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Infection and Immunity, December 2009, p. 5676-5681, Vol. 77, No. 12
0019-9567/09/$08.00+0     doi:10.1128/IAI.00856-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Rhodococcus equi Virulence-Associated Protein A Is Required for Diversion of Phagosome Biogenesis but Not for Cytotoxicity{triangledown}

Kristine von Bargen,1,{dagger} Marco Polidori,1,{dagger} Ulrike Becken,1 Gitta Huth,1 John F. Prescott,2 and Albert Haas1*

Cell Biology Institute, University of Bonn, Bonn, Germany,1 Department of Pathobiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada2

Received 29 July 2009/ Returned for modification 5 September 2009/ Accepted 11 September 2009

Rhodococcus equi is a gram-positive facultative intracellular pathogen that can cause severe bronchopneumonia in foals and AIDS patients. Virulence is plasmid regulated and is accompanied by phagosome maturation arrest and host cell necrosis. A replacement mutant in the gene for VapA (virulence-associated protein A), a major virulence factor of R. equi, was tested for its activities during macrophage infection. Early in infection, phagosomes containing the vapA mutant did not fuse with lysosomes and did not stain with the acidotropic fluor LysoTracker similar to those containing virulent wild-type R. equi. However, vapA mutant phagosomes had a lower average pH. Late in infection, phagosomes containing the vapA mutant were as frequently positive for LysoTracker as phagosomes containing plasmid-cured, avirulent bacteria, whereas those with virulent wild-type R. equi were still negative for the fluor. Macrophage necrosis after prolonged infection with virulent bacteria was accompanied by a loss of organelle staining with LysoTracker, suggesting that lysosome proton gradients had collapsed. The vapA mutant still killed the macrophages and yet did not affect the pH of host cell lysosomes. Hence, VapA is not required for host cell necrosis but is required for neutralization of phagosomes and lysosomes or their disruption. This is the first report of an R. equi mutant with altered phagosome biogenesis.

* Corresponding author. Mailing address: Cell Biology Institute, University of Bonn, Ulrich-Haberland-Str. 61a, 53121 Bonn, Germany. Phone: 49 (0) 228 736340. Fax: 49 (0) 228 736304. E-mail: albert.haas{at}uni-bonn.de

{triangledown} Published ahead of print on 21 September 2009.

Editor: A. J. Bäumler

{dagger} K.V.B. and M.P. contributed equally to this study.

Infection and Immunity, December 2009, p. 5676-5681, Vol. 77, No. 12
0019-9567/09/$08.00+0     doi:10.1128/IAI.00856-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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