Structure-activity relationship of gelatinase biosynthesis-activating pheromone of Enterococcus faecalis

The expression of pathogenicity-related extracellular proteases, namely, gelatinase and serine protease, in Enterococcus faecalis is positively regulated by a quorum-sensing system mediated by an autoinducing peptide called gelatinase biosynthesis-activating pheromone (GBAP). GBAP is an 11-amino-acid-residue cyclic peptide containing a lactone linkage. To study the structure-activity relationship of GBAP, we synthesized a series of GBAP analogues and evaluated their activities by a gelatinase-inducing assay and newly developed receptor-binding assays in which fluorescence-labeled peptides bound onto the FsrC-overexpressing Lactococcus lactis cell surface were observed by fluorescent microscopy and quantified by using a fluorophotometer. Alanine-scanning analysis of GBAP showed that the entire ring region was involved in the GBAP agonist activity, while side chains of the tail region were not strictly recognized. The alanine substitution of Phe⁷ or Trp¹⁰ almost abolished their receptor-binding abilities and GBAP agonist activities, suggesting that these two aromatic side chains are strongly involved in receptor interaction and activation. Furthermore, the Trp¹⁰ substitution with natural and unnatural aromatic amino acids, except pentafluorophenylalanine, caused no loss of agonist activity. This suggested the importance of a negative electrostatic potential created by an π-electron cloud on the aromatic ring surface. Structural analysis of GBAP with nuclear magnetic resonance spectroscopy revealed that the ring region adopted a hairpin-like fold and was tightly packed into a compact form. The side chain of Trp¹⁰ was partially buried in the core structure, contributing to the stabilization of the compact form, while that of Phe⁷ was extended from the core structure into the solvent and was probably directly involved in receptor binding.     

粪肠球菌Fsr群体感应系统的感染调控

群体感应(quorum sensing,QS)是细菌间的一种通讯方式,是控制微生物信号传递的重要机制,它使细菌能够感知周围环境中其他细菌的存在,并对其密度的变化做出快速反应,在调控基因表达和生物膜形成过程中起着重要作用。作为粪肠球菌(Enterococcus faecalis,E. faecalis)特有的Fsr QS系统,通过调控E. faecalis毒力因子及生物膜等的表达,从而引起机体的各种感染。本综述通过阐述E. faecalis Fsr QS系统在介导毒力因子的表达及生物膜的形成中的作用,为E. faecalis所引起的感染提供理论基础。     

Chemical synthesis and biological activity of the gelatinase biosynthesis-activating pheromone of Enterococcus faecalis and its analogs.

An 11-residue peptide lactone, termed the gelatinase biosynthesis-activating pheromone (GBAP), triggers the production of the pathogenicity-related extracellular proteases, gelatinase and serine protease, in Enterococcus faecalis. In this study, we synthesized GBAP and its analogs and examined their gelatinase biosynthesis-inducing activity. This study on the structure-activity relationship shows that a lactone ring was indispensable for the activity.     

Cell-associated pheromone peptide (cCF10) production and pheromone inhibition in Enterococcus faecalis

In Enterococcus faecalis, the peptide cCF10 acts as a pheromone, inducing transfer of the conjugative plasmid pCF10 from plasmid-containing donor cells to plasmid-free recipient cells. In these studies, it was found that a substantial amount of cCF10 associates with the envelope of the producing cell. Pheromone activity was detected in both wall and membrane fractions, with the highest activity associated with the wall. Experiments examining the effects of protease inhibitor treatments either prior to or following cell fractionation suggested the presence of a cell envelope-associated pro-cCF10 that can be processed to mature cCF10 by a maturase or protease. A pCF10-encoded membrane protein, PrgY, was shown to prevent self-induction of donor cells by reducing the level of pheromone activity in the cell wall fraction.     

PrgU: a suppressor of sex pheromone toxicity in Enterococcus faecalis

Upon sensing of the peptide pheromone cCF10, Enterococcus faecalis cells carrying pCF10 produce three surface adhesins (PrgA, PrgB or Aggregation Substance, PrgC) and the Prg/Pcf type IV secretion system and, in turn, conjugatively transfer the plasmid at high frequencies to recipient cells. Here, we report that cCF10 induction is highly toxic to cells sustaining a deletion of prgU, a small orf located immediately downstream of prgB on pCF10. Upon pheromone exposure, these cells overproduce the Prg adhesins and display impaired envelope integrity, as evidenced by antibiotic susceptibility, misplaced division septa and cell lysis. Compensatory mutations in regulatory loci controlling expression of pCF10‐encoded prg/pcf genes, or constitutive PrgU overproduction, block production of the Prg adhesins and render cells insensitive to pheromone. Cells engineered to overproduce PrgB, even independently of other pCF10‐encoded proteins, have severely compromised cell envelopes and strong growth defects. PrgU has an RNA‐binding fold, and prgB‐prgU gene pairs are widely distributed among E. faecalis isolates and other enterococcal and staphylococcal species. Together, our findings support a model in which PrgU proteins represent a novel class of RNA‐binding regulators that act to mitigate toxicity accompanying overproduction of PrgB‐like adhesins in E. faecalis and other clinically‐important Gram‐positive species.     

Revised model for Enterococcus faecalis fsr quorum-sensing system: the small open reading frame fsrD encodes the gelatinase biosynthesis-activating pheromone propeptide corresponding to staphylococcal agrd

Gelatinase biosynthesis-activating pheromone (GBAP) is an autoinducing peptide involved in Enterococcus faecalis fsr quorum sensing, and its 11-amino-acid sequence has been identified in the C-terminal region of the 242-residue deduced fsrB product (J. Nakayama et al., Mol. Microbiol. 41:145-154, 2001). In this study, however, we demonstrated the existence of fsrD, encoding the GBAP propeptide, which is in frame with fsrB but is translated independently of fsrB. It was also demonstrated that FsrB', an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme to generate GBAP from FsrD. This revised model is consistent with the staphylococcal agr system.     

Identification of new sex pheromone plasmids in Enterococcus faecalis.

Summary We describe the identification of the following new sex pheromone plasmids inEnterococcus faecalis: a haemolysin-bacteriocin plasmid, pIP964; three R plasmids, pIP1017, pIP1438 and pIP1440; and two cryptic conjugative plasmids, pIP1141 and pMV120. The identification was based on the formation of cell aggregates on filter membranes during conjugation, on efficient transfer in broth matings, and on a positive clumping reaction of cells carrying these plasmids. In addition these plasmids hybridized with DNA probes specific for sex pheromone-induced structural genes encoding surface proteins required for conjugative transfer of the plasmids.     

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.     

A lipoprotein signal peptide encoded by the staphylococcal conjugative plasmid pSK41 exhibits an activity resembling that of Enterococcus faecalis pheromone cAD1.

The traH gene of the staphylococcal conjugative plasmid pSK41 has recently been shown to encode a lipoprotein (N. Firth, K. P. Ridgway, M. E. Byrne, P. D. Fink, L. Johnson, I. T. Paulsen, and R. A. Skurray, Gene 136:13-25, 1993). Here we report that traH encodes a product recognized as a pheromone by Enterococcus faecalis cells harboring the conjugative plasmid pAD1. The mature traH product is not essential for this phenomenon, as expression of pheromone-like activity was found to require sequences encoding only the pro-TraH signal peptide.     

Structural basis and specificity of acyl-homoserine lactone signal production in bacterial quorum sensing

Synthesis and detection of acyl-homoserine lactones (AHLs) enables many gram-negative bacteria to engage in quorum sensing, an intercellular signaling mechanism that activates differentiation to virulent and biofilm lifestyles. The AHL synthases catalyze acylation of S-adenosyl-L-methionine by acyl-acyl carrier protein and lactonization of the methionine moiety to give AHLs. The crystal structure of the AHL synthase, EsaI, determined at 1.8 A resolution, reveals a remarkable structural similarity to the N-acetyltransferases and defines a common phosphopantetheine binding fold as the catalytic core. Critical residues responsible for catalysis and acyl chain specificity have been identified from a modeled substrate complex and verified through functional analysis in vivo. A mechanism for the N-acylation of S-adenosyl-L-methionine by 3-oxo-hexanoyl-acyl carrier protein is proposed.     

Synthesis of 'clickable' acylhomoserine lactone quorum sensing probes: unanticipated effects on mammalian cell activation

Alkynyl- and azido-tagged 3-oxo-C₁₂-acylhomoserine lactone probes have been synthesized to examine their potential utility as probes for discovering the mammalian protein target of the Pseudomonas aeruginosa autoinducer, 3-oxo-C₁₂-acylhomoserine lactone. Although such substitutions are commonly believed to be quite conservative, from these studies, we have uncovered a drastic difference in activity between the alkynyl- and azido-modified compounds, and provide an example where such structural modification has proved to be much less than conservative.     

Production of an endo-beta-N-acetylglucosaminidase activity mediates growth of Enterococcus faecalis on a high-mannose-type glycoprotein

Enterococcus faecalis is associated with a high proportion of nosocomial infections; however, little is known of the ability of this organism to proliferate in vivo. The ability of RNase B, a model glycoprotein with a single N-glycosylation site occupied by a family of high-mannose-type glycans (Man(5)- to Man(9)-GlcNAc(2)), to support growth of E. faecalis was investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of RNase B demonstrated a reduction in the molecular mass of this glycoprotein during bacterial growth. Further analysis by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry revealed that this mass shift was due to the degradation of all high-mannose-type glycoforms to a single N-linked N-acetylglucosamine residue. High-pH anion-exchange chromatography analysis during exponential growth demonstrated the presence of RNase B-derived glycans in the culture supernatant, indicating the presence of an endoglycosidase activity. The free glycans were eluted with the same retention times as those generated by the action of Streptomyces plicatus endo-beta-N-acetylglucosaminidase H on RNase B. The cleavage specificity was confirmed by MALDI-TOF analysis of the free glycans, which showed glycan species containing only one N-acetylglucosamine residue. No free glycans were detectable after 5 h of bacterial growth, and we have subsequently demonstrated the presence of mannosidase activity in E. faecalis, which releases free mannose from RNase B-derived glycans. We propose that this deglycosylation of glycoproteins containing high-mannose-type glycans and the subsequent degradation of the released glycans by E. faecalis may play a role in the survival and persistence of this nosocomial pathogen in vivo.     

A cysteine-rich extracellular protein containing a PA14 domain mediates quorum sensing in Dictyostelium discoideum

Much remains to be understood about quorum-sensing factors that allow cells to sense their local density. Dictyostelium discoideum is a simple eukaryote that grows as single-celled amoebae and switches to multicellular development when food becomes limited. As the growing cells reach a high density, they begin expressing discoidin genes. The cells secrete an unknown factor, and at high cell densities the concomitant high levels of the factor induce discoidin expression. We report here the enrichment of discoidin-inducing complex (DIC), an ~400-kDa protein complex that induces discoidin expression during growth and development. Two proteins in the DIC preparation, DicA1 and DicB, were identified by sequencing proteolytic digests. DicA1 and DicB were expressed in Escherichia coli and tested for their ability to induce discoidin during growth and development. Recombinant DicB was unable to induce discoidin expression, while recombinant DicA1 was able to induce discoidin expression. This suggests that DicA1 is an active component of DIC and indicates that posttranslational modification is dispensable for activity. DicA1 mRNA is expressed in vegetative and developing cells. The mature secreted form of DicA1 has a molecular mass of 80 kDa and has a 24-amino-acid cysteine-rich repeat that is similar to repeats in Dictyostelium proteins, such as the extracellular matrix protein ecmB/PstA, the prespore cell-inducing factor PSI, and the cyclic AMP phosphodiesterase inhibitor PDI. Together, the data suggest that DicA1 is a component of a secreted quorum-sensing signal regulating discoidin gene expression during Dictyostelium growth and development.     

PldB, a putative phospholipase D homologue in Dictyostelium discoideum mediates quorum sensing during development

Quorum sensing, also known as cell-density sensing in the unicellular eukaryote Dictyostelium discoideum, is required for efficient entry into the differentiation and development segment of its life cycle. Quorum sensing is accomplished by simultaneously secreting and sensing the glycoprotein Conditioned Medium Factor, or CMF. When the density of starving cells is high, CMF levels are high, which leads to aggregation followed by development. Here, we describe the role of pldB, a gene coding for a putative phospholipase D (PLD) homologue, in quorum sensing. We find that in submerged culture, adding butanol, an inhibitor of PLD-catalyzed phosphatidic acid production, allows cells to bypass the requirement for CMF mediated quorum sensing and aggregate at low cell density. Deletion of pldB mimics the presence of butanol, allowing cells to aggregate at low cell density. pldB- cells also initiate and finish aggregation rapidly. Analysis of early developmental gene expression in pldB- cells reveals that the cyclic AMP receptor cAR1 is expressed at higher levels earlier than in wild-type cells, which could explain the rapid aggregation phenotype. As would be predicted, cells overexpressing pldB are unable to aggregate even at high cell density. Adding CMF to these pldB- overexpressing cells does not rescue aggregation. Both of these phenotypes are cell autonomous, as mixing a small number of pldB- cells with wild-type cells does not cause the wild-type cells to behave like pldB- cells.     

Inhibition of quorum sensing in Serratia marcescens AS-1 by synthetic analogs of N-acylhomoserine lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C(6)-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C(9)-CPA), had a strong inhibitory effect on prodigiosin production. C(9)-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C(9)-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C(6)-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C(9)-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Evidence of quorum sensing in the rumen ecosystem: detection of N-acyl homoserine lactone autoinducers in ruminal contents

Acyl-homoserine lactone (AHL) based quorum-sensing systems are widespread among gram-negative bacteria, particularly in association with plants and animals. As yet, there have been no reports of AHL signaling in the anaerobic rumen environment, an ecosystem of great complexity in which cell-cell signaling is likely to occur. We detected multiple AHL autoinducers in the rumen contents of 6 out of 8 cattle fed a representative selection of diets. The signals were not associated with feed. Surprisingly, no pure cultures produced AHLs in vitro when grown under the laboratory conditions we tested. Our observations suggest that either (a) a factor specific to the rumen ecosystem is required for the rumen isolates we tested to produce AHLs or (b) a strain (or strains) that we were not able to culture but which grows to a high cell density in the rumen produces the AHLs we detected.     

Induction of autolysis in Enterococcus faecalis S-47 by peptide AS-48

In addition to its bactericidal mode of action, the peptide antibiotic AS-48 exhibits a bacteriolytic effect on Enterococcus faecalis S-47 that is associated with autolysin activation. Bacteriolysis induced by the antibiotic can be modulated by addition of EDTA, divalent cations and autolysin activators (trypsin) or inhibitors (cardiolipin), suggesting that topologic regulation of the autolysins is involved in the process. In addition, inhibitors of protein and RNA synthesis interfere markedly with bacteriolysis, as do ionophores and the ATPase inhibitor DCCD, suggesting the participation of an internal messenger in autolysin activation in the presence of AS-48.