Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi

Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 9 0-kb plasmids encoding a highly immunogenic virulence-associated protein (V apA). The objective of this study was to determine the role of the 85-kb pl asmid and VapA in the intracellular survival and virulence of R equi. Clini cal isolates containing the plasmid and expressing VapA efficiently replica ted within mouse macrophages in vitro, while plasmid-cured derivatives of t hese organisms did not multiply intracellularly, An isolate harboring the l arge plasmid also replicated in the tissues of experimentally infected mice , whereas its plasmid-cured derivative was rapidly cleared. All foals exper imentally infected with a plasmid containing clinical isolate developed sev ere bronchopneumonia, whereas the foals infected,vith its plasmid cured der ivative remained asymptomatic and free of visible lung lesions. By day 14 p ostinfection, lung bacterial burdens had increased considerably in foals ch allenged with the plasmid-containing clinical isolate. In contrast, bacteri a could no longer be cultured from the lungs of foals challenged with the i sogenic plasmid-cured derivative. A recombinant, plasmid-cured derivative e xpressing wild-type levels of VapA failed to replicate in macrophages and r emained avirulent for both mice and foals. These results show that the 85-k b plasmid of R. equi is essential for intracellular replication within macr ophages and for development of disease in the native host, the foal. Howeve r, expression of VapA alone is not sufficient to restore the virulence phen otype.