The sex pheromone plasmids in Enterococcus faecalis are one of the most efficient conjugative plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10 ؊1 transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of plasmid pCF10 and the analysis of the transcription profile of plasmid genes, relative to conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis.The advent of microarray technology allows for a comprehensive analysis of gene expression patterns associated with various biological processes, providing insights into complex regulatory networks. One of the most complex processes is the transfer of large portions of genetic material from a donor cell into a recipient cell by means of conjugation. The plasmids of the sex pheromone family in Enterococcus faecalis are among the most efficient bacterial conjugation systems known (16). The family consists of over 20 plasmids and shows extensive sequence homologies (28). E. faecalis strains can host several of these plasmids. This is exemplified by strain V583, the first vancomycin-resistant isolate in the United States (45), chosen for genome sequencing by The Institute for Genomic Research (TIGR; www.tigr.org). V583 contains two sex pheromone plasmids with homology to the well-characterized pAD1 (pTEF1) and pCF10 (pTEF2) plasmids, respectively. The complete sequences for the pheromone plasmids pAD1 and pAM373 became available recently (14,19). Analysis of the sequences of this group of plasmids allows comparisons and insights into the evolution of these elements.Although the sex pheromone plasmids can be disseminated among enterococcal populations very efficiently, plasmid transfer is highly regulated and only induced by recipient cells in close proximity to plasmid donors. The recipient cells secret 7-to 8-amino-acid-long hydrophobic sex pheromones that are bound by a plasmid-encoded binding protein (44, 51). The pheromone is then taken up into the cell (32) and releases a transcriptional block of the PrgX/TraA family of repressors (5). One of the early transcripts after induction encodes for the surface protein aggregation substance (AS) (9). Expression of AS results in tight physical contact between donor and recipient, allows for plasmid transfer rates of up to 10 Ϫ1 transconjugants/donor (16), and is nece...

Human parotid secretory protein (PSP; BPIF2A) is predicted to be structurally similar to bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. Based on the locations of known antimicrobial peptides in the latter two proteins, potential active peptides in the PSP sequence were identified. One such peptide, GL13NH 2 (PSP residues 141 to 153) was shown previously to interfere with LPS binding and agglutinate bacteria without bactericidal activity. By introducing three additional positively charged lysine residues, the peptide was converted to the novel bactericidal cationic peptide GL13K (MIC for Pseudomonas aeruginosa, 8 g/ml [5.6 M]). We investigated the antibiofilm activity of GL13K against static, monospecies biofilms of P. aeruginosa PAO1. Two-hour exposure of a 24-h biofilm to 64 g/ml (44.8 M) GL13K reduced biofilm bacteria by 10 2 , and 100 g/ml (70 M) GL13K reduced bacteria by 10 3 . Similar results could be achieved on 48-h-old biofilms. Lower concentrations of GL13K (32 g/ml [22.4 M]) were successful in reducing biofilm cell numbers in combination with tobramycin. This combination treatment also achieved total eradication of the biofilm in a majority (67.5%) of tested samples. An alanine scan of GL13K revealed the importance of the leucine residue in position six of the peptide sequence, where replacement led to a loss of antibiofilm activity, whereas the impact of replacing charged residues was less pronounced. Bacterial metalloproteases were found to partially inactivate GL13K but not a D amino acid version of the peptide.

GL13NH2 is a bacteria-agglutinating peptide derived from the sequence of the salivary protein parotid secretory protein (PSP, BPIFA2, SPLUNC2, C20orf70). The peptide agglutinates both Gram negative and Gram positive bacteria, and shows anti-lipopolysaccharide activity in vitro and in vivo. However, GL13NH2 does not exhibit bactericidal activity. To generate a more cationic peptide with potential bactericidal activity, three amino acid residues were replaced with lysine residues to generate the peptide GL13K. In this report, the antibacterial and anti-inflammatory activities of GL13K were characterized. GL13K had lost the ability to agglutinate bacteria but gained bactericidal activity. Substitution of individual amino acids in GL13K with alanine did not restore bacterial agglutination. GL13K was bactericidal against Pseudomonas aeruginosa, Streptococcus gordonii and Escherichia coli but not Porphyromonas gingivalis. Unlike the agglutinating activity of GL13NH2, the bactericidal activity of GL13K against Pseudomonas aeruginosa was retained in the presence of saliva. Both GL13NH2 and GL13K exhibited anti-lipopolysaccharide activity. In GL13K, this activity appeared to depend on a serine hydroxyl group. GL13K protected mice from lipopolysaccharide- induced sepsis and the peptide exhibited a low level of hemolysis, suggesting that it may be suitable for in vivo application.

Enterococcus faecalis has become one of the most notable nosocomial pathogens in the last decade. Aggregation substance (AS) on the sex pheromone plasmids of E. faecalis has been implicated as a virulence factor in several model systems. We investigated the AS-encoding plasmid pCF10 for its ability to increase virulence in a rabbit endocarditis model. Cells containing pCF10 increased the virulence in the model significantly, as assessed by an increase in aortic valve vegetation size. The results confirmed in vivo induction of the normally tightly controlled AS. In addition to the expression of AS when E. faecalis cells were in contact with plasma, plasmid transfer of the tetracycline resistance-carrying plasmid was also activated in vitro and in vivo. In vivo, plasmid transfer reached remarkable frequencies of 8 ؋ 10 ؊2 to 9 ؋ 10 ؊2 . These values are comparable to the highest frequencies ever observed in vitro. Cells harboring pCF10 had a significant survival advantage over plasmid-free cells indicated by pCF10 present in two-thirds of the recipient population. Plasma induction was dependent on the presence of the plasmid-encoded PrgZ protein, indicating the requirement of the pheromonesensing system in the induction process. The data suggested that the mechanism of in vivo induction may involve interference of plasma with the normal function of the pheromone peptide and its inhibitor. Aggregation substance (AS) is a 137-kDa surface protein encoded on sex pheromone plasmids of Enterococcus faecalis.This protein mediates strong binding between E. faecalis cells as manifested by the formation of visible cell aggregates preceding the conjugative transfer of a sex pheromone plasmid (14). The plasmid transfer response is initiated by 7-to 8-amino-acid-long hydrophobic peptides secreted by potential recipient cells (16,32). These peptides are part of signal sequences of chromosomally encoded lipoproteins (10) and lead to induction of AS expression.Asc10, the AS of the sex pheromone plasmid pCF10 (encoding tetracycline resistance) (17), is induced by the 7-aminoacid peptide cCF10 (32) encoded by the ccfA gene. The donor cell binds the peptide (37), employing a specific plasmid-encoded receptor, PrgZ, an OppA homologue (Fig. 1). The peptide is then transported into the cell via the oligopeptide permease system (29), leading to the expression of AS and subsequent plasmid transfer. Since expression of AS and the other plasmid transfer machinery is presumably a very energyconsuming process, it is not surprising that expression of AS is tightly regulated. This ensures that the nonmotile enterococcal cells expressing transfer functions are in close proximity for cell contact to occur between mating partners. pCF10 donor cells are faced with the dilemma that they secrete unaltered amounts of cCF10 (33), necessitating a complex scheme to prevent autoinduction by their own pheromones. A schematic overview of the events controlling AS expression is given in Fig. 1. Secreted cCF10 may accumulate to approximately 8 pg/ml (33). To...