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.
Highlights
Burkholderia pseudomallei is a Gram-negative, soil dwelling bacillus
To investigate which of these pathways may be responsible for colocalization of B. pseudomallei with light chain 3 (LC3) in infected RAW 264.7 cells, we used transmission electron microscopy (TEM) to view sections prepared from cells infected with wild-type bacteria (Figure 3)
These analyses showed that B. pseudomallei was primarily present either free in the cytoplasm (Figure 3A), or within single-membrane phagosomes (Figure 3B)
Summary
Burkholderia pseudomallei is a Gram-negative, soil dwelling bacillus It is the causative agent of melioidosis, a fatal infection of many animal species and humans and is endemic in tropical and subtropical areas of the world [1,2]. Once in the cytoplasm bacteria can replicate and induce actin polymerization at one pole of the bacterium, facilitating intracellular motility [10]. This actin-based motility is considered to facilitate bacterial spreading into adjacent cells leading to the formation of multinucleated giant cells (MNGC), which have been observed both in cultured cell lines and the tissues of patients [11]
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