Modulation of biogenesis of the Francisella tularensis subsp. novicida-containing phagosome in quiescent human macrophages and its maturation into a phagolysosome upon activation by IFN-γ

Abstract

SummaryFrancisella tularensis is a highly virulent facultative intracellular pathogen that has been categorized as a class A bioterrorism agent, and is classified into four subsp, tularensis, holarctica , mediasiatica and novicida . Although the ability of F. tularensis subsp. novicida to cause tularemia in mice is similar to the virulent subsp. tularensis and holarctica, it is attenuated in humans. It is not known whether attenuation of F. tularensis subsp. novicida in humans is resulting from a different route of trafficking within human macrophages, compared with the tularensis or holarctica subsp. Here we show that in quiescent human monocytes-derived macrophages (hMDMs), the F. tularensis subsp. novicida containing phagosome (FCP) matures into a late endosome-like stage that acquires the late endosomal marker LAMP-2 but does not fuse to lysosomes. This modulation of phagosome biogenesis by F. tularensis is followed by disruption of the phagosome at 4-12 h and subsequent bacterial escape into cytoplasm where the organism replicates. In IFN-g g g g -activated hMDMs, intracellular replication of F. tularensis is completely inhibited, and is associated with failure of the organism to escape from the phagosome into the cytoplasm for up to 24 h after infection. In IFN-g g g g -activated hMDMs, the FCPs acquire the lysosomal enzymes Cathepsin D, which is excluded in quiescent hMDMs. When the lysosomes of IFN-g g g gactivated hMDMs are preload with Texas Red Ovalbumin or BSA-gold, the FCPs acquire both lysosomal tracers. In contrast, both lysosomal tracers are excluded from the FCPs within quiescent hMDMs. We conclude that although F. tularensis subsp. novicida is attenuated in humans, it modulates biogenesis of its phagosome into a late endosome-like compartment followed by bacterial escape into the cytoplasm within quiescent hMDMs, similar to the virulent subsp. tularensis . In IFN-g g g g -activated hMDMs, the organism fails to escape into the cytoplasm and its phagosome fuses to lysosomes, similar to inert particles.

A Francisella novicida pdpA mutant exhibits limited intracellular replication and remains associated with the lysosomal marker LAMP-1

“…The phagosome subsequently gains late endosomal markers such as Rab7, CD63, LAMP-1 and LAMP-2 (Clemens et al, 2004;Santic et al, 2005a). This late endosome-like compartment acquires the proton vacuolar ATPase pump and becomes transiently acidified but does not associate with lysosomal markers, such as cathepsin D (Clemens et al, 2004;Santic et al, 2005aSantic et al, , 2008. Francisella is thus able to prevent phagosomal-lysosomal fusion, and within 4 h of uptake actively breaks down the phagosomal membrane in order to escape into the host-cell cytosol and replicate.…”

“…Approximately 20 min after macrophage engulfment, the Francisellacontaining phagosome acquires the early endosomal markers EEA-1 and Rab5 (Clemens et al, 2004). The phagosome subsequently gains late endosomal markers such as Rab7, CD63, LAMP-1 and LAMP-2 (Clemens et al, 2004;Santic et al, 2005a). This late endosome-like compartment acquires the proton vacuolar ATPase pump and becomes transiently acidified but does not associate with lysosomal markers, such as cathepsin D (Clemens et al, 2004;Santic et al, 2005aSantic et al, , 2008.…”

“…In vitro studies have shown that F. tularensis subverts the normal endocytic pathway of host macrophages and grows rapidly within these cells (Santic et al, 2005a). Approximately 20 min after macrophage engulfment, the Francisellacontaining phagosome acquires the early endosomal markers EEA-1 and Rab5 (Clemens et al, 2004).…”

A comprehensive transposon mutant library of Francisella novicida , a bioweapon surrogate

“…holarctica strain (LVS) are commonly used as surrogates for subsp. tularensis in virulence studies using animal models (8,9). Both strains cause tularemialike diseases in rodents, and F. novicida is particularly virulent toward mice (8,10).…”

Inhalation of Francisella novicida ΔmglA causes replicative infection that elicits innate and adaptive responses but is not protective against invasive pneumonic tularemia

“…Due to its low infectious dose and airborne transmissibility, F. tularensis is considered a potential bioweapon [3]. The pathogenicity of F. tularensis is not completely understood but its intracellular parasitism of macrophages involves escape from the phagosome prior to lysosomal fusion [4]. The organism also escapes neutrophil phagosomes after inhibiting the respiratory burst [5].…”