The Burkholderia pseudomallei Type III Secretion System and BopA Are Required for Evasion of LC3-Associated Phagocytosis

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal infectious disease endemic in tropical regions worldwide, and especially prevalent in southeast Asia and northern Australia. This intracellular pathogen can escape from phagosomes into the host cytoplasm, where it replicates and infects adjacent cells. We previously demonstrated that, in response to B. pseudomallei infection of macrophage cell line RAW 264.7, a subset of bacteria co-localized with the autophagy marker protein, microtubule-associated protein light chain 3 (LC3), implicating autophagy in host cell defence against infection. Recent reports have suggested that LC3 can be recruited to both phagosomes and autophagosomes, thereby raising questions regarding the identity of the LC3-positive compartments in which invading bacteria reside and the mechanism of the autophagic response to B. pseudomallei infection. Electron microscopy analysis of infected cells demonstrated that the invading bacteria were either free in the cytosol, or sequestered in single-membrane phagosomes rather than double-membrane autophagosomes, suggesting that LC3 is recruited to B. pseudomallei-containing phagosomes. Partial or complete loss of function of type III secretion system cluster 3 (TTSS3) in mutants lacking the BopA (effector) or BipD (translocator) proteins respectively, resulted in delayed or no escape from phagosomes. Consistent with these observations, bopA and bipD mutants both showed a higher level of co-localization with LC3 and the lysosomal marker LAMP1, and impaired survival in RAW264.7 cells, suggesting enhanced killing in phagolysosomes. We conclude that LC3 recruitment to phagosomes stimulates killing of B. pseudomallei trapped in phagosomes. Furthermore, BopA plays an important role in efficient escape of B. pseudomallei from phagosomes.

Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade

“…In contrast to obvious requirements for the Bsa T3SS in vitro and in vivo (2), to our knowledge no effectors have been definitively shown to be required for invasion, replication in nonphagocytic cells, cell-cell spread, MNGC formation, or virulence in animals. BopA, a suspected T3SS Bsa substrate, is reported to facilitate survival and evasion of autophagy in phagocytic cells (11), but bopA mutants are not significantly attenuated in mice (2). The precise mechanism of T3SS-mediated endosome escape is unknown for any intracellular pathogen; however, it could conceivably be a function of translocon insertion in the endosomal membrane and occur in the absence of additional effectors.…”

“…The third, T3SS Bsa , is homologous to the Shigella MxiSpa and Salmonella SPI-1 T3SSs and is highly conserved in Bp, Bt, and Bm (1, 2). T3SS Bsa is required for virulence in hamster and murine models of pathogenesis (2) and has been implicated in invasion of epithelial cells, escape from endosomes, intracellular survival, and evasion of autophagy (11). In addition, Bp encodes six type VI secretion systems (T6SSs) (12).…”

Burkholderia pseudomallei survival in lung epithelial cells benefits from miRNA-mediated suppression of ATG10

“…Previous studies have reported that autophagic evasion of B. pseudomallei is an active process relying on TTSS effector, BopA, which shows 23% amino acid identity to the Shigella TTSS effector, IcsB. 12,22 In Shigella, IcsB competitively binds to its surface protein VirG to inhibit the autophagic process; ATG5 recognizes VirG. 8 Therefore, in a manner analogous to IcsB, it appears unlikely that BopA is the key factor to interfere with ATG10 expression upon B. pseudomallei infection.…”

Autophagy as a macrophage response to bacterial infection

“…Thus, only a small portion of S. flexneri in the cytosol are recognized by autophagy via polyubiquitination and p62/NDP52 recruitment in a process that requires IcsA-mediated actin polymerization and the assembly of bacteria-containing cages from septin, a component of the host cytoskeleton (23,78). Other bacteria such as B. pseudomallei may be killed by LAP, but can escape into the cytosol, a process facilitated by the T3SS effector BopA (34). Once in the cytosol B. pseudomallei escape recognition by the autophagic machinery using a mechanism yet to be elucidated (34,35).…”

“…Other bacteria such as B. pseudomallei may be killed by LAP, but can escape into the cytosol, a process facilitated by the T3SS effector BopA (34). Once in the cytosol B. pseudomallei escape recognition by the autophagic machinery using a mechanism yet to be elucidated (34,35).…”