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Infection and Immunity, June 2004, p. 3204-3217, Vol. 72, No. 6
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.6.3204-3217.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Virulent and Avirulent Strains of Francisella tularensis Prevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

Daniel L. Clemens,* Bai-Yu Lee, and Marcus A. Horwitz

Division of Infectious Diseases, Department of Medicine, School of Medicine, University of California—Los Angeles, Los Angeles, California 90095-1688

Received 25 November 2003/ Returned for modification 30 December 2003/ Accepted 13 February 2004

Francisella tularensis, the agent of tularemia, is an intracellular pathogen, but little is known about the compartment in which it resides in human macrophages. We have examined the interaction of a recent virulent clinical isolate of F. tularensis subsp. tularensis and the live vaccine strain with human macrophages by immunoelectron and confocal immunofluorescence microscopy. We assessed the maturation of the F. tularensis phagosome by examining its acquisition of the lysosome-associated membrane glycoproteins (LAMPs) CD63 and LAMP1 and the acid hydrolase cathepsin D. Two to four hours after infection, vacuoles containing live F. tularensis cells acquired abundant staining for LAMPs but little or no staining for cathepsin D. However, after 4 h, the colocalization of LAMPs with live F. tularensis organisms declined dramatically. In contrast, vacuoles containing formalin-killed bacteria exhibited intense staining for all of these late endosomal/lysosomal markers at all time points examined (1 to 16 h). We examined the pH of the vacuoles 3 to 4 h after infection by quantitative immunogold staining and by fluorescence staining for lysosomotropic agents. Whereas phagosomes containing killed bacteria stained intensely for these agents, indicating a marked acidification of the phagosomes (pH 5.5), phagosomes containing live F. tularensis did not concentrate these markers and thus were not appreciably acidified (pH 6.7). An ultrastructural analysis of the F. tularensis compartment revealed that during the first 4 h after uptake, the majority of F. tularensis bacteria reside within phagosomes with identifiable membranes. The cytoplasmic side of the membranes of ~50% of these phagosomes was coated with densely staining fibrils of ~30 nm in length. In many cases, these coated phagosomal membranes appeared to bud, vesiculate, and fragment. By 8 h after infection, the majority of live F. tularensis bacteria lacked any ultrastructurally discernible membrane separating them from the host cell cytoplasm. These results indicate that F. tularensis initially enters a nonacidified phagosome with LAMPs but without cathepsin D and that the phagosomal membrane subsequently becomes morphologically disrupted, allowing the bacteria to gain direct access to the macrophagic cytoplasm. The capacity of F. tularensis to alter the maturation of its phagosome and to enter the cytoplasm is likely an important element of its capacity to parasitize macrophages and has major implications for vaccine development.

* Corresponding author. Mailing address: Division of Infectious Diseases, Department of Medicine, UCLA School of Medicine, CHS 37-121, 10833 LeConte Ave., Los Angeles, CA 90095-1688. Phone: (310) 825-9324. Fax: (310) 794-7156. E-mail: dclemens{at}mednet.ucla.edu.

Editor: D. L. Burns

Infection and Immunity, June 2004, p. 3204-3217, Vol. 72, No. 6
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.6.3204-3217.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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