Antibodies to a surface-exposed, N-terminal domain of aggregation substance are not protective in the rabbit model of Enterococcus faecalis infectiveendocarditis

The aggregation substance (AS) surface protein from Enterococcus faecalis h as been implicated as an important virulence factor for the development of infective endocarditis. To evaluate the role of antibodies specific for Asc 10 (the AS protein from the conjugative plasmid pCP10) in protective immuni ty to infective endocarditis, an N-terminal region of Asc10 lacking the sig nal peptide and predicted to be surface exposed (amino acids 44 to 331: AS( 44-331)) was cloned with a C-terminal histidine tag translational fusion an d expressed from Escherichia coli. N-terminal amino acid sequencing of the purified protein revealed the correct sequence, and rabbit polyclonal antis era raised against AS(44-331) reacted specifically to Asc10 expressed from E.faecalis OG1SSp, but not to other proteins as judged by Western blot anal ysis. Using these antisera, flow cytometry analysis dem onstrated that anti bodies to AS(44-331) bound to a surface-exposed region of Asc10. Furthermor e, antibodies specific for AS(44-331) were opsonic for E. faecalis expressi ng Asc10 in vitro but not for cells that did not express Asc10. New Zealand White rabbits immunized with AS(44-331) were challenged intravenously with E,faecalis cells constitutively expressing Asc10 in the rabbit model of ex perimental endocarditis, Highly immune animals did not show significant dif ferences in clearance of organisms from the blood or spleen or in formation of vegetations on the aortic valve, in comparison with nonimmune animals. Although in vivo expression of Asc10 was demonstrated by immunohistochemist ry, these experiments provide evidence that immunity to Asc10 does not play a role in protection from experimental infective endocarditis due to E, fa ecalis and may have important implications for the development of immunolog ical approaches to combat enterococcal endocarditis.