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Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species
Published on: October 15, 2013
An electronmicroscopic study of the interaction of Burkholderia pseudomallei and human macrophages
Sakthi A Nathan1, S D Puthucheary
1Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Malaysia.
Abstract:
B. pseudomallei has been shown to persist intracellularly in melioidosis patients until reactivated by decreasing immunocompetence. We have shown by transmission electron microscopy the internalization of B. pseudomallei by human macrophages via conventional phagocytosis enclosed within membrane-bound vacuoles or phagosomes. Ferritin labeled lysosomes provided evidence of phagosome-lysosome fusion. Ingested bacilli were designated as "intact" or "damaged" on the basis of their ultrastructural features. An intact bacterium was seen with low electron opaque central nuclear region surrounded by dense bacterial cytoplasm, bounded externally by bacterial plasma membrane and cell wall. In contrast, B. pseudomallei were considered damaged when seen with cavitation within the central nuclear region, separation of bacterial cytoplasm from the cell wall, herniation of cytoplasmic contents and lamination of bacterial cell wall and its surrounding electron transparent zone. Our observations indicate that the microbicidal mechanism(s) in B. pseudomallei-infected macrophages failed to ensure complete clearance of the organism and this failure probably facilitates intracellular persistence and proliferation, and this may be one of the survival strategies adopted by this organism.
Insights
Burkholderia pseudomallei (B. pseudomallei) survives inside human macrophages, evading immune clearance mechanisms. This intracellular persistence, characterized by damaged or intact bacteria, facilitates melioidosis reactivation.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Burkholderia pseudomallei (B. pseudomallei) is known to persist within host cells, leading to chronic melioidosis.
- Decreased immunocompetence can trigger the reactivation of latent B. pseudomallei infections.
Purpose of the Study:
- To investigate the intracellular fate of B. pseudomallei within human macrophages.
- To elucidate the mechanisms underlying B. pseudomallei's intracellular survival and persistence.
Main Methods:
- Transmission electron microscopy (TEM) was used to visualize B. pseudomallei internalization by macrophages.
- Phagosome-lysosome fusion was assessed using ferritin-labeled lysosomes.
- Bacterial ultrastructure was analyzed to differentiate between intact and damaged B. pseudomallei.
Main Results:
- B. pseudomallei was internalized by human macrophages through conventional phagocytosis, forming membrane-bound vacuoles (phagosomes).
- Evidence of phagosome-lysosome fusion was observed, indicating potential microbicidal activity.
- A significant proportion of ingested B. pseudomallei exhibited ultrastructural damage, while others remained intact.
- Macrophage microbicidal mechanisms were insufficient for complete clearance of B. pseudomallei.
Conclusions:
- The failure of macrophages to completely eliminate B. pseudomallei supports its intracellular persistence.
- Intracellular survival and proliferation of B. pseudomallei may represent a key survival strategy.
- Understanding these mechanisms is crucial for developing effective treatments against melioidosis.
