Rab32 GTPase, as a direct target of miR-30b/c, controls the intracellular survival of Burkholderia pseudomallei by regulating phagosome maturation

Burkholderia pseudomallei is a gram-negative, facultative intracellular bacterium, which causes a disease known as melioidosis. Professional phagocytes represent a crucial first line of innate defense against invading pathogens. Uptake of pathogens by these cells involves the formation of a phagosome that matures by fusing with early and late endocytic vesicles, resulting in killing of ingested microbes. Host Rab GTPases are central regulators of vesicular trafficking following pathogen phagocytosis. However, it is unclear how Rab GTPases interact with B. pseudomallei to regulate the transport and maturation of bacterial-containing phagosomes. Here, we showed that the host Rab32 plays an important role in mediating antimicrobial activity by promoting phagosome maturation at an early phase of infection with B. pseudomallei. And we demonstrated that the expression level of Rab32 is increased through the downregulation of the synthesis of miR-30b/30c in B. pseudomallei infected macrophages. Subsequently, we showed that B. pseudomallei resides temporarily in Rab32-positive compartments with late endocytic features. And Rab32 enhances phagosome acidification and promotes the fusion of B. pseudomallei-containing phagosomes with lysosomes to activate cathepsin D, resulting in restricted intracellular growth of B. pseudomallei. Additionally, Rab32 mediates phagosome maturation depending on its guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding state. Finally, we report the previously unrecognized role of miR-30b/30c in regulating B. pseudomallei-containing phagosome maturation by targeting Rab32 in macrophages. Altogether, we provide a novel insight into the host immune-regulated cellular pathway against B. pseudomallei infection is partially dependent on Rab32 trafficking pathway, which regulates phagosome maturation and enhances the killing of this bacterium in macrophages.
Author summary Burkholderia pseudomallei is a gram-negative intracellular bacterium and the etiological agent of melioidosis. Little is known about the host innate immune system, which is engaged in a continuous battle against this pathogen and may contribute to the outcomes of melioidosis. Recently, Rab32, a Rab GTPase was shown to be a critical regulator of a host defense pathway against intracellular bacterial pathogens. However, the exact mechanism of how Rab32 contributes to the restriction of intracellular pathogens is not completely understood. In this study, we determined that the infection of macrophages with B. pseudomallei resulted in the upregulation of Rab32 expression through the inhibition of miR-30b/30c expression. Subsequently, Rab32 is recruited to the B. pseudomallei-containing phagosomes and promotes the fusion of the phagosomes with lysosomes, which results in the increased exposure of B. pseudomallei to lysosomal acid hydrolases CTSD, thus limiting the intracellular growth of B. pseudomallei at an early phase of infection in macrophages. Our findings establish for the first time that Rab32 plays an important role in suppressing the intracellular replication of B. pseudomallei by modulating phagosome maturation in macrophages, providing a new insight into the host defense mechanisms against B. pseudomallei infection.