Cytosolic Access of Mycobacterium tuberculosis: Critical Impact of Phagosomal Acidification Control and Demonstration of Occurrence In Vivo

Abstract

Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving—as recently confirmed—escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies.

Virulence Factors and Pathogenicity of Mycobacterium

“…ESX-1 allows cytosolic contact and mediates vacuoles rupture [47]; the protein intervenes in host cell lysis in a contact dependent way, producing gross membrane disruptions [48]. DNA transfer through conjugation is also a function of ESX-1 system [49]; a phenomenon called "distributive conjugal transfer" that describes a genetic exchange between recipient and donor is dependent of ESX-1 in M. smegmatis [50].…”

Interplay of human macrophages and Mycobacterium tuberculosis phenotypes

“…Although we did not analyze phagosomal rupture, rapid host cell death within 24 h of infection is indicative of early escape or phagosomal membrane disintegration, which others have shown to occur within hours of infection, even with Mtb cultivated in detergent [184,215]. Inhibition of phagolysosomal acidification was suggested to be a prerequisite for rupture [184].…”

“…Retention of Coronin 1 (TACO) Coronin 1 (TACO) Coronin 1 (TACO) Coronin 1 (TACO) on the Mtb-containing phagosome also contributes to phagosomal maturation arrest by interference with calcium-dependentsignaling [181], and in Mycobacterium marinum, loss of ESAT6 secretion not only attenuates the bacteria, but also inhibits phagolysosomal fusion [182]. An involvement of ESAT6 or ESX-1 in Mtb-induced inhibition of phagosomal acidification was investigated but not conclusive [183], but inhibition of phagolysosomal acidification is a prerequisite for the ESX-1-mediated escape of Mtb into the cytosol [184].…”

“…Still controversial, it was argued that phagosomal membrane disintegration during Mtb infection constitutes a feature of cell death rather than a true virulence mechanism with the aim of escaping phagolysosomal effectors and accessing the nutrient-rich cytosol for replication. Anyhow, there is evidence that phagosomal rupture precedes host cell death [217], and phagosomal escape has been demonstrated in vivo in mice [184].…”

“…Inhibition of phagolysosomal acidification was suggested to be a prerequisite for rupture [184]. Controversially, upregulation of the expression of ESX-1 components in acidic pH was reported, which could constitute an ultimate escape mechanism from acidified phagolysosomes [413], while other studies report downregulation during macrophage infection [244,272].…”