examine the potential of M. avium infection of a nonprofessional phagocyte, the murine fibroblast cell line (L929 cells) are infected with strain 101 (serotype 1) of M. avium. The duplication time for the intracellular bacteria was approximately 36 hr. Progressive intracellular growth ultimately resulted in the destruction of infected cells (after approximately 12 days in culture). Supernatant obtained from infected L929 cells contained interferon-β (IFNβ) and granulocyte macrophage colony stimulating factor (GM-CSF) (50±12 ng/10⁵ cells and 63±18 pg/10⁵ cells, respectively, by 18 hr). IFNβ could be detected by 3 hr after infection, while GM-CSF was first detected by 6 hr. Release of IFNβ by infected L929 cells could subsequently stimulate NK cells, but not macrophages, to lyse infected L929 cells. These data using L929 cells suggest that M. avium may invade fibroblasts during the course of the infection and that fibroblast infection may trigger NK cell-mediated cytotoxicity against the infected fibroblasts.

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Assessment of the invasiveness of rapidly-growing nonpigmented mycobacteria with the fibroblast microcolony assay
2006, Enfermedades Infecciosas y Microbiologia Clinica
Se evaluó la capacidad invasiva de aislamientos clínicos y de colección de varias especies de micobacterias no pigmentadas de crecimiento rápido mediante el análisis de las microcolonias en cultivo de fibroblastos diploids humanos, y se comparó la misma con el fenotipo de colonias en medio de Middlebrook 7H11 y con el significado clínico de los aislamientos.
Se analizaron 29 cepas de: Mycobacterium chelonae (8), M. fortuitum (6), M. peregrinum (5), M. abscessus (5), M. mucogenicum (4) y M. septicum (1), tanto clínicamente significativas como de colección. Se evaluó el fenotipo liso o rugoso de las colonias en Middlebrook 7H11. Se evaluó la capacidad de invasion intracelular mediante el ensayo de microcolonias en fibroblastos descrito por Shepard. Las características cuantitativas se compararon con el test de la t de Student y las características cualitativas con el test exacto de Fisher.
No se encontraron diferencias significativas entre colonias de fenotipos distintos o cepas con diferentes significados clínicos, excepto en dos aislamientos de M. chelonae de bacteriemia relacionados con catéteres, que presentaron microcolonias alargadas en el cultivo celular. Las cepas de M. fortuitum y M. peregrinum presentaron microcolonias más grandes y de apariencia desflecada, mientras que las de M. chelonae, M. abscessus o M. mucogenicum, presentaron unas colonias más pequeñas y de apariencia redondeada.
La capacidad invasiva está presente solamente en unas pocas cepas de micobacterias, y esta característica no aparece en la mayoría de las cepas aisladas de infecciones en humanos y en ninguna de las que no fueron clínicamente significativas, por lo que no sería una característica esencial para que los diversos aislamientos sean causa de enfermedad en humanos.
The invasive capacity of rapidly-growing nonpigmented mycobacteria strains was evaluated by means of a fibroblast microcolony assay and related to the colony phenotype on Middlebrook 7H11 and to the clinical significance of the isolates.
Twenty-nine strains [Mycobacterium chelonae (8), M. fortuitum (6), M. peregrinum (5), M. abscessus (5), M. mucogenicum (4) and M. septicum (1)], proceeding from a bacterial collection and clinical isolates, were evaluated. The smooth or rough phenotype of the colonies was assessed in Middlebrook 7H11 medium. Intracellular invasiveness was determined by the fibroblast-microcolony assay described by Shepard. Quantitative culture characteristics were compared with tudent's t-test, and qualitative characteristics with Fisher's exact test.
No significant differences were found between colonies with different phenotypes or strains having a different clinical significance, except for two strains of Mycobacterium chelonae isolated from cases of catheter-related bacteremia, which showed elongated microcolonies. M. fortuitum and M. peregrinum strains showed larger microcolonies than M. chelonae, M. abscessus and M. mucogenicum, and displayed a fluffy appearance, while the latter two strains showed rounded olonies.
Very few strains of mycobacteria had invasive capacity and the majority of strains isolated from human infections do not show this characteristic; hence this trait is not essential for mycobacteria to cause infection in humans.
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Citation Excerpt :
Alveolar and interstitial macrophages were the most common positive cells, but positivity was also seen in type II pneumocytes, endothelial cells, and fibroblasts, and particularly in cells located in or close to the subpleural fibrotic bands with variable deposition of carbon particles. Several species of pathogenic mycobacteria including M avium, M marinum, and M tuberculosis, have been shown to invade and replicate within a variety of nonprofessional phagocytes, such as fibroblasts, endothelium, and human epithelial cells.17–22 It has always seemed paradoxical that M leprae is routinely seen inside a variety of cell types in vivo,23 whereas M tuberculosis, which readily invades such cells in vitro, has been said to be exclusively extracellular or within macrophages and macrophage-derived cell types in vivo.
A third of the world's population has latent infection with Mycobacterium tuberculosis, and in areas of low endemicity, most cases of active tuberculosis arise as a result of reactivation of latent bacilli. We sought to establish the cellular location of these latent organisms to facilitate their elimination.
We applied in-situ PCR to sections of macroscopically normal lung tissue from 13 individuals from Ethiopia and 34 from Mexico who had died from causes other than tuberculosis. Sections of lung tissue from six Norwegian individuals (ie, individuals from a non-endemic population) acted as negative controls, and six Ethiopian tuberculosis cases acted as positive controls.
Control necropsy samples from the Norwegian individuals were all negative by in-situ PCR and conventional PCR, whereas all samples from known Ethiopian tuberculosis cases were positive by both methods. However, in macroscopically normal lung tissue from Ethiopian and Mexican individuals without tuberculous lesions, the in-situ PCR revealed five of 13 and ten of 34 positive individuals, respectively. These results were confirmed by conventional PCR with extracted DNA. Positive cells included alveolar and interstitial macrophages, type II pneumocytes, endothelial cells, and fibroblasts.
M tuberculosis can persist intracellularly in lung tissue without histological evidence of tuberculous lesions. M tuberculosis DNA is situated not only in macrophages but also in other non-professional phagocytic cells. These findings contradict the dominant view that latent organisms exist in old classic tuberculous lesions, and have important implications for strategies aimed at the elimination of latent and persistent bacilli.
Expression of the green fluorescent protein (GFP) in Mycobacterium avium as a tool to study the interaction between Mycobacteria and host cells
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Mycobacterium aviumis an intracellular pathogen able to invade and survive within macrophages and mucosal epithelial cells. The study of the interaction betweenM. aviumand host cells is important to establish the mechanisms of pathogenesis of the infection. One of the limitations of microscopic study of intracellularM. aviumis the difficulty of identifying isolated bacilli within cells. As a general strategy to visualize and analyse the influence ofM. aviumon intracellular trafficking, we cloned the gene encoding the green fluorescent protein (GFP) inM. avium. A vector was constructed by cloning the cDNA for the GFP of the bioluminescent jellyfishAequorea victoriainto anEscherichia coli/Mycobacteriashuttle vector (pMV261). The recombinant plasmid (pWES-4) was transformed into bothE. coliandM. aviumstrain 104 (serovar 1). The transformants were screened for strong expression of the GFP. TransformedM. aviumwere clearly visible inside human macrophages and epithelial cells using fluorescence microscopy. These transformedM. aviumshould provide a useful tool for further study of intracellular behaviour.
Comparison of the ability of Mycobacterium avium, M. smegmatis and M. tuberculosis to invade and replicate within HEp-2 epithelial cells
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Previous studies have demonstrated that mycobacteria can interact with epithelial cells, a property which can be important for establishing infection. In this study we investigated comparatively the ability of Mycobacterium avium, M. tuberculosis and M. smegmatis to invade and multiply within HEp-2 epithelial cells.
The ability to invade and to multiply intracellularly in HEp-2 cells was examined using a virulent strain of M. avium, a virulent (H37Rv) and an attenuated (H37Ra) strain of M. tuberculosis and a strain of M. smegmatis. The locus responsible for M. avium invasion was also cloned in Escherichia coli and M. smegmatis.
It was observed that M. avium invaded HEp-2 cells with greater efficiency than M. tuberculosis and M. smegmatis, while the H37Rv strain of M. tuberculosis was more efficient in invading HEp-2 than H37Ra and M. smegmatis. Both M. avium and M. tuberculosis were capable of multiplying within HEp-2 cells, while M. smegmatis was not. E. coli K12 and M. smegmatis were transformed with M. avium DNA. The invasive locus of M. avium provided E. coli K12 and M. smegmatis strains S5M101-1 and S5M101-2 with the ability to invade HEp-2 epithelial cells. Transformed M. smegmatis strains were able to grow intracellularly.
‘Virulent’ strains of M. avium and M. tuberculosis were shown to invade and to multiply within HEp-2 epithelial cells. This property was transferred to E. coli K12 and M. smegmatis by transformation with the invasive locus of M. avium. The ability of certain strains of mycobacteria to invade epithelial cells (bronchial, alveolar, intestinal) may represent an important phenotypic characteristic and could be directly related to pathogenicity.
Des études antérieures ont démontré que les mycobactéries peuvent interagir avec les cellules épithéliales, propriété qui pourrait s'avérer importante dans l'installation de l'infection. Dans la présente étude a été comparée la capacité de Mycobacterium avium, M. tuberculosis et M. smegmatis d'envahir et de se multiplier à l'intérieur des cellules épithéliales HEp-2.
La capacité d'envahir des cellules HEp-2 et de se reproduire de façon intracellulaire a été examinée en utilisant une souche virulente de M. avium, des souches virulentes (H37Rv) et atténuées (H37Ra) de M. tuberculosis et une souche de M. smegmatis. Le locus responsable de l'envahissement de M. avium a été en outre cloné dans Escherichia coli et M. smegmatis.
Il a été observé que M. avium a envahi les cellules HEp-2 avec davantage d'efficacité que M. tuberculosis et M. smegmatis, tandis que la souche H37Rv de M. tuberculosis était plus efficace dans l'envahissement de HEp-2 que H37Ra et M. smegmatis. M. avium et M. tuberculosis étaient capables de se reproduire à l'intérieur des cellules HEp-2 alors que M. smegmatis ne l'était pas. E. coli K12 et M. smegmatis ont été transformés par l'ADN M. avium. Le locus d'invasion de M. avium a conféré sur les souches E. coli K12 et M. smegmatis S5M101-1 et S5M101-2 la possibilité d'envahir les cellules épithéliales HEp-2. Les souches modifiées de M. smegmatis étaient également capables de croître à l'intérieur des cellules.
Il a été démontré que des souches ‘virulentes’ de M. avium et M. tuberculosis étaient capables d'envahir et de se reproduire à l'intérieur des cellules épithéliales HEp-2. Cette propriété a été transférée à E. coli K12 et à M. smegmatis par une transformation du locus invasif de M. avium. La capacité qu'ont certaines souches mycobactériennes d'envahir des cellules épithéliales (bronchiques, alvéolaires, intestinales) pourrait représenter une caractéristique phénotypique importante et pourrait être directement liée à la pathogénicité.
Estudios previos han demostrado que las micobacterias pueden tener una interacción con las células epiteliales. Esta propiedad puede ser importante para la instalación de la infección. En este estudio investigamos comparativamente la capacidad de Mycobacterium avium, M. tuberculosis y M. smegmatis para invadir y multiplicarse en las células epiteliales HEp-2.
Se analizó la capacidad de invasión y de multiplicación intracelular en las células HEp-2, utilizando una cepa virulenta de M. avium, una cepa virulenta (H37Rv) y otra atenuada (H37Ra) de M. tuberculosis y una cepa de M. smegmatis. Además, el locus responsable para la invasión de M. avium fue clonado en Escherichia coli y M. smegmatis.
Se observó que M. avium invadía las células HEp-2 con mayor eficiencia que M. tuberculosis y M. smegmatis, mientras que la cepa H37Rv de M. tuberculosis fue más eficiente para invadir las células HEp-2 que la cepa H37Ra y que M. smegmatis. Ambos, M. tuberculosis y M. avium fueron capaces de multiplicarse dentro de las células HEp-2, mientras que M. smegmatis no lo fue. E. coli K12 y M. smegmatis fueron transformados con ADN de M. avium. El locus de invasión de M. avium confirió a E. coli K12 y a M. smegmatis, cepas S5M101-1 y S5M101-2, la capacidad de invadir las células epiteliales HEp-2. Además, las cepas transformadas de M. smegmatis fueron capaces de crecer al interior de las células.
Las cepas ‘virulentas’ de M. avium y M. tuberculosis se mostraron capaces de invadir y de multiplicarse dentro de las células epiteliales HEp-2. Esta propiedad fue transferida a E. coli y a M. smegmatis por transformación del locus de invasión de M. avium. La capacidad de ciertas cepas de micobacterias para invadir las células epiteliales (bronquiales, alveolares, intestinales) pueden representar una característica fenotípica importante y puede estar directamente relacionada con la patogenicidad.
Treatment and developmental therapeutics of Mycobacterium avium complex (MAC) infections
1994, International Journal of Antimicrobial Agents
Opportunistic infections associated with AIDS pose very serious problems because of their exceedingly high morbidity and mortality. For their part, Mycobacterium avium and M. intracellulare, two organisms belonging to M. avium complex (MAC), are the most often isolated bacteria from AIDS patients. Although in recent years, some progress has been made, by and large, there is no viable drug and /or combinations of drugs effective against MAC, especially in AIDS patients. Conventional therapies with multiple drug combinations involving isoniazid, rifampin, ethambutol, streptomycin, ethionamide, and cycloserine are still widely used, as well as prophylactic treatment with rifabutin. The use of clofazimine and ciprofloxacin has also been reported. Studies on new quinolones (sparfloxacin, difloxacin, WIN 57273), macrolides, folate antagonists and antimcobacterial anitbiotics are being actively pursued along with novel strategies involving drugs inhibiting mycobacterial cell wall biosynthesis. The role of various cytokines in enhancing host immune defenses against MAC infections is also discussed.

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¹: This work was supported by Grant 5 RO1 AI 25769-03 from the National Institute of Allergy and Infectious Diseases.

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Infection of “nonprofessional phagocytes” with Mycobacterium avium complex1

Cell. Immunol.

Cell. Immunol.

Adv. Immunol.

Rev. Microbiol.

Rev. Infect. Dis.

Clin. Res.

Infect. Immun.

Infect. Immun.

J. Infect. Dis.

J. Infect. Dis.

Ann. Intern. Med.

J. Clin. Microbiol.

J. Med. Biol. Res.

J. Immunol.

J. Leukocyte Biol.

J. Infect. Dis.

Ann Intern. Med.

J. Immunol.

Eur. J. Immunol.

J. Infect. Dis.

J. Immunol.

Antimicrob. Agents Chemother.

J. Leukocyte Biol.

J. Immunol.