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Infection and Immunity, February 2007, p. 1017-1024, Vol. 75, No. 2
0019-9567/07/$08.00+0     doi:10.1128/IAI.00914-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Oxidant Generation by Single Infected Monocytes after Short-Term Fluorescence Labeling of a Protozoan Parasite{triangledown} ,{dagger}

Haeok K. Chang,1 Colin Thalhofer,2 Breck A. Duerkop,3,{ddagger} Joanna S. Mehling,3 Shilpi Verma,2 Kenneth J. Gollob,4 Roque Almeida,5 and Mary E. Wilson1,2,3,6,7*

Departments of Internal Medicine,1 Microbiology,3 Epidemiology,6 Interdisciplinary Immunology Program, University of Iowa,2 VA Medical Center, Iowa City, Iowa 52242,7 Universidade Federal de Minas Gerais, Bela Horizonte, MG,4 Universidade Federal da Bahia, Salvador, BA, Brazil5

Received 8 June 2006/ Returned for modification 18 July 2006/ Accepted 10 November 2006

Leishmania spp. are intracellular protozoa residing in mononuclear phagocytes. Leishmania organisms are susceptible to microbicidal responses generated in response to phagocytosis. Assuming that both phagocyte and parasite populations are heterogeneous, it is advantageous to examine the response of individual cells phagocytosing living parasites. Because Leishmania spp. lose virulence during the raising of transfectants, we developed a method to label live Leishmania chagasi short-term with fluorescent dyes. Up to six parasite divisions were detected by flow cytometry after labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE), dioctadecyl-tetramethylindo carbocyanine perchlorate, or chloromethyl tetramethylrhodamine. Labeled parasites entered mononuclear phagocytes as determined by confocal and time-lapse microscopy. Dihydroethidium (DHE) was used to detect macrophage-derived oxidants generated during phagocytosis. Presumably Leishmania organisms are opsonized with host serum/tissue components such as complement prior to phagocytosis. Therefore, we investigated the effects of opsonization and found that this increased the efficiency of CFSE-labeled parasite entry into monocytes (84.6% ± 8.8% versus 20.2% ± 3.8% monocytes infected; P < 0.001). Opsonization also increased the percentage of phagocytes undergoing a respiratory burst (66.0% ± 6.3% versus 41.0% ± 8.3% of monocytes containing CFSE-labeled parasites; P < 0.001) and the magnitude of oxidant generation by each infected monocyte. Inhibitor data indicated that DHE was oxidized by products of the NADPH oxidase. These data suggest that opsonized serum components such as complement lead to more efficient entry of Leishmania into their target cells but at the same time activate the phagocyte oxidase to generate microbicidal products in infected cells. The parasite must balance these positive and negative survival effects in order to initiate a viable infection.

* Corresponding author. Mailing address: Department of Internal Medicine, SW34-GH, 200 Hawkins Dr., Iowa City, IA 52242. Phone: (319) 356-3169. Fax: (319) 384-7208. E-mail: mary-wilson{at}uiowa.edu.

{triangledown} Published ahead of print on 21 November 2006.

Editor: W. A. Petri, Jr.

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

{ddagger} Present address: University of Washington, Department of Microbiology, Seattle, WA 98195.

Infection and Immunity, February 2007, p. 1017-1024, Vol. 75, No. 2
0019-9567/07/$08.00+0     doi:10.1128/IAI.00914-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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