Carboxyl-Terminal Protease Regulates Brucella suis Morphology in Culture and Persistence in Macrophages and Mice

Abstract

ABSTRACT The putative carboxyl-terminal processing protease (CtpA) of Brucella suis 1330 is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. The B. suis CtpA protein shared up to 77% homology with CtpA proteins of other bacteria. A CtpA-deficient Brucella strain (1330Δ ctpA ), generated by allelic exchange, produced smaller colonies on enriched agar plates and exhibited a 50% decrease in growth rate in enriched liquid medium and no growth in salt-free enriched medium compared to the wild-type strain 1330 or the ctpA -complemented strain 1330Δ ctpA [pBB ctpA ]. Electron microscopy revealed that in contrast to the native coccobacillus shape of wild-type strain 1330, strain 1330Δ ctpA possessed a spherical shape, an increased cell diameter, and cell membranes partially dissociated from the cell envelope. In the J774 mouse macrophage cell line, 24 h after infection, the CFU of the strain 1330Δ ctpA declined by approximately 3 log 10 CFU relative to wild-type strain 1330. Nine weeks after intraperitoneal inoculation of BALB/c mice, strain 1330Δ ctpA had cleared from spleens but strain 1330 was still present. These observations suggest that the CtpA activity is necessary for the intracellular survival of B. suis . Relative to the saline-injected mice, strain 1330Δ ctpA -vaccinated mice exhibited 4 to 5 log 10 CFU of protection against challenge with virulent B. abortus strain 2308 or B. suis strain 1330 but no protection against B. melitensis strain 16 M. This is the first report correlating a CtpA deficiency with cell morphology and attenuation of B. suis .