摘要：

Phagosome maturation is a key innate immune response involving interactions of phagosomes with the endosomal system. These interactions result in the creation of a destructive, antimicrobial phagolysosome compartment. How this process is regulated is not entirely known, and intracellular pathogens such as Mycobacterium tuberculosis (Mtb) inhibit phagosome maturation as a survival strategy. We examined phagosome maturation from the perspective of two factors, one host- and one pathogen-derived. First, we determined whether the class IA phosphatidylinositol 3-kinase (PI3K), p110α, contributes to maturation regulation. Of the various PI3Ks, only hVps34 is known to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in p110α, we discovered that this PI3K isoform controls maturation processes beyond Rab7 acquisition, leading to delivery of lysosomal markers. Phagosomes from p110α knockdown cells were markedly deficient in LAMP-1, LAMP-2 and β-galactosidase, and could not fuse with lysosomes. Despite lacking lysosomal components, p110α deficient phagosomes recruited Rab7 and its effectors RILP and HOPS components Vps16 and Vps41, suggesting that in addition to Rab7, p110α is required for phagolysosome formation. We also examined how Mtb mediates phagosome maturation arrest by screening an Mtb genomic library for factors able to disrupt yeast vacuolar protein sorting (VPS). Since VPS is homologous to mammalian endosomal trafficking, factors that inhibit VPS might also arrest phagosome maturation. Four proteins able to disrupt yeast VPS were identified in this screen, two hypothetical proteins, Rv0900 and Rv1268c, the P-type ATPase Rv0425c, and PE-PGRS62. To study its effects on macrophage function, we generated M. smegmatis able to express PE-PGRS62. Murine macrophages infected with this construct had arrested phagosome maturation, displaying decreased Rab7 and LAMP-1 recruitment. Infected macrophages also expressed 2-3 fold less iNOS protein when compared to cells infected with control bacteria. Loss of PE-PGRS62 expression in M. marinum resulted in greater iNOS levels, and complementation of the mutant with PE-PGRS62 restored the ability to inhibit iNOS expression. Marked differences in colony morphology were also seen in M. smegmatis expressing PE-PGRS62 and in the M. marinum PE-PGRS62 transposon mutant. Our results suggest that PE-PGRS62 supports mycobacterial virulence via inhibition of phagosome maturation and iNOS expression.

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