Effects of biofilm growth on plasmid copy number and expression of antibiotic resistance genes in Enterococcus faecalis.

Biofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity in Enterococcus faecalis cells carrying plasmid pCF10. In this study, we examined whether biofilm growth affected the copy number and expression of antibiotic resistance determinants for several plasmids with diverse replication systems. Four different E. faecalis plasmids, unrelated to pCF10, demonstrated increased copy number in biofilm cells. In biofilm cells, we also observed increased transcription of antibiotic resistance genes present on these plasmids. The increase in plasmid copy number correlated with increased plating efficiency on high concentrations of antibiotics. Single-cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells. Regrowth of harvested biofilm cells in liquid culture resulted in a rapid reduction of plasmid copy number to that observed in the planktonic state. These results suggest a possible mechanism by which biofilm growth could reduce susceptibility to antibiotics in clinical settings.

An amino-terminal domain of Enterococcus faecalis aggregation substance is required for aggregation, bacterial internalization by epithelial cells and binding to lipoteichoic acid.

Aggregation substance (AS), a plasmid-encoded surface protein of Enterococcus faecalis, plays important roles in virulence and antibiotic resistance transfer. Previous studies have suggested that AS-mediated aggregation of enterococcal cells could involve the binding of this protein to cell wall lipoteichoic acid (LTA). Here, a method to purify an undegraded form of Asc10, the AS of the plasmid pCF10, is described. Using this purified protein, direct binding of Asc10 to purified E. faecalis LTA was demonstrated. Equivalent binding of Asc10 to LTA purified from INY3000, an E. faecalis strain that is incapable of aggregation, was also observed. Surprisingly, mutations in a previously identified aggregation domain from amino acids 473 to 683 that abolished aggregation had no effect on LTA binding. In frame deletion analysis of Asc10 was used to identify a second aggregation domain located in the N-terminus of the protein from amino acids 156 to 358. A purified Asc10 mutant protein lacking this domain showed reduced LTA binding, while a purified N-terminal fragment from amino acids 44-331 had high LTA binding. Like the previously described aggregation domain, the newly identified Asc10((156-358)) aggregation domain was also required for efficient internalization of E. faecalis into HT-29 enterocytes. Thus, Asc10 possess two distinct domains required for aggregation and eukaryotic cell internalization: an N-terminal domain that promotes binding to LTA and a second domain located near the middle of the protein.

Peptide pheromone-induced transfer of plasmid pCF10 in Enterococcus faecalis: probing the genetic and molecular basis for specificity of the pheromone response.

The tetracycline resistance plasmid pCF10 represents a class of unique mobile genetic elements of the bacterial genus Enterococcus, whose conjugative transfer functions are inducible by peptide sex pheromones excreted by potential recipient cells. These plasmids play a significant role in the dissemination of virulence and antibiotic resistance genes among the enterococci, which have become major nosocomial pathogens. Pheromone response by plasmid-carrying donor cells involves specific import of the peptide signal molecule, and subsequent interaction of the signal with one or more intracellular regulatory gene products. The pheromones are chromosomally encoded hydrophobic octa- or hepta-peptides, and different families of homologous plasmids encode the ability to respond to each pheromone. Among the four pheromone-responsive plasmids that have been characterized in some detail, there is considerable conservation in the genes encoding pheromone sensing and regulatory functions, and the peptides themselves show considerable similarity. In spite of this, there is extremely high specificity of response to each peptide, with virtually no "cross-induction" of transfer of non-cognate pheromone plasmids by the pheromones. This communication reviews the evidence for this specificity and discusses current molecular and genetic approaches to defining the basis for specificity.

Analysis of functional domains of the Enterococcus faecalis pheromone-induced surface protein aggregation substance.

Pheromone-inducible aggregation substance (AS) proteins of Enterococcus faecalis are essential for high-efficiency conjugation of the sex pheromone plasmids and also serve as virulence factors during host infection. A number of different functions have been attributed to AS in addition to bacterial cell aggregation, including adhesion to host cells, adhesion to fibrin, increased cell surface hydrophobicity, resistance to killing by polymorphonuclear leukocytes and macrophages, and increased vegetation size in an experimental endocarditis model. Relatively little information is available regarding the structure-activity relationship of AS. To identify functional domains, a library of 23 nonpolar 31-amino-acid insertions was constructed in Asc10, the AS encoded by the plasmid pCF10, using the transposons TnlacZ/in and TnphoA/in. Analysis of these insertions revealed a domain necessary for donor-recipient aggregation that extends further into the amino terminus of the protein than previously reported. In addition, insertions in the C terminus of the protein also reduced aggregation. As expected, the ability to aggregate correlates with efficient plasmid transfer. The results also indicated that an increase in cell surface hydrophobicity resulting from AS expression is not sufficient to mediate bacterial aggregation.

Antibodies to a surface-exposed, N-terminal domain of aggregation substance are not protective in the rabbit model of Enterococcus faecalis infective endocarditis.

The aggregation substance (AS) surface protein from Enterococcus faecalis has been implicated as an important virulence factor for the development of infective endocarditis. To evaluate the role of antibodies specific for Asc10 (the AS protein from the conjugative plasmid pCF10) in protective immunity to infective endocarditis, an N-terminal region of Asc10 lacking the signal peptide and predicted to be surface exposed (amino acids 44 to 331; AS(44-331)) was cloned with a C-terminal histidine tag translational fusion and expressed from Escherichia coli. N-terminal amino acid sequencing of the purified protein revealed the correct sequence, and rabbit polyclonal antisera raised against AS(44-331) reacted specifically to Asc10 expressed from E. faecalis OG1SSp, but not to other proteins as judged by Western blot analysis. Using these antisera, flow cytometry analysis demonstrated that antibodies to AS(44-331) bound to a surface-exposed region of Asc10. Furthermore, antibodies specific for AS(44-331) were opsonic for E. faecalis expressing Asc10 in vitro but not for cells that did not express Asc10. New Zealand White rabbits immunized with AS(44-331) were challenged intravenously with E. faecalis cells constitutively expressing Asc10 in the rabbit model of experimental endocarditis. Highly immune animals did not show significant differences in clearance of organisms from the blood or spleen or in formation of vegetations on the aortic valve, in comparison with nonimmune animals. Although in vivo expression of Asc10 was demonstrated by immunohistochemistry, these experiments provide evidence that immunity to Asc10 does not play a role in protection from experimental infective endocarditis due to E. faecalis and may have important implications for the development of immunological approaches to combat enterococcal endocarditis.

Dominant-negative mutants of prgX: evidence for a role for PrgX dimerization in negative regulation of pheromone-inducible conjugation.

PrgX negatively regulates prgQ transcriptional readthrough in the pheromone-inducible enterococcal conjugative plasmid pCF10. We isolated and characterized 13 dominant-negative prgX mutants, all of which mapped in either the N- or the C-terminus of PrgX. In all mutants, the in vivo level of Qa RNA, an antisense RNA to prgQ RNA, was greatly reduced. When oligomerization of PrgX was tested with a phage lambda cI repressor fusion system, the oligomerization domain was found to be between amino acid residues 78 and 280. When histidine-tagged PrgX (His-PrgX) was purified by nickel column chromatography from a strain also expressing PrgX, PrgX was co-purified with His-PrgX. Although PrgX was expressed at a much higher level than His-PrgX, an approximately equal amount of PrgX was co-purified. Pheromone induction greatly decreased the co-purification of PrgX. Based on these data, we propose that both the N- and the C-terminal domains of PrgX are required for PrgX positive autoregulation and for the repression of prgQ transcription readthrough. In vivo, PrgX exists as a dimer, and dimerization is mediated by the central region of PrgX.

Inducible expression of Enterococcus faecalis aggregation substance surface protein facilitates bacterial internalization by cultured enterocytes.

Aggregation substance (AS) is an Enterococcus faecalis surface protein that may contribute to virulence. Using a recently described system for controlled expression of AS in E. faecalis and the heterologous host Lactococcus lactis, experiments were designed to assess the effect of AS on bacterial internalization by HT-29 and Caco-2 enterocytes. AS expression was associated with increased internalization of E. faecalis by HT-29 enterocytes and of L. lactis by HT-29 and Caco-2 enterocytes. Compared to enterocytes cultivated under standard conditions, either cultivation in hypoxia or 1-h pretreatment of enterocytes with calcium-free medium resulted in increased internalization of both E. faecalis and L. lactis (with and without AS expression). Also, AS expression augmented these increases when E. faecalis was incubated with pretreated HT-29 enterocytes and when L. lactis was incubated with pretreated Caco-2 and HT-29 enterocytes. These data indicated that AS might facilitate E. faecalis internalization by cultured enterocytes.

Improved vectors for nisin-controlled expression in gram-positive bacteria.

A set of shuttle vectors, able to replicate in Escherichia coli and in gram-positive bacteria, containing a nisin-inducible promoter (PnisA) and genes encoding NisR and NisK, the two-component signaling mechanism for activating transcription from PnisA in the presence of nisin, was constructed. To test these vectors, Enterococcus faecalis pCF10 plasmid genes prgX, prgY, and prgZ, which respectively encode cytosolic, integral membrane, and cell surface proteins, were cloned downstream of PnisA. Increased protein expression, in the presence of nisin, was demonstrated by Western blot analysis.

Regulation of intron function: efficient splicing in vivo of a bacterial group II intron requires a functional promoter within the intron.

Conjugative transfer of the Lactococcus lactis plasmid pRS01 requires splicing of a group II intron, Ll.ltrB, for accurate translation of the mRNA for the exon gene ltrB. The protein product of ltrB is a conjugative relaxase, essential for pRS01 transfer. Using a molecular technique for the identification of transcription initiation sites in bacteria, a functional promoter within Ll.ltrB was identified upstream from the gene for the intron-encoded protein (IEP) LtrA. LtrA is required for efficient splicing of Ll.ltrB in vivo. Mutation of the ltrA promoter dramatically reduced the steady-state level of ltrA mRNA, LtrA, intron splicing and conjugation in L. lactis. These effects could be relieved by expression in trans of the ltrA gene cloned under the control of an inducible promoter. These results suggest that the ltrA mRNAs are translated inefficiently. We hypothesize that this bacterial intron, in contrast to previously studied group II introns in eukaryotes, requires a promoter within the intron to regulate ltrA expression and to produce an adequate level of the protein in the cell for efficient splicing.

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.

Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30 S subunits.

Ribosomal protein S7 nucleates folding of the 16 S rRNA 3' major domain, which ultimately forms the head of the 30 S ribosomal subunit. Recent crystal structures indicate that S7 lies on the interface side of the 30 S subunit, near the tRNA binding sites of the ribosome. To map the functional surface of S7, we have tagged the protein with a Protein Kinase A recognition site and engineered alanine substitutions that target each exposed, conserved residue. We have also deleted conserved features of S7, using its structure to guide our design. By radiolabeling the tag sequence using Protein Kinase A, we are able to track the partitioning of each mutant protein into 30 S, 70 S, and polyribosome fractions in vivo. Overexpression of S7 confers a growth defect, and we observe a striking correlation between this phenotype and proficiency in 30 S subunit assembly among our collection of mutants. We find that the side chain of K35 is required for efficient assembly of S7 into 30 S subunits in vivo, whereas those of at least 17 other conserved exposed residues are not required. In addition, an S7 derivative lacking the N-terminal 17 residues causes ribosomes to accumulate on mRNA to abnormally high levels, indicating that our approach can yield interesting mutant ribosomes.

Heterologous inducible expression of Enterococcus faecalis pCF10 aggregation substance asc10 in Lactococcus lactis and Streptococcus gordonii contributes to cell hydrophobicity and adhesion to fibrin.

Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis and Streptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of E. faecalis and L. lactis but not on that of S. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactis revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.

Analysis of expression of prgX, a key negative regulator of the transfer of the Enterococcus faecalis pheromone-inducible plasmid pCF10.

Conjugative transfer of pCF10, a plasmid found in Enterococcus faecalis, is induced by the peptide pheromone cCF10 and the donor-recipient aggregation is mediated by PrgB. Expression of prgB in pCF10 is negatively regulated by PrgX. The prgX gene is adjacent to prgQ which provides the promoter for prgB expression. The prgX and prgQ genes are transcribed in opposite directions. A deletion spanning nucleotides+259 to +398 from the prgQ transcription initiation site abolished the prgX gene products at both RNA and protein levels. An RNA, named Qa, was found to be transcribed in the antisense direction in the prgQ region. The transcription start site and the promoter were found to be almost identical with those of the traD determinant in pAD1, another pheromone-responsive plasmid. The first inverted repeat sequence in prgQ, IRS1, was required for the full activity of the Qa promoter. Although the size of the major Qa RNA detected by Northern blot analysis was too short (ca 120 nt) to be an mRNA for PrgX protein, the transcription from the Qa promoter was shown to proceed through to prgX. Transcriptional fusion analyses showed that the transcription of prgX requires one or more trans elements. Moreover, deletion of prgX greatly reduced the level of the Qa RNA and abolished transcription of prgX. Although transcription initiation from the Qa promoter was not increased by PrgX, transcriptional readthrough to prgX was increased by the protein. Based on these data, we conclude that transcription of prgX is initiated from the Qa promoter in prgQ, and PrgX autoregulates its transcription either by mediating transcriptional readthrough or increasing mRNA stability. The PrgX protein, prgX RNA, and the Qa RNA were downregulated by cCF10 induction; however, prgX gene products were still detected for at least 40 minutes after induction.

Enterococcus faecalis aggregation substance promotes opsonin-independent binding to human neutrophils via a complement receptor type 3-mediated mechanism.

Enterococcus faecalis aggregation substance (AS) mediates efficient adhesion between bacteria, thereby facilitating plasmid exchange as an integral part of a bacterial sex pheromone system. We examined the interaction of AS-bearing E. faecalis with human neutrophils (PMNs), an important component of the host defense system. AS promoted a markedly increased opsonin-independent bacterial binding to PMNs. Adhesion was dependent on the expression of the enterococcal Asc10 protein, which contains two Arg-Gly-Asp (RGD) sequences, and addition of exogenous RGD-containing peptides inhibited AS-mediated binding by 66%. AS-mediated adhesion was inhibited by 85% by anti-human complement receptor type 3 (CR3) monoclonal antibodies or by use of PMNs from a patient with leukocyte adhesion deficiency. However, AS-bearing E. faecalis cells were unable to bind to CHO-Mac-1 cells, expressing functionally active CR3, suggesting the potential need for additional PMN surface receptors for bacterial adhesion. Monoclonal antibodies against integrin-associated protein (CD47) and L-selectin, both of which may interact with CR3 and bind to ligands on E. faecalis, also inhibited AS-dependent binding. The non-opsonic binding of E. faecalis to PMNs may play an important role in this organism's pathogenesis.

Enterococcus faecalis bearing aggregation substance is resistant to killing by human neutrophils despite phagocytosis and neutrophil activation.

Enterococcus faecalis aggregation substance (AS) mediates efficient bacterium-bacterium contact to facilitate plasmid exchange as part of a bacterial sex pheromone system. We have previously determined that AS promotes direct, opsonin-independent binding of E. faecalis to human neutrophils (PMNs) via complement receptor type 3 and other receptors on the PMN surface. We have now examined the functional consequences of this bacterium-host cell interaction. AS-bearing E. faecalis was phagocytosed and internalized by PMNs, as determined by deconvolution fluorescence microscopy. However, these bacteria were not killed by PMNs, and internalized bacteria excluded propidium iodide, indicating intact bacterial membranes. Resistance to killing occurred despite activation of PMNs, as indicated by an increase in both functional and total surface Mac-1 expression, shedding of L-selectin, and an increase in PMN extracellular superoxide and phagosomal oxidant production. Deconvolution fluorescence microscopy also revealed that phagosomes containing AS-bearing bacteria were markedly larger than phagosomes containing opsonized E. faecalis, suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. PMN phagosomal pH was significantly higher after ingestion of nonopsonized AS-bearing E. faecalis than after that of opsonized bacteria. The novel ability of AS to promote intracellular survival of E. faecalis inside PMNs suggests that AS may be a virulence factor used by strains of E. faecalis.

Group II introns and expression of conjugative transfer functions in lactic acid bacteria.

The homologous lactococcal conjugative elements pRS01 and the sex factor of Lactococcus lactis strain 712 both contain a Group II intron within a gene believed to encode a conjugative relaxase enzyme. This enzyme is responsible for nicking of DNA at the origin of transfer (oriT) sequence of the sex factor DNA to initiate the strand transfer process. Group II introns have been studied in eukaryotes, and several of these elements in yeast mitochondrial genes have received considerable attention. These introns are relatively large in size and generally encode a protein within the intron sequence. In addition to splicing activity. Group II introns are mobile genetic elements. The intron-encoded proteins (IEPs) contain endonuclease and reverse transcriptase domains believed to play an enzymatic role in genetic mobility reactions, while a putative maturase domain is thought to promote splicing by stabilizing the folding of the intron RNA into an active ribozyme structure which carries out the splicing reaction. The lactococcal introns represent the first examples of Group II introns shown to be functional in vivo in prokaryotes. Because of the advantages of a bacterial system for genetic and molecular studies, the Ll.ltrB intron from pRS01 has attracted the attention of several laboratories interested in Group II intron biology. Recently, it has been shown that the system can be adapted to function in Escherichia coli (although at somewhat reduced efficiency). In addition, it has been recently proven that the best studied form of mobility, the homing of the intron into an intronless allele of the cognate exon gene, occurs via an RNA intermediate and does not require DNA homology or generalized host recombination functions. Current efforts are analysis of the role Ll.ltrB splicing in regulating expression of pRS01 conjugation functions. The lactococcal Group II introns represent the first demonstrated genetically mobile prokaryotic retroelements, and they also have considerable potential as genetic engineering tools for Lactic Acid Bacteria (LAB) and other organisms.

An origin of transfer (oriT) on the conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3.

Previous analysis of the Tra1 region of the conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3 suggested that an origin of transfer (oriT) was present. Deletion derivatives of this cloned Tra1 region were assayed for mobilization in the presence of the wild-type pRS01 element in trans. The pRS01 oriT was localized to a 446-nucleotide segment in the intergenic region between open reading frames ltrD and ltrE. Sequence analysis of this region revealed a cluster of direct and inverted repeat structures characteristic of oriT regions associated with other conjugative systems.

Aggregation and binding substances enhance pathogenicity in rabbit models of Enterococcus faecalis endocarditis.

We investigated the importance of enterococcal aggregation substance (AS) and enterococcal binding substance (EBS) in rabbit models of Enterococcus faecalis cardiac infections. First, American Dutch belted rabbits were injected intraventricularly with 10(8) CFU and observed for 2 days. No clinical signs of illness developed in animals given AS- EBS- organisms, and all survived. All rabbits given AS- EBS+ organisms developed signs of illness, including significant pericardial inflammation, but only one of six died. All animals given AS+ EBS- organisms developed signs of illness, including pericardial inflammation, and survived. All rabbits given AS+ EBS+ organisms developed signs of illness and died. None of the rabbits receiving AS+ EBS+ organisms showed gross pericardial inflammation. The lethality and lack of inflammation are consistent with the presence of a superantigen. Rabbit and human lymphocytes were highly stimulated in vitro by cell extracts, but not cell-free culture fluids, of AS+ EBS+ organisms. In contrast, cell extracts from AS- EBS- organisms weakly stimulated lymphocyte proliferation. Culture fluids from human lymphocytes stimulated with AS+/EBS+ enterococci contained high levels of gamma interferon and tumor necrosis factor alpha (TNF-alpha) and TNF-beta, which is consistent with functional stimulation of T-lymphocyte proliferation and macrophage activation. Subsequent experiments examined the abilities of the same strains to cause endocarditis in a catheterization model. New Zealand White rabbits underwent transaortic catheterization for 2 h, at which time catheters were removed and animals were injected with 2 x 10(9) CFU of test organisms. None of the animals given AS- EBS- organisms developed vegetations or showed autopsy evidence of tissue damage. Rabbits given AS- EBS+ or AS+ EBS- organisms developed small vegetations and had splenomegaly at autopsy. All rabbits given AS+ EBS+ organisms developed large vegetations and had splenomegaly and lung congestion at autopsy. Similar experiments that left catheters in place for 3 days revealed that all rabbits given AS- EBS- or AS+ EBS+ organisms developed vegetations, but animals given AS+ EBS+ organisms had larger vegetations and autopsy evidence of lung congestion. These experiments provide direct evidence that these two cell wall components play an important role in the pathogenesis of endocarditis as well as in conjugative plasmid transfer.

A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron.

The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion. Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli. The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein. As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression in E. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.