Analysis of functional domains of the Enterococcus faecalis pheromone-induced surface protein aggregation substance

Abstract

Pheromone-inducible aggregation substance (AS) proteins of Enterococcus faecalis are essential for high-efficiency conjugation of the sex pheromone plasmids and also serve as virulence factors during host infection. A number of different functions have been attributed to AS in addition to bacterial cell aggregation, including adhesion to host cells, adhesion to fibrin, increased cell surface hydrophobicity, resistance to killing by polymorphonuclear leukocytes and macrophages, and increased vegetation size in an experimental endocarditis model. Relatively little information is available regarding the structure-activity relationship of AS. To identify functional domains, a library of 23 nonpolar 31-amino-acid insertions was constructed in Asc10, the AS encoded by the plasmid pCF10, using the transposons TnlacZ/in and TnphoA/in. Analysis of these insertions revealed a domain necessary for donor-recipient aggregation that extends further into the amino terminus of the protein than previously reported. In addition, insertions in the C terminus of the protein also reduced aggregation. As expected, the ability to aggregate correlates with efficient plasmid transfer. The results also indicated that an increase in cell surface hydrophobicity resulting from AS expression is not sufficient to mediate bacterial aggregation.

FIG. 1

(A) The positions of insertion mutations in prgB are shown on a linear map of the gene. Each mutation consists of an in-frame 31-amino-acid insertion. The insertions at r2760 and r3183 are in frame but in the reverse orientation, while the insertion at s3599 is out of frame and produces a stop codon. Structural map: SS, signal sequence; helix, predicted N-terminal helix domain; variable, unconserved AS region; essential, aggregation domain identified by Muscholl-Silberhorn; and cleavage, site of cleavage that produces the characteristic N-terminal 78-kDa fragment. (B) The mutant Asc10 proteins were expressed using the nisin-inducible Asc10 expression vector pMSP7517.

FIG. 2

Western blot analysis of surface extracts from the Asc10 insertion mutants. The Western blot utilized a polyclonal antibody generated against the N terminus of Asc10. An equivalent amount of protein was added to each lane. The laddering pattern is typical of AS protein preparations, as they have high instability. Migration of molecular mass standard marker proteins is shown at the left of the blot, and the 137-kDa full-length Asc10 and 78-kDa fragment are indicated by arrows. 7517, Asc10⁺; 3535, vector control.

FIG. 3

Aggregation of the mutants was measured by spectrophotometry and flow cytometry. Representative data for Asc10⁺ (7517), the vector control (3535), a functional mutant (1299), intermediate mutant (r2760), and a nonaggregating mutant (3102) are shown. UR, upper right. (A). The aggregation of the mutants was measured by flow cytometry as a percentage of the induced populations in the (UR) quadrant (B). The OD₆₀₀ of induced cultures after 1 h of settling was also determined (C). A decrease in OD₆₀₀ indicates aggregation. #, P < 0.05 from 7517; @, P < 0.1 from 7517; ∗, P < 0.05 from 3535; $, P < 0.1 from 3535.

FIG. 4

Insertion mutants 2049 and 2421 have near-wild-type aggregation levels when grown at 30°C as indicated by spectrophotometry (A). Western blot analysis of surface extracts indicated much more reactive protein on these cells when they were grown at 30°C (B). Migration of molecular mass standard marker proteins is shown to the left of the blot, and the 137-kDa full-length and 78-kDa Asc10 fragments are indicated by arrows.

FIG. 5

The plasmid transfer levels of each mutant were determined and were expressed as transconjugants/donor (tc/donor) (A). Plasmid transfer levels are compared with the ability to aggregate (B). UR, upper right.

FIG. 6

Cell surface hydrophobicity of each mutant as a percentage of cells that were extracted with hexadecane (A). Comparison of hydrophobicity with aggregation for each mutant can be seen in the scatter plot (B). UR, upper right.

FIG. 7

Functional domains identified in this study that disrupted aggregation (top) and hydrophobicity (bottom). All proteins generated from these insertions were expressed on the cell surface. The two mutants with increased stability at 30°C are indicated (∗). Amino acids preceding the insertions indicate the mutants (nucleotide residues are given in parentheses). The Extended Agg domain was identified by the mutations in this paper and by the previously identified aggregation functional domain (21). Note that the N-terminal aggregation domain extends into the variable region. C-terminal insertions that disrupt aggregation are hypothesized to play a structural role. Many mutants that are unable to aggregate still increase cell surface hydrophobicity.