The aggregation substance (AS) of Enterococcus faecalis, encoded on sex pheromone plasmids, is a surfacebound glycoprotein that mediates aggregation between bacteria thereby facilitating plasmid transfer. Sequencing of the pAD1-encoded Asa1 revealed that this surface protein contains two RGD motifs which are known to ligate integrins. Therefore, we investigated the influence of AS on the interaction of E. faecalis with human monocyte-derived macrophages which constitutively express ␤ 2 integrins (e.g., CD18). AS was found to cause a greater-than-fivefold increase in enterococcal adherence to macrophages and a greater-than-sevenfold increase in phagocytosis. Adherence was mediated by an interaction between the RGD motif and the integrin CD11b/CD18 (complement receptor type 3) as demonstrated by inhibition studies with monoclonal antibodies and RGD peptide. AS-bearing enterococci were significantly more resistant to macrophage killing during the first 3 h postinfection, probably due to inhibition of the respiratory burst as indicated by reduced concentrations of superoxide anion.Enterococci are gram-positive cocci which inhabit the gastrointestinal tract as well as the vagina and the oral cavity. Enterococcus faecalis accounts for 90% of human enterococcal infections, the most common being urinary tract infections, followed by abdominal infections, wound infections, bacteremia, and infective endocarditis (31, 39). Although infections due to E. faecalis have increased substantially during the last 10 years, the understanding of virulence mechanisms is still limited (24). One of the postulated virulence factors is the aggregation substance (AS), a sex pheromone plasmid-encoded surface protein which promotes the conjugative transfer of sex pheromone plasmids by formation of mating aggregates between donor and recipient cells (6,13,52). DNA sequencing of the structural gene for the pAD1-encoded AS revealed the presence of two Arg-Gly-Asp (RGD) sequences (16); RGD is a well-known motif recognized by a family of eukaryotic receptors, the integrins (38). Integrins consist of noncovalently linked ␣ and ␤ chains and are expressed on leukocytes, thrombocytes, endothelium, and various epithelial cells (21, 37, 42). Our group first suggested an interaction of AS with integrins, since we found that AS augmented adherence to porcine renal tubular cells which could be inhibited competitively by an RGD-Ser (RGDS) peptide (26). This hypothesis was corroborated by in vitro experiments with human polymorphonuclear leukocytes (PMN) which demonstrated that AS promotes opsonin-independent binding of E. faecalis via a ␤ 2 integrinmediated mechanism (46). It is assumed that many enterococcal infections are endogenous, originating from the intestinal tract (25, 51). Wells et al. speculated that macrophages may serve as a vehicle facilitating translocation from the intestinum into the lymph system and bloodstream (49, 50). However, this can occur only if enterococci are able to survive within macrophages. Indeed, Gentry-Weeks et al. demons...

Bacterial oligopeptide permeases are membrane-associated complexes of five proteins belonging to the ABC-transporter family, which have been found to be involved in obtaining nutrients, cell-wall metabolism, competence, and adherence to host cells. A lambda library of the strain CS101 group A streptococcal (GAS) genome was used to sequence 10,192 bp containing the five genes oppA to oppF of the GAS opp operon. The deduced amino acid sequences exhibited 50-84% homology to pneumococcal AmiA to AmiF sequences. The operon organization of the five genes was confirmed by transcriptional analysis and an additional shorter oppA transcript was detected. Insertional inactivation was used to create serotype M49 strains which did not express either the oppA gene or the ATPase genes, oppD and oppF. The mutation in oppA confirmed that the additional shorter oppA transcript originated from the opp operon and was probably due to an intra-operon transcription terminator site located downstream of oppA. While growth kinetics, binding of serum proteins, and attachment to eukaryotic cells were unaffected, the oppD/F mutants showed reduced production of the cysteine protease, SpeB, and a change in the pattern of secreted proteins. Thus, the GAS opp operon appears to contribute to both protease production and export/processing of secreted proteins.

Streptococcus agalactiae is a leading cause of neonatal sepsis and meningitis. Adherence to extracellular matrix proteins is considered an important factor in the pathogenesis of infection, but the genetic determinants of this process remain largely unknown. We identified and sequenced a gene which codes for a putative lipoprotein that exhibits significant homology to the streptococcal LraI protein family. Mutants of this locus were demonstrated to have substantially reduced adherence to immobilized human laminin. The nucleotide sequence of the gene was subsequently designated lmb (laminin binding) and shown to be present in all of the common serotypes of S. agalactiae. To determine the role of Lmb in the adhesion of S. agalactiaewild-type strains to laminin, a recombinant Lmb protein harboring six consecutive histidine residues at the C terminus was cloned, expressed, and purified from Escherichia coli. Preincubation of immobilized laminin with recombinant Lmb significantly reduced adherence of the wild-type strain O90R to laminin. These results indicate that Lmb mediates the attachment of S. agalactiae to human laminin, which may be essential for the bacterial colonization of damaged epithelium and translocation of bacteria into the bloodstream.

The adhesion of gram-positive bacteria to extracellular matrix (ECM) proteins is regarded as an important determinant of pathogenicity. A comparison of the adhesion of Streptococcus agalactiae strain O90R to different ECM proteins showed that the most pronounced binding could be observed for immobilized fibrinogen. To investigate the genetic determinants of S. agalactiae fibrinogen binding, a pGhost9:ISS1 mutant library was screened for mutants displaying reduced agglutination of fibrinogen-coated latex beads. A putative two-component signal transduction system was identified and designated rgfBDAC. It comprises genes encoding a putative response regulator of 218 amino acids and a putative histidine kinase of 426 amino acids. Comparison of the deduced proteins with the GenBank database revealed a significant similarity to quorum-sensing systems of gram-positive pathogens. Transcription analysis of the rgf locus showed that the encoding genes are located on one transcript. To further characterize the influence of the putative histidine kinase encoded in the rgf locus on the adhesion of S. agalactiae to immobilized fibrinogen, a targeted mutant of rgfC was generated. In comparison to the wild-type strain this mutant demonstrated altered fibrinogen binding capacities depending on bacterial cell density. Transcription analysis of secreted and surface-localized S. agalactiae proteins in the wild type and the rgfC mutant strain revealed that mRNA levels of the C5a peptidase gene scpB were increased in the mutant strain while the transcription of the secreted CAMP factor gene cfb was unaffected by this mutation. Based on these results, we hypothesize that rgf regulates the expression of bacterial cell surface components.Streptococcus agalactiae (group B streptococcus) is an important human pathogen causing neonatal pneumonia, sepsis, and meningitis and severe infections in immunocompromised adult patients. It has the ability to survive and multiply in various host compartments requiring the expression and coordinate regulation of multiple pathogenicity factors. Several group B streptococcal molecules important for pathogenicity and virulence have been identified and characterized. However, the regulation and control of these factors are poorly understood, and a typical two-component regulatory system that controls their expression has not yet been described.In gram-positive bacteria, peptide-based signal transduction appears to be the preferred mechanism to sense and respond to population density and coordinate the expression of molecules that are important for pathogenicity. A comprehensive review of bacterial quorum sensing has been published recently (6). Peptide-controlled two-component signal transduction systems regulate the production of pathogenicity factors in Staphylococcus aureus (20), competence of streptococci (10), and bacteriocin production in lactic acid bacteria (7). The agrBDCA (accessory gene regulator) locus encodes the quorum-sensing system that controls the expression of S. aureus virulence factor...

Ochrobactrum anthropi, formerly "Achromobacter" CDC group Vd, is a nonfermentative, nonfastidious gram-negative bacillus, that only recently has been given attention as a potential human pathogen. Over a 2-year period, we observed four patients with multiple blood cultures that were positive for the organism. The patients had acute leukemia as underlying disease, and presented with clinical and microbiologic features consistent with catheter-related bacteremia. In three of the patients the infection initially appeared to be unrelated to chemotherapy-associated profound neutropenia and occurred early after, or was the reason for, hospital admission. The antimicrobial susceptibility of the isolates varied: unlike previously reported cases, resistance in some of our isolates included aminoglycosides, newer fluoroquinolones, and trimethoprim-sulfamethoxazole. Despite in vitro susceptibility to imipenem in initial isolates, treatment of two patients with this agent obviously failed to eradicate the organism, and the patients either relapsed with bacteremia shortly after discontinuation of treatment or remained persistently febrile and bacteremic. O. anthropi appears to be increasingly recognized as a human opportunist pathogen associated with intravascular catheters and unpredictable multiple antibiotic resistance.

The aggregation substance of Enterococcus faecalis increased bacterial adherence to and internalization by epithelial cells originating from the colon and duodenum but not by cells derived from the ileum. However, enterococcal translocation through monolayers of intestinal epithelium was not observed.Enterococcus faecalis, a gram-positive facultative anaerobic bacterium, belongs to the normal flora of the intestinal tract and is also found in the vaginal vault and the oral cavity. Enterococci have increasingly gained attention as pathogens, since they have become the fourth leading cause of nosocomial infections in the United States (8). E. faecalis frequently causes local or systemic infections, such as urinary tract and abdominal infections, wound infections, bacteremia, and endocarditis (15). It is assumed that many enterococcal infections are endogenous, resulting from bacterial translocation from the intestinal lumen to extraintestinal sites (16,31).Surprisingly little is known about the virulence factors of E. faecalis. One of the potential virulence factors is the aggregation substance (AS), an adhesin encoded by inducible sex pheromone plasmids (for a review, see reference 5). The AS, a signal peptide-containing protein, appears as a hair-like structure on the cell surface and is incorporated primarily into the ''old'' parts of the cell wall (30). It has been demonstrated that this adhesin is responsible for bacterium-bacterium contact during conjugative transfer of sex pheromone plasmids (5).The AS contains two Arg-Gly-Asp motifs which are known to be recognized by integrins, a family of eukaryotic cell surface receptors (24). Since integrins have been reported to be expressed on intestinal epithelial cells (2), we hypothesized that the AS promotes adherence and possibly invasion into these cells, thereby enabling enterococci to translocate through the intestinal epithelial barrier.(This work was presented in part at the 98th General Meeting of the American Society for Microbiology, Atlanta, Ga., 17 to 21 May 1998.)Three different E. faecalis strains (Table 1), kindly provided by R. Wirth (Institute of Microbiology, University of Regensburg, Regensburg, Germany), were used to study the effect of the AS on adherence, internalization, and translocation through intestinal epithelial cells. The bacteria were cultured in Todd-Hewitt medium (Difco, Augsburg, Germany) supplemented with erythromycin (20 g/ml) for growth of the plasmid-containing strains (OG1X/pAM721 and OG1X/pAM944). Since E. faecalis strains expressing the AS spontaneously clump, all inocula were sonicated (Branson W-450 Sonifier; 80 W, 20 s, continuously) to obtain single-cell suspensions. Salmonella enterica serovar Typhimurium ATCC 14028 and Escherichia coli HB101 grown in Luria-Bertani medium were used as positive and negative controls, respectively. For the experiments described below, the bacteria were harvested in mid-log phase and suspended in tissue culture medium without supplements, which served as the infection medium. Appropriate ...

Components of bacterial peptidoglycans have potent biological activities, including adjuvant effects, cytotoxicity, and induction of sleep. Mixtures of peptidoglycan components also induce inflammation in the lung, subarachnoid space, and joint, but the structural requirements for activity are unknown. Using a rabbit model for meningitis, we determined the biological activities of 14 individual muramyl peptides constituting > 90% of the peptidoglycan of the gram-negative pediatric pathogen Haemophilus influenzae. Upon intracisternal inoculation, most of the muropeptides induced leukocytosis in cerebrospinal fluid (CSF), influx of protein into CSF, or brain edema, alone or in combination. The disaccharide-tetrapeptide, the major component of all gram-negative peptidoglycans, induced CSF leukocytosis and protein influx at doses as low as 0.4 ,gg (0.42 nM). Modification of the N-acetyl muramic acid or substitution of the alanine at position four in the peptide side chain decreased leukocytosis but enhanced brain edema. As the size ofthe muropeptide increased, the inflammatory activity decreased. Muropeptides carrying the diaminopimelyl-diaminopimelic acid cross-link specifically induced cytotoxic brain edema. These findings significantly expand the spectrum of biological activities of natural muramyl peptides and provide the basis for a structure-activity relationship for the inflammatory properties of bacterial muropeptides. (J. Clin. Invest. 1993.92:297-302.)

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