Abstract
M protein is thought to contribute to the ability of non-opsonized group A and group G streptococci (GAS and GGS, respectively) to resist phagocytosis by polymorphonuclear leukocytes. In previous studies, correlation between M protein expression and phagocytosis was determined by incubating these pathogens in human blood and comparing colony-forming bacterial counts prior to and after exposure to blood (direct bactericidal assay; DBA). Here, we report the application of flow cytometry to measure GAS and GGS resistance to phagocytosis. The results of the assays were in complete agreement with those from DBAs. Nevertheless, flow cytometry was regarded as superior to DBA because of its speed and potential uses for quantitative studies. In addition, the use of anti-CD1 1b monoclonal antibody for granulocyte staining guaranteed a non-compromized granulocyte function. The optimized protocol for flow cytometry presented here could be utilized to directly measure the involvement of individual protein types in bacterial resistance to phagocytosis
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References
Bassoe CF, Laerum OD, Solberg CO, Haneberg B (1983) Phagocytosis of bacteria by human leukocytes measured by flow cytometry. Soc Exp Biol Med 174:182–188
Bettelheim P (1989) Summary of the results of the functional analyses presented at the conference. In: Knapp W (ed) Leucocyte Typing IV, White Cell Differentiation Antigens. Oxford University Press, Oxford, pp 891–893
Bisno AL, Craven DE, McCabe WR (1987) M proteins of group G streptococci isolated from bacteremic human infections. Infect Imun 55:753–757
Cantinieaux B, Hariga C, Courtoy P, Hupin J, Fondu P (1989) Staphylococcus aureus phagocytosis: a new cytofluorometric method using FITC and paraformaldehyde. J Immunol Methods 121:203–208
Cleary P, Retnoningrum D (1994) Group A streptococcal immunoglobulin-binding proteins: adhesions, molecular mimicry or sensory proteins? Trends Microbiol 2:131–136
Collins CM, Kimura A, Bisno AL (1992) Group G streptococcal M protein exhibits structural features analogous to those of class I M protein of group A streptococci. Infect Immun 60:3689–3696
Drevet DA, Campbell PA (1991) Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria. J Immunol Methods 142:31–38
Hed J, Hallden G, Johansson SGO, Larsson P (1987) The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J Immunol Methods 101: 119–125
Jones KF, Fischetti VA (1987) Biological and immunochemical identity of M protein on group G streptococci with M protein on group A streptococci. Infect Immun 55:502–506
Lancefield RC (1957) Differentiation of group A streptococci with a common R antigen into three serological types, with special reference to the bactericidal test. J Exp Med 106:525–544
Lancefield RC (1962) Current knowledge of type-specific M antigens of group A streptococci. J Immunol 89:307–313
Martin E, Bhakdi S (1991) Quantitative analysis of opsonophagocytosis and of killing of Candida albicans by human peripheral blood leukocytes by using flow cytometry. J Clin Microbiol 29:2013–2023
Martin E, Bhakdi S (1992) Flow cytometric assay for quantifying opsonophagocytosis and killing of Staphylococcus aureus by peripheral blood leukocytes. J Clin Microbiol 30:2246–2255
Peterson PK, Verhoef J, Schmeling D, Quie PG (1977) Kinetics of phagocytosis and bacterial killing by human polymorphonuclear leukocytes and monocytes. J Infect Dis 136:502–509
Rothe G, Emmendörffer A, Oser A, Roesler J, Valet G (1991) Flow cytometric measurement of the respiratory burst activity of phagocytes using dihydrorhodamine 123. J Immunol Methods 138:133–135
Schnitzler N, Podbielski A, Baumgarten G, Mignon M, Kaufhold A (1995) M or M-like protein gene polymorphisms in human group G streptococci. J Clin Microbiol 33:356–363
Simpson WJ, Robbins JC, Cleary PP (1987) Evidence for group A-related M protein genes in human but not animal-associated group G streptococcal pathogens. Microb Pathog 3:339–350
Sunder-Plassmann G, Wagner L, Apperl A, Hruby K, Balcke P (1989) Modulation of neutrophil respiratory burst and chemotaxis by the myeloid panel mAB. In: Knapp W (ed) Leucocyte typing IV, white cell differentiation antigens. Oxford University Press, Oxford, pp 896–899
Verhoef J, Peterson PK, Quie PG (1977) Kinetics of Staphylococcus opsonization, attachment, ingestion and killing by human polymorphonuclear leukocytes: a quantitative assay using [3H]thymidine-labeled bacteria. J Immunol Methods 14:303–311
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Schnitzler, N., Haase, G., Büssing, A. et al. Measuring resistance to phagocytosis of group A and G streptococci: comparison of direct bactericidal assay and flow cytometry. Med Microbiol Immunol 184, 17–22 (1995). https://doi.org/10.1007/BF00216785
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DOI: https://doi.org/10.1007/BF00216785