An assay to quantitate the binding of Rhodococcus equi to macrophages.
Abstract: A Rhodococcus equi radiobinding assay has been developed using organisms labeled with 3H-uracil. These labeled organisms resemble their unlabeled counterparts with respect to colony morphology, viability, and buoyant density. Bacteria routinely incorporate between 5 x 10(-3) and 5 x 10(-2) counts per minute per colony forming unit (cfu) which in this assay allows the detection of fewer than 0.2 cfu per macrophage. Once incorporated, greater than 90% of the label remains bacterial associated for at least 4 h postlabeling. The majority of the label is trichloroacetic acid precipitable, partitions into the aqueous phase following phenol/chloroform extraction and is ethanol precipitable. RNAse treatment of the ethanol precipitate abolishes label trichloroacetic acid precipitation. This radiolabeling technique has been used to quantitate the attachment of R. equi to both murine peritoneal and equine alveolar macrophages adherent to 13 mm glass coverslips. R. equi binding is dose dependent, saturable, and specific to macrophages. Further, binding is enhanced in the presence of fresh serum. Inhibition of radiolabeled bacterial binding can be obtained by competition with cold R. equi. This radiolabeled binding assay represents a crucial step in identifying the receptors on macrophages involved in the recognition of R. equi and may help to provide information on how macrophages recognize intracellular bacteria in general.
Publication Date: 1992-05-01 PubMed ID: 1632069DOI: 10.1016/0165-2427(92)90055-uGoogle Scholar: Lookup
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- Comparative Study
- Journal Article
Summary
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The research article discusses the creation of an assay for measuring the binding of the bacterium Rhodococcus equi to macrophages, which are types of immune cells. The study utilizes a radiolabeling technique using 3H-uracil labeled bacteria to identify and potentially learn how macrophages recognize these and potentially other intracellular bacteria.
Development of the Assay
- The researchers developed a Rhodococcus equi radiobinding assay by labeling the bacteria with 3H-uracil, a form of radioactive uracil.
- The labeled bacteria maintained their natural behaviors, including colony morphology, viability, and buoyant density, similar to the unlabeled bacteria.
- Bacteria could incorporate the radiolabel at sufficient levels to allow the detection of fewer than 0.2 colony forming units (cfu) per macrophage in the developed assay.
- Post-labeling, over 90% of the radiolabel remained associated with the bacteria for at least 4 hours, ensuring minimal loss during the assay run-time.
Assay Performance and Observations
- The radiolabeling technique studied the attachment of R. equi to murine peritoneal and equine alveolar macrophages adherent to 13mm glass coverslips.
- R. equi binding was found to be dose-dependent, saturable, and specific to macrophages, meaning the number of bacteria that can attach to a macrophage follows a certain pattern and does not surpass a certain limit.
- The binding rate was found to be enhanced in the presence of fresh serum. The experiment showed that non-radiolabeled R. equi could inhibit the binding of the radiolabeled ones indicating a competition for similar binding sites.
Implications and Potential Applications
- This radiolabeled binding assay proves instrumental in identifying the receptors on macrophages that are involved in recognizing R. equi.
- The data collected from this research will open up further research toward the understanding of how macrophages recognize intracellular bacteria in general.
Cite This Article
APA
Hondalus MK, Sweeney CR, Mosser DM.
(1992).
An assay to quantitate the binding of Rhodococcus equi to macrophages.
Vet Immunol Immunopathol, 32(3-4), 339-350.
https://doi.org/10.1016/0165-2427(92)90055-u Publication
Researcher Affiliations
- Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140.
MeSH Terms
- Animals
- Bacterial Adhesion / immunology
- Colony Count, Microbial
- Female
- Horses / microbiology
- Macrophages / microbiology
- Macrophages, Alveolar / microbiology
- Mice
- Mice, Inbred BALB C
- Peritoneal Cavity
- Radioligand Assay / veterinary
- Rhodococcus equi / immunology
Citations
This article has been cited 4 times.- Miranda-CasoLuengo AA, Miranda-CasoLuengo R, Lieggi NT, Luo H, Simpson JC, Meijer WG. A real-time impedance based method to assess Rhodococcus equi virulence.. PLoS One 2013;8(3):e60612.
- Hondalus MK, Diamond MS, Rosenthal LA, Springer TA, Mosser DM. The intracellular bacterium Rhodococcus equi requires Mac-1 to bind to mammalian cells.. Infect Immun 1993 Jul;61(7):2919-29.
- Hondalus MK, Mosser DM. Survival and replication of Rhodococcus equi in macrophages.. Infect Immun 1994 Oct;62(10):4167-75.
- McNeil MM, Brown JM. The medically important aerobic actinomycetes: epidemiology and microbiology.. Clin Microbiol Rev 1994 Jul;7(3):357-417.
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