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Rhodococcus equi can survive a phagolysosomal environment in macrophages by suppressing acidification of the phagolysosome

Kiminori Toyooka¹, Shinji Takai² and Teruo Kirikae¹

¹Department of Infectious Diseases, Research Institute, International Medical Center of Japan, Toyama 1-21-1, Shinjuku, Tokyo 162-8655, Japan ²Department of Animal Hygiene, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan

Correspondence Teruo Kirikae tkirikae{at}ri.imcj.go.jp

Received March 11, 2005
Accepted July 26, 2005

Rhodococcus equi is one of the most important causes of pneumonia in foals and has emerged as a significant opportunistic pathogen of immunosuppressed hosts such as human immunodeficiency virus-infected patients. Virulent R. equi harbouring an 85 kb plasmid, but not the avirulent form lacking the plasmid, has the ability to survive in macrophages. However, the survival mechanism is not known. In the present study, morphological interactions were observed between virulent or plasmid-cured avirulent R. equi and phagolysosomes in murine macrophage-like J774.1 cells by immunocytological methods. The J774.1 cells phagocytosed virulent and avirulent bacteria to a similar extent, and both bacteria replicated in single membrane vacuoles at similar rates up to 6 h after infection. Thereafter, the virulent bacteria continued to grow, whereas the avirulent bacteria stopped growing. When the infected cells were stained with phagosomal and lysosomal markers and observed with a confocal fluorescence microscope, the majority of phagosomes containing these bacteria were fused with lysosomes. Neither R. equi organism has the ability to hinder phagosome-lysosome fusion. The acidity in phagolysosomes containing R. equi was examined by staining with LysoTracker Red DND-99, an acidotropic probe. The phagolysosomes containing virulent organisms were not acidic as compared with avirulent organisms. Over 90 % of the phagolysosomes containing avirulent R. equi were stained with LysoTracker 6 h after infection, whereas less than 50 % of those containing virulent R. equi were stained. Furthermore, when the supernatant obtained from a virulent R. equi culture was added to the cell cultures, the acidity of acidic compartments in macrophages was reduced. The authors conclude that some substance(s) produced by virulent R. equi suppress acidification in phagolysosomes, and help R. equi survival and replication in the bactericidal environment.

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