Biochemical, immunological and ultrastructural characterization of aggregation substances encoded by Enterococcus faeclis sex‐pheromone plasmids

Abstract

The sex-pheromone system of Enterococcus faecalis can be viewed as a unique and highly efficient plasmid-collection mechanism. The contact needed for transfer of the conjugative sexpheromone plasmids is mediated by an adhesin, called aggregation substance, which is encoded by these plasmids. We show here that for 17 of the 18 sex-pheromone plasmids (pAM373 being the exception) described to date, their adhesins are immunologically related to each other. In each case, we observed the presence of an N-terminal fragment of about 78 kDa in addition to the 137-kDa form of mature aggregation substance. The cross-reactions were different for the various plasmids. In the case of pPDl the 78-kDa fragment reacted only weakly. The aggregation substance encoded by sex-pheromone plasmid PAD1 (Asal) was characterized in detail. The conditions used for SDSI PAGE had a drastic influence on the migration behavior of mature aggregation substance and differently migrating, interconvertible forms were identified. Preliminary data indicate that Asal might be a glycoprotein. Antibodies were isolated which are directed against the N-and C-terminal parts of aggregation substance. They showed about the same reactivity on Western blots; however, only antibodies directed against the N-terminal part of the aggregation substance could inhibit the bacterial cell/cell contact. The reactions of the two antibody preparations with induced cells of E. faecalis was analyzed by transmission electron microscopy. The results indicated that especially the N-terminal part of aggregation substance is exposed on the cell surface of E. faecalis; the C-terminal part seems to be much less exposed.The sex-pheromone system of Enterococcus faecalis was discovered by the group of D. B. Clewell (Dunny et al., 1978). A first model to its function was given in 1979 (Dunny et al., 1979); it is, with slight modifications, still valid today (see Clewell and Weaver, 1989;Dunny, 1990, for recent reviews). According to this model, the sex-pheromone system can be viewed as a highly efficient plasmid-collection mechanism which is triggered by so-called sex pheromones (small linear peptides) excreted by plasmid-free recipient strains. Donors do not excrete sex peromone(s) corresponding to their plasmid(s) ; plasmid-free recipients are expected to excrete all existing sex pheromones. As a response to sex pheromones, the donor strains synthesize an adhesin (called aggregation substance) which is of proteinaceous nature and appears as hair-like structures (Galli Correspondence to R. Wirth, Lehrstuhl fur Mikrobiologie, Ludiwg-Maximilians-Universitat Miinchen,

The sex pheromone system of Enterococcus faecalis

“…These authors also noted, however, proteins of 130, 153 and 157 kDa after induction. Meanwhile we demonstrated that these higher-molecular-mass proteins are different forms of ASAl migrating differently in SDSPAGE (Hirt et al, 1993) and that the low-molecular-mass proteins to OrjjL represent an N-terminal fragment of the adhesin (depending on the extraction procedures for ASAl different amounts of the mature 137-kDa adhesin or its prominent 74/78-kDa Nterminal fragment are isolated). The situation was also complicated (but see below for explanation) by the fact that the pPD1-encoded gene pd78 does not show similarity to asal, and its gene product PD78 was reported to be important for cell aggregation and mating (Nakayama et al, 1990).…”

“…(d) The structural gene for aggregation substance is present in all plasmids except pAM373 ; in addition, polyclonal antibodies against ASAl react with induced cells carrying the various sex pheromone plamids, again with pAM373 as an exception. (Details of these data can be found in Galli and Wirth, 1991;Weidlich et al, 1992;Hirt et al, 1993).…”

In Vivo Induction of Virulence and Antibiotic Resistance Transfer in Enterococcus faecalis Mediated by the Sex Pheromone-Sensing System of pCF10

“…AS of E. faecalis is highly conserved among sex pheromone plasmids (25) and was assumed to be a contributor to virulence of this important nosocomial pathogen after sequencing studies revealed the presence of two RGD motifs encoded in the AS protein (20,21,27). Kreft and coworkers demonstrated increased adhesion of AS-expressing cells to cultured renal tubular cells (28), which could be inhibited somewhat by the addition of RGD-containing peptides.…”

Analysis of Functional Domains of the Enterococcus faecalis Pheromone-Induced Surface Protein Aggregation Substance

“…Secondary structural analysis yields little information with the exception of a predicted alpha-helix domain from amino acids 200 to 280 (36). Isolation of AS yields both a full-length version of the protein (137 kDa) and a specific, 78-kDa, N-terminal cleavage product (9). Scanning electron microscopy of Asa1 on the cell surface suggests that the N terminus of the protein is more exposed than the C terminus (9).…”

“…Isolation of AS yields both a full-length version of the protein (137 kDa) and a specific, 78-kDa, N-terminal cleavage product (9). Scanning electron microscopy of Asa1 on the cell surface suggests that the N terminus of the protein is more exposed than the C terminus (9). Finally, the only structural analysis of AS done to date found that an aggregation domain of Asa1 from amino acids 525 to 617 (of the mature protein with the signal sequence removed) exists and that the C terminus plays no essential role in aggregation (20).…”