Virulent and Avirulent Strains ofFrancisella tularensisPrevent Acidification and Maturation of Their Phagosomes and Escape into the Cytoplasm in Human Macrophages

Abstract

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 ofF. tularensissubsp.tularensisand the live vaccine strain with human macrophages by immunoelectron and confocal immunofluorescence microscopy. We assessed the maturation of theF. tularensisphagosome 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 liveF. tularensiscells acquired abundant staining for LAMPs but little or no staining for cathepsin D. However, after 4 h, the colocalization of LAMPs with liveF. tularensisorganisms 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 liveF. tularensisdid not concentrate these markers and thus were not appreciably acidified (pH 6.7). An ultrastructural analysis of theF. tularensiscompartment revealed that during the first 4 h after uptake, the majority ofF. tularensisbacteria 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 liveF. tularensisbacteria lacked any ultrastructurally discernible membrane separating them from the host cell cytoplasm. These results indicate thatF. tularensisinitially 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 ofF. tularensisto 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.