Florence Y. An

Enterococcal plasmid transfer: sex pheromones, transfer origins, relaxases, and the Staphylococcus aureus issue.

Certain conjugative plasmids in Enterococcus faecalis encode a mating response to peptide sex pheromones encoded on the chromosome of potential recipient (plasmid-free) strains. The pheromone precursors correspond to the precursors of surface lipoproteins with the mature peptides coming from the last 7-8 residues of the related signal sequences. Processing that gives rise to the pAD1-related peptide involves a chromosome-encoded metalloprotease (Eep) that is believed to operate within the cytoplasmic membrane. Mutations in the determinants for cAD1 and cAM373, cad and camE, respectively, do not affect cell viability; and when the related plasmid is present, the pheromone response is normal. A cAM373-like activity is produce by Staphylococcus aureus, but the corresponding lipoprotein determinant (camS) is unrelated to the enterococcal determinant (camE). pAD1 has two origins of transfer, oriT1 and oriT2 and encodes a relaxase (TraX), which has been shown to specifically nick in oriT2. pAM373 has a site, oriT, that is similar to oriT2 of pAD1. Both sites (oriT2 of pAD1 and oriT of pAM373) have a series of short direct repeats (5-6 bp with 5-6 bp-spacings) adjacent to a long inverted repeat (140 bp). The direct repeats differ significantly and confer specificity to the two systems. pAD1 and pAM373 are both able to mobilize the nonconjugative plasmid pAMalpha1, which encodes two relaxases that are involved in transfer. Relevant information concerning the possible movement of vancomycin resistance from E. faecalis to S. aureus in a clinical environment is discussed.

Identification of the cAD1 Sex Pheromone Precursor in Enterococcus faecalis

The Enterococcus faecalis virulence plasmid pAD1 encodes a mating response induced by exposure to an octapeptide sex pheromone, cAD1, secreted by plasmid-free enterococci. The determinant for the pheromone in E. faecalis FA2-2, designated cad, was found to encode a 309-amino-acid lipoprotein precursor with the last 8 residues of its 22-amino acid signal sequence representing the cAD1 moiety. The lipoprotein moiety contained two 77-amino-acid repeats (70% identity) separated by 45 residues. The nonisogenic E. faecalis strain V583 determinant encodes a homologous precursor protein, but it differs at two amino acid positions, both of which are located within the pheromone peptide moiety (positions 2 and 8). Construction of a variant of strain FA2-2 containing the differences present in V583 resulted in cells that did not produce detectable cAD1. The mutant appeared normal under laboratory growth conditions, and while significantly reduced in recipient potential, when carrying pAD1 it exhibited a normal mating response. A mutant of FA2-2 with a truncated lipoprotein moiety appeared normal with respect to recipient potential and, when carrying plasmid DNA, donor potential. A gene encoding a protein designated Eep, believed to be a zinc metalloprotease, had been previously identified as required for pheromone biosynthesis and was believed to be involved in the processing of a pheromone precursor. Our new observation that the pAD1-encoded inhibitor peptide, iAD1, whose precursor is itself a signal sequence, is also dependent on Eep is consistent with the likelihood that such processing occurs at the amino terminus of the cAD1 moiety.

NUCLEOTIDE SEQUENCE OF THE SEX PHEROMONE INHIBITOR (IAD1) DETERMINANT OF ENTEROCOCCUS FAECALIS CONJUGATIVE PLASMID PAD1

The determinant for the peptide sex pheromone inhibitor iAD1 (iad) on the hemolysin/bacteriocin plasmid pAD1 of Enterococcus faecalis was sequenced. The sequence reveals a 22-amino-acid precursor with the carboxyl-terminal 8 residues corresponding to iAD1. It appears that iAD1 is a component of its own signal sequence.

Isolation and structure of the Streptococcus faecalis sex pheromone, cAM373

The Streptococcus faecalis sex pheromone, cAM373, which induces a mating response of donor cells harboring plasmid pAM373 and is also produced by Staphylococcus aureus, was isolated and its structure determined. Supernatant from an overnight culture of a recipient strain was subjected to successive purification procedures, and 4.4 μg cAM373 was obtained. The isolated pheromone showed activity at a concentration as low as 5 × 10−11 M. Sequence analysis indicated that cAM373 was a heptapeptide, H‐Ala‐Ile‐Phe‐Ile‐Leu‐Ala‐Ser‐OH, and that its M r was 733. A synthetic replicate of the peptide showed the same biological activity and chromatographic behavior as the native cAM373.

Streptococcus faecalis sex pheromone (cAD1) response: Evidence that the peptide inhibitor excreted by pAD1-containing cells may be plasmid determined

Streptococcus faecalis strains harboring the conjugative plasmid pAD1 excrete a small peptide, iAD1, which inhibits the sex pheromone cAD1. Studies making use of the host strain Streptococcus faecium 9790, which normally does not excrete peptide pheromones, suggest that iAD1 may be determined directly by pAD1.

HtpG, the Porphyromonas gingivalis HSP-90 homologue, induces the chemokine CXCL8 in human monocytic and microvascular vein endothelial cells.

CXCL8 (interlukin 8, IL‐8) has a diverse spectrum of biological activities including T cell, neutrophil and basophil chemotactic properties. It is produced by a wide variety of cell types and plays a significant role in the initiation of the acute inflammatory response. During inflammation, CXCL8 attracts and activates leukocytes at the site of infection leading to leukocyte infiltration, which can lead to tissue damage. Porphyromonas gingivalis, an aetiological agent of periodontitis, induces production of CXCL8 from several types of cells via its LPS and outer membrane proteins. Bacterial chaperones elicit a strong pro‐inflammatory response in cells of the innate immune system. In P. gingivalis the htpG gene codes for the homologue of human Hsp90, a chaperone that associates with transcription factors, hormone receptors and protein kinases, affecting signal transduction pathways. CXCL8 mRNA and CXCL8 protein production was induced in monocytic/human microvascular vein endothelial cells treated with P. gingivalis cells or rHtpG protein. Blocking of receptors CD91 and TLR4 reduced the production of CXCL8 by rHtpG either using receptor‐specific antibody or by siRNA silencing. Pre‐incubation of P. gingivalis rHtpG preparations with human anti‐HtpG significantly inhibited CXCL8 production. A P. gingivalis HtpG disruption mutant also induced less CXCL8 mRNA and protein. These results suggest that P. gingivalis HtpG might be involved in CXCL8‐mediated immunopathogenesis.

Tn917 transposase. Sequence correction reveals a single open reading frame corresponding to the tnpA determinant of Tn3-family elements

A nucleotide sequence correction on the Enterococcus faecalis transposon Tn917 indicates that what was formerly thought to be two open reading frames (ORF5 and ORF6) is actually one reading frame (ORF5). The latter exhibits homology with the Tn3-family transposase determinants known as tnpA.

Sex Pheromones and Plasmid Transfer in Streptococcus Faecalis: A Pheromone, cAM373, Which is Also Excreted by Staphylococcus Aureus

Recipient strains of Streptococcus faecalis excrete small, peptide sex pheromones which induce donors harboring certain conjugative plasmids to synthesize a proteinaceous adhesin that facilitates the formation of mating aggregates. (For a recent review of this phenomenon, see Ref. 2.) Referred to as “aggregation substance,” the adhesin has been revealed as a unique antigen that uniformly coats the donor cell surface (14,25). Self aggregation of donors also occurs; for this reason the pheromones are also referred to as clumping inducing agents (CIAs). It has been proposed that “aggregation substance” binds specifically to a “binding substance” present on the surface of both donor and recipient cells (6,7).

Sex Pheromones and Plasmid Transfer in Streptococcus faecalis

INTRODUCTION Within a highly evolved multicellular organism, communication between distally located cells occurs by means of a variety of different chemical signals. These hormonal substances play important roles in development, maintenance, and fertility and are also involved in coordinating immune and neurological responses to external stimuli. Chemical communication can also occur between individual organisms, and the molecules representing these signals are referred to as pheromones (Karlson and Luscher 1959). Well-known examples of pheromones in higher systems include those serving as sex attractants in insects and certain vertebrates. Communication between unicellular organisms is, by definition, via pheromones, and the involvement of such substances in certain stages of development is well known or has been implicated in a number of microbial species (Ensign 1978; Hirosawa and Wolk 1979; Kaiser et al. 1979; Biro et al. 1980; Aaronson 1981; Stephens et al. 1982). Chemical substances that are excreted and facilitate mating between two organisms are called sex pheromones; these compounds and their behavior have been studied in fungi, algae (Kochert 1978), and ciliates (Nanney 1977). Much attention has been focused on the yeast α - and a -factors and the mating phenomena associated with their pheromonal activities (Manney et al. 1981; Kurjan and Herskowitz 1982; Sprague et al. 1983). Although extracellular compounds that affect competence for transformation are well known in Streptococcus pneumoniae and S. sanguis (Lacks 1977), substantive evidence for bacterial sex pheromones that promote conjugative transfer of genetic material has been reported only in S. faecalis (discussed below). Sex-related chemotactic factors in Escherichia ...