Aggregation Substance Research Articles

Regulation of aggregation substance expression by bacterial and host factors.

Enterococcus faecalis is a gram positive member of the intestinal tract normal flora which can cause septicemia, urinary tract infections and endocarditis when introduced into other body sites by intravenous needles, catheters or surgery (reviewed by 3). Treatment of these diseases is complicated by high levels of antibiotic resistance encoded on plasmids which can be transferred at high frequency between Enterococci by pheromome inducible conjugation. We use the expression of Aggregation Substance (AS) encoded by the conjugative plasmid, pCF10, following induction by the pheromone, cCF10, as a model for pheromone inducible conjugation. cCF10 is made by both donor and recipient cells (7) and can have two effects on E. faecalis cells. When cCF10 is produced by a recipient cell and internalized by a donor cell containing pCF10, cCF10 acts as a pheromone and initiates conjugative transfer of the plasmid to the recipient cell (6, reviewed by 1, 8). If the cCF10 is produced and detected by the same donor cell it has autocrine function which allows for replication of the conjugative plasmid without the induction of transfer (Leonard et al., submitted for publication).

ThetraAGene of theEnterococcus faecalisConjugative Plasmid pPD1 Encodes a Negative Regulator for the Pheromone Response

TheEnterococcus faecalisconjugative plasmid pPD1 (59 kb) produces an aggregation substance in response to pheromone. ThetraAgene (962 bp) is a member of a gene cluster involved in the regulation of the pPD1 pheromone response. A chimericE. faecalissuicide plasmid containing a 762-bp DNA fragment from the middle portion of thetraAgene was constructed. The pPD1traAwas disrupted by integration of this chimeric plasmid via homologous recombination. TheE. faecalisstrain containing pPD1 with the disruptedtraAexhibited dry colony morphology, constitutive clumping, and an ability to transfer at high frequencies in a short (10-min) mating period, indicating that thetraAproduct of pPD1 is a negative regulator for the pheromone response.

Frequency of Aggregation Substance and Cytolysin Genes among Enterococcal Endocarditis Isolates

Enterococcus faecalisblood isolates were probed for the serine protease activator of cytolysin (cylA) and aggregation substance (asa1), traits that generally reside on pheromone-responsive plasmids, to determine how commonly these genotypes were associated with disease. In dot blot assays, no significant difference was found in the frequency ofasa1for blood isolates [55 of 103 (54%)] and isolates recovered from stool [9 of 14 (60%);P> 0.1, χ2test]. In contrast,cylAoccurred more frequently among bacteremia isolates [34 of 68 (50%)] than endocarditis [4 of 35 (11%)] or stool isolates (0 of 14;P< 0.001; χ2test). However, when the clonality of isolates was accounted for, the frequency ofasa1andcylAamong unrelated strains was not significantly different among the three sets of strains (P> 0.2, χ2test). The lack of enrichment forasa1orcylAamong clonally unrelatedE. faecalisbloodstream isolates fails to support a role for plasmid-encoded aggregation substance or cytolysin in the transition from bacteremia to endocarditis. Clonally related, cytolytic strains, however, demonstrated an increased propensity to cause bloodstream infection.

Incidence of hemolysin, gelatinase, and aggregation substance among enterococci isolated from patients with endocarditis and other infections and from feces of hospitalized and community-based persons.

The presence of hemolysin, gelatinase, and aggregation substance (by use of a probe known to hybridize to most pheromone-responsive plasmids) was determined in 192 isolates of Enterococcus faecalis from patients with endocarditis or other infections and fecal isolates from hospitalized patients or healthy volunteers, and in 86 non-E. faecalis isolates. Hemolysin was more common in nonendocarditis clinical isolates and in hospital fecal isolates (37% and 31%, respectively) than among endocarditis and community fecal isolates (16% and 20%, respectively). Gelatinase and aggregation substance, respectively, were found in 54% and 52% of isolates from endocarditis, in 58% and 72% of isolates from other infections, in 62% and 56% of hospital fecal isolates, and in 27% and 30% of fecal isolates from healthy volunteers. All 86 non-E. faecalis enterococcal isolates were negative for these traits. The absence of hemolysin, gelatinase, or the aggregation substance gene in > 45% of endocarditis E. faecalis isolates suggests that while these traits may play a role in virulence, other properties are also important.

Transcriptional analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions.

The prgB gene encodes aggregation substance (Asc10) which is essential for transfer of the pheromone-inducible conjugative plasmid pCF10 in Enterococcus faecalis. The prgQ and prgS regions, located 4 kb upstream of prgB, are required for the expression of prgB. Complementation studies indicated that the prgQ region functions in cis and in an orientation-dependent manner relative to the prgB gene (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). Analysis of transcriptional fusions in this study, using a promoterless lacZ gene in several locations between prgQ and prgB, confirmed that the prgQ region does not carry a promoter for the expression of prgB and that prgB does not comprise an operon with prgA (which encodes the surface exclusion protein Sec10), the gene immediately upstream from prgB. Northern (RNA) blot analysis demonstrated that two distinct transcripts (Qs RNA and QL RNA), much larger than the prgQ gene, were expressed in the prgQ region. QS RNA was produced constitutively, whereas QL RNA was produced inducibly by pheromone. The lack of any other open reading frame in QL RNA and significant sequence complementarity between the 3' end of QL RNA and the promoter region of prgB suggested that the functional products of the prgQ region might be RNA molecules rather than proteins. A mutation in prgS completely abolished the production of QL RNA. A model for transcriptional activation of prgB is presented.

A plasmid-encoded surface protein on Enterococcus faecalis augments its internalization by cultured intestinal epithelial cells.

Aggregation substance, a plasmid-encoded Enterococcus faecalis surface protein, plays a role in mediating the formation of mating aggregates, resulting in plasmid transfer. The role of aggregation substance in the internalization of E. faecalis by cultured intestinal epithelial cells, namely HT-29 cells, was analyzed. It was associated with a significant increase in endocytosis of E. faecalis by HT-29 cells: Numbers of internalized enterococci were fewer than of an invasive strain of Listeria monocytogenes, similar to Salmonella typhimurium and another L. monocytogenes strain, and greater than relatively noninvasive strains of E. faecalis, Proteus mirabilis, and Escherichia coli. Electron microscopy confirmed aggregation substance on the surface of strains interacting with the enterocyte microvillous surface, and intracellular enterococci were localized within membrane-bound vacuoles in the enterocyte cytoplasm. Thus, aggregation substance may facilitate E. faecalis internalization by host epithelial cells.

Virulence of enterococci

Enterococci are commensal organisms well suited to survival in intestinal and vaginal tracts and the oral cavity. However, as for most bacteria described as causing human disease, enterococci also possess properties that can be ascribed roles in pathogenesis. The natural ability of enterococci to readily acquire, accumulate, and share extrachromosomal elements encoding virulence traits or antibiotic resistance genes lends advantages to their survival under unusual environmental stresses and in part explains their increasing importance as nosocomial pathogens. This review discusses the current understanding of enterococcal virulence relating to (i) adherence to host tissues, (ii) invasion and abscess formation, (iii) factors potentially relevant to modulation of host inflammatory responses, and (iv) potentially toxic secreted products. Aggregation substance, surface carbohydrates, or fibronectin-binding moieties may facilitate adherence to host tissues. Enterococcus faecalis appears to have the capacity to translocate across intact intestinal mucosa in models of antibiotic-induced superinfection. Extracellular toxins such as cytolysin can induce tissue damage as shown in an endophthalmitis model, increase mortality in combination with aggregation substance in an endocarditis model, and cause systemic toxicity in a murine peritonitis model. Finally, lipoteichoic acid, superoxide production, or pheromones and corresponding peptide inhibitors each may modulate local inflammatory reactions.

Bacteriocin plasmid pMB1 of Enterococcus faecalis: identification of the cell aggregation substance after induction by sex pheromone.

Strains of Enterococcus faecalis carrying the bacteriocinogenic plasmids pMB1 or pMB1.1 exhibit a clumping response to culture supernatants of different enterococcal strains. Antibodies raised against cells induced by a homologous pheromone recognize two surface proteins of 152 and 72.5 kDa (the second one is possibly the degradation product of the first), respectively. These antigens are very similar to those found in induced cells of E. faecalis OGIRF(pAM211) as shown by the cross-reaction of the immune sera obtained in this work. We propose that the 152-kDa protein corresponds to the aggregation substance coded by plasmids pMB1 and pMB1.1. Moreover, antibodies raised against induced cells are able to block the pheromone-induced clumping response. When the cells induced to form aggregates by pheromones were examined under a scanning electron microscope they showed a surface layer of hairlike structures.

The sex pheromone system of Enterococcus faecalis. More than just a plasmid-collection mechanism?

The sex pheromone system of Enterococcus faecalis was discovered by observing a clumping reaction of E. faecalis strains during conjugative transfer of plasmids. It was found that only a special type of E. faecalis plasmids, the so-called sex pheromone plasmids, are transferred via this mechanism. Various experiments, especially by the group of D. B. Clewell, led to the formulation of a model describing how the sex pheromone system works. Small linear peptides, the so-called sex pheromones, are excreted by strains not possessing the corresponding sex pheromone plasmid. Donor strains harboring the plasmid do not produce the corresponding sex pheromone; they react to the presence of the peptide by production of a plasmid-encoded adhesin, the so-called aggregation substance. This adhesin allows contact between the non-motile mating partners; after conjugative transfer of the plasmid, the former recipient possesses and replicates the new plasmid. Thereby the population of E. faecalis strains is shifted to a high percentage of donor strains. This is especially true because a donor strain will still excrete sex pheromones corresponding to plasmids it does not harbor; therefore, such a strain can also function as recipient for other sex pheromone plasmids it does not possess. Various aspects of this unique plasmid collection mechanism have been studied during the last few years. The data indicate that, with the exception of pAM373, all sex pheromone plasmids possess one DNA region which is highly similar to and codes for the adhesin. It is also becoming more and more clear that regulatory functions/proteins are not conserved between different sex pheromone plasmids. Induction of adhesin synthesis needs the action of a regulatory cascade composed of unique features; at the moment we are just beginning to understand this cascade. By sequencing the first structural gene for one of those adhesins, we realized that the aggregation substance might act also as an adhesin for eucaryotic cells, probably by interaction with integrins. At least in the case of the in vitro cultured pig kidney tubulus cell line LLC-PK1 this idea could be verified. An interesting aspect of the sex pheromone system of E. faecalis is its evolution. I will discuss the idea that two different components, both of which well might contribute to virulence of the opportunistic pathogenic bacterium, were combined in the species E. faecalis to result in this unique plasmid collection system.

The pathogenicity of enterococci.

In order to produce infection, enterococci must be able to colonize host tissues, resist the host's non-specific and immune defence mechanisms and produce pathological changes. With regard to colonization of host tissues, adherence assays have shown that enterococci can attach to intestinal and urinary tract epithelial cells and heart cells by means of adhesins expressed on the bacterial surface. The expression of these adhesins by enterococci has further been shown to be affected by bacterial growth conditions. In addition, the adherence of Enterococcus faecalis to renal tubular cells in vitro is enhanced if the organisms produce aggregation substance, a proteinaceous surface material that aggregates donor and recipient bacteria to facilitate plasmid transfer. Bacterial growth conditions also affect the interaction of enterococci with polymorphonuclear leucocytes (PMNLs), with serum-grown organisms showing less association with PMNLs than organisms grown in broth. Efficient killing of enterococci by PMNLs in vitro requires the presence of serum complement proteins and is enhanced by anti-enterococcal antibodies. Enterococci produce a number of factors that may be associated with pathological changes in the host. Both sex pheromones and plasmid-encoded pheromone inhibitors produced by E. faecalis are chemotactic for PMNLs in vitro, and may mediate, at least in part, the inflammatory response often associated with enterococcal infection. E. faecalis may also produce a plasmid-encoded haemolysin, which is associated with increased severity of infection. In addition, enterococci are capable of inducing platelet aggregation and tissue factor-dependent fibrin production, which may be relevant to the pathogenesis of enterococcal endocarditis.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocarditis

A rabbit endocarditis model was utilized to evaluate the virulence conferred by the conjugative plasmid pAD1 with the following strains: Enterococcus faecalis plasmid-free FA2-2 and FA2-2 containing plasmids pAD1 (hemolysin and aggregation substance positive), pAM9058 (insertional inactivation of hemolysin), and pAM944 or pAM947 (insertional inactivation of aggregation substance). All isolates were similar in ability to produce endocarditis. Mean vegetation weight was greater in animals inoculated with strains that produced aggregation substance (P < 0.01). Mortality was significantly increased in animals given FA2-2 containing pAD1 compared with those given all other strains (P < 0.01). These results suggest that the combination of hemolysin and aggregation substance is associated with increased mortality and that vegetation weight is associated with production of aggregation substance in experimental E. faecalis endocarditis.

A microbial model for visualization of surface proteins using cryoimmobilization and high-resolution cryo-SEM

Numerous studies on high resolution visualization of surface membrane proteins have utilized colloidal gold markers in conjunction with SEM, TEM, and by freeze-fracture cytochemistry using conventional or fracture flip methods, but in each case only information on the distribution of the marker complex, and not the actual structure of individual surface protein(s) was obtained.To investigate the detection of individual surface proteins, we have used a microbial model to examine surface proteins involved in conjugation or the transfer of DNA inEnterococcus faecalis. Two surface proteins encoded by the pCF10 plasmid, aggregation substance (Asc10; globular shape) and surface exclusion protein (Sec10; helical shape), mediate the aggregation of donor and recipient cells and prevent aggregation of like donor cells, respectively. Isogenic strains of E. faecalis that constitutively express neither Asc10 or Sec10 (pWM401) or both (pINY1801) were processed by 1) conventional chemical fixation, dehydration, critical point drying with CO2, ion beam sputtering with a discontinuous platinum film (&lt;1 nm), and examination in a Hitachi S-900 FESEM operated at low voltage (&lt;3 keV), or 2) cryoimmobilization of cells by high pressure freezing (Balzers HPM 010), mounting in a Gatan cryoholder followed by etching for 60 minutes at −100°C, cryocoating by planar magnetron sputtering (Balzers MED 010), and examination at −100°C in the S-900 FESEM.

Sex pheromone plasmid pAD1-encoded aggregation substance of Enterococcus faecalis is positively regulated in trans by traE1.

Sex-pheromone-plasmid-bearing strains of Enterococcus faecalis react with sex pheromone to induce the expression of an adhesin, the so-called aggregation substance, on their cell surface. Here we show that, by complementation studies, for sex-pheromone plasmid pAD1, expression of the structural gene asa1, coding for an aggregation substance, is mediated by a diffusible factor encoded on pAD1. We were able to demonstrate that a small open reading frame, traE1, is sufficient for transcription of the operon containing asa1. A model for expression of asa1 under the influence of the positive regulator is presented, which is supported by our observation that regulation involves an all-or-nothing induction phenomenon, leading to cells either fully expressing asa1 or not at all.

Biochemical, immunological and ultrastructural characterization of aggregation substances encoded by Enterococcus faecalis sex-pheromone plasmids.

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 sex-pheromone 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 pPD1 the 78-kDa fragment reacted only weakly. The aggregation substance encoded by sex-pheromone plasmid pAD1 (Asa1) was characterized in detail. The conditions used for SDS/PAGE had a drastic influence on the migration behavior of mature aggregation substance and differently migrating, interconvertible forms were identified. Preliminary data indicate that Asa1 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.

Performance of a Gyratory Sifter. 1st Report. Experimental Study.

Experimental study of gyratory sifters, which are used to remove aggregate or alien substances, is presented. The effects of operating conditions, such as the bed depth of powder, the sieve revolving speed, and the size and shape of the sieve, on sifting capacity or sifting rate are investigated. The motion of the powder bed on a screening surface is also observed. Experimental results show that the sifting rate is very sensitive to the bed depth. There is an optimum bed depth at which the sifting rate is maximized. The sifting rate of a circular sieve is higher that of a rectangular sieve. The sifting capacity increases with the revolving speed.

Transcriptional control of sex‐pheromone‐inducible genes on plasmid pAD1 of Enterococcus faecalis and sequence analysis of a third structural gene for (pPD1 ‐encoded) aggregation substance

The expression of several neighbouring genes on plasmid pAD1 that are necessary for conjugation depend on induction with sex pheromone cAD1. Analyses of transcripts by Northern blot hybridization demonstrated that the genes sea1 (encoding surface exclusion protein) and asa1 (encoding aggregation substance) are transcribed independently. Both genes are organized in different operons together with neighbouring open reading frames of unknown function. Several transcripts could be identified for sea1 and asa1. Their transcriptional start sites were determined by primer extension experiments, confirming the results of the Northern blot experiments. We also could identify sea1- and iad- (encoding an inhibitory peptide counteracting sex pheromone cAD1) specific transcripts which are expressed constitutively, but to a lower extent relative to induced conditions. In addition, we localized the asp1 gene coding for aggregation substance of sex pheromone plasmid pPD1 and determined its DNA sequence, which was found to be highly homologous to asa1 (aggregation substance gene of pAD1) and prgB (aggregation substance gene of pCF10). The structural genes were found to be organized more or less identically on the three sex-pheromone plasmids pAD1, pCF10, and pPD1, and to be highly conserved. Regions supposed to be of crucial importance for regulatory functions, however, were found to differ. We also could identify some conserved DNA motifs which might be potential target sites for transcriptional regulators. In combination these data allowed us to formulate a model for the regulation of sex-pheromone-inducible genes of plasmid pAD1. Its main statement is that only in the presence of cAD1 can the gene traE1 be transcribed. The positive regulatory factor TraE1 then can trigger expression of the structural genes sea1 and asa1.

Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis.

During conjugative transfer of sex pheromone plasmids of Enterococcus faecalis a so-called surface exclusion protein reduces the frequency with which these plasmids are transferred to cells already possessing the same plasmid. We report here the DNA sequence of a 3.8 kb fragment of the sex pheromone plasmid pAD1 containing the structural gene sea1 for surface exclusion protein and a small open reading frame (ORF) upstream of sea1. Surface exclusion protein Sea1 was found to be highly homologous to the surface exclusion protein Sec10 encoded by the sex pheromone plasmid pCF10. Hybridization studies with DNA probes derived from the structural gene sea1 demonstrated that, with the exception of pAM373, all known sex pheromone plasmids carry a homologous gene. These studies also indicated that the genetic organization is similar in these plasmids, with the structural gene for surface exclusion protein being located 5' to that for aggregation substance.

Aggregation substance of Enterococcus faecalis mediates adhesion to cultured renal tubular cells

The sex pheromone system of Enterococcus faecalis is a unique, highly efficient plasmid collection mechanism for this species. A crucial role in this system is played by an adhesin called aggregation substance which enables the cell-cell contact between donor and recipient strains. The existence of the amino acid motif Arg-Gly-Asp-Ser in the adhesin prompted us to look for a possible binding of E. faecalis cells expressing aggregation substance to eucaryotic cells. We were able to show that the adhesin mediated binding to cultured renal tubular cells (porcine cell line LLC-PK1) via light microscopic, electron microscopic, and enzyme-linked immunosorbent assay-based studies. Synthesis of the adhesin was induced by some component(s) of serum. These data are interpreted to mean that aggregation substance is an adhesin mediating not only cell-cell contact between different E. faecalis strains but also binding of E. faecalis to eucaryotic cells, and therefore it might contribute to virulence.

C-terminal identification of AD74, a proteolytic product of Enterococcus faecalis aggregation substance: application of liquid chromatography/mass spectrometry.

Sexual aggregation involved in conjugative transfer of Enterococcus faecalis plasmid pAD1 is enhanced by the sex pheromone cAD1, which is excreted from recipient cells. A membrane-anchored 137 kDa protein is a pAD1-encoded aggregation substance designated asal, which is responsible for cell-cell contact and leads to the aggregation of cells. An AD74 protein is a proteolytic product corresponding to the N-terminal half of asal. The C-terminal of AD74 was identified as lysine at position 510 (K-510) by liquid chromatography/mass spectrometry (LC/MS): it indicates that asal is cleaved specifically between K-510 and G-511.

Role of the pheromone-inducible surface protein Asc10 in mating aggregate formation and conjugal transfer of the Enterococcus faecalis plasmid pCF10

The high transfer frequency of pheromone-inducible conjugative plasmids of Enterococcus faecalis in liquid culture is due in part to the formation of mating aggregates. These aggregates result from the interaction of two surface components, aggregation substance (AS), which is plasmid encoded, and the chromosomally encoded binding substance (BS). In the accompanying paper (S.-M. Kao, S. B. Olmsted, A. S. Viksnins, J.C. Gallo, G. M. Dunny, J. Bacteriol, 173:7650-7664, 1991), the sequence of the prgB gene encoding the AS molecule (Asc10) produced by pheromone-induced cells carrying plasmid pCF10 is presented. Here we report the results of genetic and immunological experiments which define the role of Asc10 in aggregation and plasmid transfer. These data indicate expression of AS on the surface of an E. faecalis cell and its binding to BS expressed on a second cell are required for the formation of a mating pair and the efficient transfer of pCF10 in liquid matings. However, the orientation of the receptors was not critical for transfer; ie., AS expressed on recipient cells could facilitate plasmid transfer via binding to BS on the donor. Our results suggest that additional (as yet unidentified) products are involved in forming the channel that ultimately serves to transfer the DNA, with AS-BS binding serving primarily to generate the initial attachment between cells. The putative prgC gene product, identified by DNA sequencing (data presented in the accompanying paper), could be involved in transfer events occurring subsequent to aggregation.