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Inactivation of macrophage Rab7 by Burkholderia cenocepacia
Kassidy K Huynh1, Jonathan D Plumb, Gregory P Downey
1Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Abstract:
Strains of the Burkholderia cepacia complex can survive within macrophages by arresting the maturation of phagocytic vacuoles. The bacteria preclude fusion of the phagosome with lysosomes by a process that is poorly understood. Using murine macrophages, we investigated the stage at which maturation is arrested and analyzed the underlying mechanism. Vacuoles containing B. cenocepacia strain J2315, an isolate of the transmissible ET12 clone, recruited Rab5 and synthesized phosphatidylinositol-3-phosphate, indicating progression to the early phagosomal stage. Despite the fact that the B. cenocepacia-containing vacuoles rarely fused with lysosomes, they could nevertheless acquire the late phagosomal markers CD63 and Rab7. Fluorescence recovery after photobleaching and use of a probe that detects Rab7-guanosine triphosphate indicated that activation of Rab7 was impaired by B. cenocepacia, accounting at least in part for the inability of the vacuole to merge with lysosomes. The Rab7 defect was not due to excessive cholesterol accumulation and was confined to the infected vacuoles. Jointly, these experiments indicate that B. cenocepacia express virulence factors capable of interfering with Rab7 function and thereby with membrane traffic.
Insights
Burkholderia cepacia complex bacteria survive in macrophages by blocking phagosome maturation. This study reveals the bacteria impair Rab7 function, preventing lysosome fusion and enabling bacterial survival.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- The Burkholderia cepacia complex (BCC) are opportunistic pathogens.
- BCC can survive inside host macrophages by preventing phagosome maturation.
- The mechanisms by which BCC arrests phagosome maturation are not fully understood.
Purpose of the Study:
- To investigate the stage of phagosome maturation arrest by BCC.
- To elucidate the molecular mechanisms underlying this arrest.
Main Methods:
- Murine macrophages were infected with B. cenocepacia strain J2315.
- Phagosome maturation markers (Rab5, phosphatidylinositol-3-phosphate, CD63, Rab7) were analyzed.
- Fluorescence recovery after photobleaching and Rab7-GTP detection were used to assess Rab7 activation.
Main Results:
- B. cenocepacia-containing vacuoles progressed to the early phagosomal stage (Rab5, PI3P+).
- Vacuoles acquired late phagosomal markers (CD63, Rab7) but rarely fused with lysosomes.
- B. cenocepacia impaired Rab7 activation, hindering lysosome fusion.
- The Rab7 defect was specific to infected vacuoles and not caused by cholesterol accumulation.
Conclusions:
- B. cenocepacia arrests phagosome maturation by interfering with Rab7 function.
- This interference with Rab7 disrupts normal membrane traffic and lysosome fusion.
- BCC employs virulence factors to manipulate host cell processes for survival.
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