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Infection and Immunity, August 1999, p. 4264-4267, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Fluorescent Labels Influence Phagocytosis of Bordetella pertussis by Human Neutrophils

Christine L. Weingart,1 Gina Broitman-Maduro,2 Gary Dean,1 Simon Newman,3 Mark Peppler,2 and Alison A. Weiss1,*

Department of Molecular Genetics, Biochemistry, and Microbiology,1 and Division of Infectious Diseases,3 University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524, and Department of Medical Microbiology and Infectious Diseases, University of Alberta, Edmonton, Alberta, Canada T6G 2H72

Received 1 March 1999/Returned for modification 23 April 1999/Accepted 20 May 1999

To explore the role of neutrophil phagocytosis in host defense against Bordetella pertussis, bacteria were labeled extrinsically with fluorescein isothiocyanate (FITC) or genetically with green fluorescent protein (GFP) and incubated with adherent human neutrophils in the presence or absence of heat-inactivated human immune serum. In the absence of antibodies, FITC-labeled bacteria were located primarily on the surface of the neutrophils with few bacteria ingested. However, after opsonization, about seven times more bacteria were located intracellularly, indicating that antibodies promoted phagocytosis. In contrast, bacteria labeled intrinsically with GFP were not efficiently phagocytosed even in the presence of opsonizing antibodies, suggesting that FITC interfered with a bacterial defense. Because FITC covalently modifies proteins and could affect their function, we tested the effect of FITC on adenylate cyclase toxin activity, an important extracellular virulence factor. FITC-labeled bacteria had fivefold-less adenylate cyclase toxin activity than did unlabeled wild-type bacteria or GFP-expressing bacteria, suggesting that FITC compromised adenylate cyclase toxin activity. These data demonstrated that at least one extracellular virulence factor was affected by FITC labeling and that GFP is a more appropriate label for B. pertussis.

* Corresponding author. Mailing address: Department of Molecular Genetics, Biochemistry, and Microbiology, 231 Bethesda Ave., ML 524, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524. Phone: (513) 558-2820. Fax: (513) 558-8474. E-mail: Alison.Weiss{at}uc.edu.

Infection and Immunity, August 1999, p. 4264-4267, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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