Interaction of fibronectin and aggregation substance promotes adherence of Enterococcus faecalis to human colon.

This in vitro study investigates the interaction between aggregation substance (AS), a virulence factor of Enterococcus faecalis, and colonic mucosal fibronectin in normal colon and colon from patients with Crohn's disease. Fibronectin was found to be overexpressed in Crohn's disease compared to normal colon. Compared to E. faecalis OG1X:pAM944 (AS-negative), E. faecalis OG1X:pAM721 (expressing AS) showed a significantly enhanced adhesion to human colonic mucosa in normal colon and in colon from patients with Crohn's disease. Double-staining of fibronectin and AS-positive enterococci showed that colocalization of bacteria and fibronectin was significantly more frequent in Crohn's disease than in normal colon. Preincubation of bacteria with soluble fibronectin caused a significant reduction in the adherence to fibronectin. In conclusion, the interaction between AS and fibronectin plays is an important factor that mediates adhesion of Enterococcus faecalis to colonic mucosa. This might be one of the mechanisms responsible for bacterial translocation of Enterococcus faecalis.

Influence of hepatocyte growth factor, epidermal growth factor, and mycophenolic acid on endothelin-1 synthesis in human endothelial cells.

BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictive peptide which plays an important pathophysiological role in ischaemic renal failure and drug-induced renal injury such as cyclosporin A (CsA)- and tacrolimus-associated nephrotoxicity. In contrast, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) seem to accelerate renal regeneration after ischaemic and drug-induced renal injury. This study aimed to investigate the influence of HGF and EGF on ET-1 synthesis in cultured human umbilical vein endothelial cells (HUVEC) and renal artery endothelial cells (RAEC). In addition, we have investigated whether mycophenolic acid (MPA), a new immunosuppressive drug, which in contrast to CsA and tacrolimus lacks nephrotoxic side effects, modulates ET-1 synthesis in endothelial cells. METHODS: ET-1 release was measured with a specific enzyme-linked immunosorbent assay. ET-1 mRNA expression was investigated by reverse transcription polymerase chain reaction. RESULTS: HGF and EGF (0.001-10 nM) exerted a significant concentration-dependent inhibitory effect on ET-1 release by HUVEC and RAEC (minimum 56.1+/-4.3% of control, n=6, mean+/-SE). The suppressive effect of HGF and EGF on ET-1 synthesis was dose-dependently antagonized by the tyrosine kinase inhibitors tyrphostin AG1478, lavendustin A and methyl 2,5-dihydroxycinnamate. Incubation of HUVEC and RAEC with MPA (2.5, 10, 25, and 50 microg/ml) for 3-5 h induced a significant reduction of ET-1 mRNA expression. After 48 h incubation with MPA (1-50 microg/ml) a significant decrease of ET-1 release and DNA content per culture well was observed, whereas ET-1 release referred to the DNA content in the corresponding culture well did not differ significantly from controls. CONCLUSIONS: The present findings demonstrate that HGF and EGF reduce ET-1 synthesis in endothelial cells via their receptor tyrosine kinase activity and suggest that the renoprotective effects of HGF and EGF might be linked to their inhibitory action on ET-1 synthesis. This study also provides evidence that, in contrast to CsA and tacrolimus, MPA does not stimulate ET-1 synthesis. This might explain the clinical observation that renal function often improves when CsA or tacrolimus is replaced by mycophenolate mofetil.

Aggregation substance-mediated adherence of Enterococcus faecalis to immobilized extracellular matrix proteins.

Aggregation substance (AS) of Enterococcus faecalis (E. faecalis), a sex pheromone plasmid encoded cell surface protein, mediates the formation of bacterial aggregates, thereby promoting plasmid transfer. The influence of pAD1-encoded AS, Asa1, on binding to immobilized extracellular matrix proteins was studied. The presence of AS increased enterococcal adherence to fibronectin more than eight-fold, to thrombospondin more than four-fold, to vitronectin more than three-fold, and to collagen type I more than two-fold (P<0.001). In contrast, binding to laminin and collagen type IV occurred independently of AS. Adherence of the constitutively AS expressing E. faecalis OG1X(pAM721) to immobilized fibronectin was found to be approximately five times higher than that of Staphylococcus aureus Cowan and approximately 30 times higher than that of Streptococcus bovis. Investigation of strains with various deletions within the structural gene of asa1 suggests that attachment to immobilized fibronectin is mainly mediated by amino acids within the variable region or by neighbouring residues. Thus, AS may promote adherence to injured epithelium and endothelium, where extracellular matrix proteins are exposed, thereby facilitating colonization and infection.

Hepatocyte growth factor is upregulated by low-density lipoproteins and inhibits endothelin-1 release.

Low-density lipoproteins (LDL) are known to cause endothelial injury and to promote the development of atherosclerotic lesions. This study demonstrates a significant concentration-dependent stimulatory effect of LDL on hepatocyte growth factor (HGF) synthesis (maximum release: 423 +/- 16% of control) and HGF receptor mRNA expression in cultured human coronary artery endothelial cells (HCAEC). HGF is a potent mitogen for endothelial cells but does not affect smooth muscle cell proliferation. In contrast, endothelin-1 (ET-1) acts as a mitogen on vascular smooth muscle cells and seems to be upregulated in coronary atherosclerosis. In this study, the basal ET-1 synthesis in HCAEC was concentration-dependently reduced by HGF (minimum: 54 +/- 3% of control). This inhibitory effect seems to be mediated via the tyrosine kinase activity of the HGF receptor c-met, since it was antagonized by the tyrosine kinase inhibitor lavendustin A. In addition, HGF also significantly reduced the LDL-stimulated ET-1 release. The LDL-induced upregulation of HGF synthesis in HCAEC and the inhibitory effect of HGF on ET-1 synthesis suggest a protective role of HGF in coronary atherosclerosis.

Aggregation substance increases adherence and internalization, but not translocation, of Enterococcus faecalis through different intestinal epithelial cells in vitro.

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.

Aggregation substance promotes adherence, phagocytosis, and intracellular survival of Enterococcus faecalis within human macrophages and suppresses respiratory burst.

The aggregation substance (AS) of Enterococcus faecalis, encoded on sex pheromone plasmids, is a surface-bound 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 beta(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.

Aggregation substance promotes colonic mucosal invasion of Enterococcus faecalis in an ex vivo model.

BACKGROUND: Bacterial translocation through the gastrointestinal tract is the crucial step in the pathogenesis of intraabdominal infections. We assessed whether aggregation substance (AS), a bacterial adhesin and virulence factor of Enterococcus faecalis, promotes bacterial translocation and colonic mucosal invasion in an ex vivo experiment. METHODS: Colonic mucosa of male Wistar rats was placed in a modified Ussing system. The mucosal side of the chamber was filled with a suspension of E. faecalis OG1X:pAM721 (AS-positive) or E. faecalis OG1X (AS-negative). The serosal side was filled with sterile Dulbecco's modified Eagle's medium. For assessment of colonic mucosal invasion the mucosal side was incubated for 2.5 h with a suspension of AS-positive or AS-negative enterococci. After being washed, a solution of gentamicin and penicillin G in tissue culture medium was added on both sides in order to kill extracellular bacteria. Subsequently, the mucosa was removed from the system, washed, lysed with Triton X-100, and homogenized. Viable intramural bacteria were quantified by plating serial dilutions of the homogenate on Todd-Hewitt broth agar plates. To quantify bacterial translocation samples which were taken at various time points from the serosal side were plated on Todd-Hewitt broth agar plates and colony forming units (CFU) were determined. RESULTS: Invasion of the AS-positive E. faecalis strain OG1X:pAM721 into the colonic mucosa was significantly higher than invasion rates of the AS-negative strain OG1X (2.88 log(10) CFU/ml vs 1.73 log(10) CFU/ml; P = 0.02). However, none of the tested strains was found to translocate from the mucosal to the serosal side within the incubation time of 4 h. CONCLUSIONS: The aggregation substance promotes invasion of E. faecalis into colonic mucosa.

Lmb, a protein with similarities to the LraI adhesin family, mediates attachment of Streptococcus agalactiae to human laminin.

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. agalactiae wild-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.

Early effect of low-dose endotoxin on rat cecal mucosa ex vivo.

It has been suggested that endotoxin triggers translocation of intestinal bacteria in vivo, either by directly damaging intestinal mucosa or by inducing a systemic inflammatory reaction that leads to mucosal disruption. To address this issue, we examined the immediate effect of extraluminal endotoxin on structure and function of isolated rat cecal mucosa without other inflammatory cells in vitro. The cecal mucosa of 12 male Wistar rats was mounted in modified Ussing chambers filled with Dulbecco's modified Eagle's medium and the ampicillin-resistant Escherichia coli HB101:K12 incubated on the mucosal side. Endotoxin was added to the submucosal side at concentrations of 1 and 10 EU/ml, respectively. Under gassing with carbogene at 37 degreesC, the potential difference across the mucosa was measured continuously. Samples of the mucosal and submucosal solutions were removed at 60, 120, and 180 min and plated out on McConkey ampicillin-agar. After 180 min, the mucosal specimens were retrieved and examined by light and scanning electron microscopy. No significant change in potential difference was observed in control or endotoxin-incubated mucosa within the observation period. Neither light nor scanning electron microscopy showed a significant change in the structure of the epithelium, mucosa, or submucosa. No significant translocation of the E. coli across the mucosa was seen. We concluded that endotoxin alone does not induce immediate structural and functional damage to rat cecal mucosa in vitro. Therefore, it seems unlikely that a short endotoxemia alone directly triggers bacterial translocation by disrupting intestinal mucosa, but rather, entotoxin induces a local and systemic inflammatory reaction that leads to mucosal disruption.

Molecular characterization of group A streptococcal (GAS) oligopeptide permease (opp) and its effect on cysteine protease production.

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.

Peptide from a prokaryotic adhesin blocks leukocyte migration in vitro and in vivo.

The integrin CD11b/CD18 promotes leukocyte extravasation during inflammation. Filamentous hemagglutinin (FHA) of Bordetella pertussis binds to CD11b/CD18, raising the possibility that peptides derived from FHA might inhibit leukocyte migration. The Arg-Gly-Asp (RGD) sequence of FHA has been suggested to modulate binding of ligands to CD11b/CD18. Peptides derived from this region inhibited adherence and transendothelial migration of neutrophils in vitro and prevented recruitment of leukocytes into the cerebrospinal fluid in an experimental model of meningitis in rabbits. The mechanism of the antiinflammatory effect may involve modulation of the activity of CD11b/CD18 through peptide interaction with the leukocyte response integrin/integrin-associated protein complex.

Inhibition of leukocyte-endothelial cell interactions and inflammation by peptides from a bacterial adhesin which mimic coagulation factor X.

Factor X (factor ten) of the coagulation cascade binds to the integrin CD11b/CD18 during inflammation, initiating procoagulant activity on the surface of leukocytes (Altieri, D.C., O.R. Etingin, D.S. Fair, T.K. Brunk, J.E. Geltosky, D.P. Hajjar, and T. S. Edgington. 1991. Science [Wash.DC]. 254:1200-1202). Filamentous hemagglutinin (FHA), an adhesin of Bordetella pertussis also binds to the CD11b/CD18 integrin (Relman D., E. Tuomanen, S. Falkow, D.T. Golenbock, K. Saukkonen, and S.D. Wright. 1990. Cell. 61:1375-1382). FHA and the CD11b/CD18 binding loops of Factor X share amino acid sequence similarity. FHA peptides similar to Factor X binding loops inhibited 125I-Factor X binding to human neutrophils and prolonged clotting time. In addition, ETKEVDG and its Factor X analogue prevented transendothelial migration of leukocytes in vitro and reduced leukocytosis and blood brain barrier disruption in vivo. Interference with leukocyte migration by a coagulation-based peptide suggests a novel strategy for antiinflammatory therapy.

Lectin domains in the toxin of Bordetella pertussis: selectin mimicry linked to microbial pathogenesis.

The pathogenesis of many infectious diseases is critically determined by prokaryotic lectins which enable differential recognition and activation of targeted eukaryotic cells. Some bacterial adhesins mimic and co-opt eukaryotic cell-cell adhesion motifs. This is illustrated by the toxin of Bordetella pertussis. Pertussis toxin mediates intoxication of eukaryotic cells by elevation of cAMP and it serves as an adhesin binding the bacteria to ciliated cells and respiratory macrophages. These activities are mediated by the lectin-like properties of the binding oligomer of the toxin. A comparison of pertussis toxin and the selectins involved in leukocyte trafficking indicates that these prokaryotic and eukaryotic C-type lectins share some element of primary sequence similarity, three dimensional structure, and biological activities. Such mimicry suggests a link between eukaryotic cell-cell adhesion motifs and microbial pathogenesis.

Peptides from pertussis toxin interfere with neutrophil adherence in vitro and counteract inflammation in vivo.

Selectins on the surface of endothelial cells initiate leukocyte rolling along the capillary walls during inflammation. The amino acid sequence 19-52 of pertussis toxin subunit S3 is strikingly similar to the sequence 15-46 of the selectins. The S3 subunit inhibits the binding of neutrophils to selectin-coated surfaces and a peptide spanning the 28-45 sequence of S3 reduces leukocyte binding to endothelial cells in vitro and inhibits leukocyte recruitment to the subarachnoid space in vivo. To identify sequences within the 28-45 S3 peptide responsible for these activities, 27 peptides derived by successive truncation of amino acids from either the amino or the carboxyl terminus were tested for anti-inflammatory activity. Truncation at five residues ablated the ability to inhibit neutrophil adherence to endothelial monolayers: valine32, alanine33, arginine36, asparagine38, and threonine43. The most active peptides were either full-length molecules (28-44, 30-45) or short peptides from both ends of the full sequence (39-45, 40-45, 41-45, 28-32). Three peptides with the strongest ability to prevent neutrophil adherence in vitro (28-44, 30-45, 40-45) reduced the cerebrospinal fluid leukocytosis in a pneumococcal meningitis model when administered intravenously. We conclude that peptides derived from a prokaryotic lectin have anti-inflammatory properties consistent with inhibition of selectin participation in leukocyte recruitment during inflammation.

Antiinflammatory effects in experimental meningitis of prokaryotic peptides that mimic selectins.

The lectin domains of two subunits of pertussis toxin, S2 and S3, share amino acid sequence similarity with the lectin domains of the eukaryotic selectin family. During inflammation, selectins appear on endothelial cells and promote recruitment of leukocytes by reversibly binding carbohydrates. Synthetic peptides representing the carbohydrate recognition domains of S2 and S3 competitively inhibited adherence of neutrophils to endothelial cells in vitro. For some peptides, this antiinflammatory effect occurred without up-regulation of the function of the leukocyte integrin CD11b/CD18. Intravenous administration of peptides to animals with meningitis disrupted recruitment of leukocytes into the cerebrospinal fluid. These findings indicate that peptides derived from prokaryotic members of the selectin family have therapeutic antiinflammatory potential.

Prokaryotic peptides that block leukocyte adherence to selectins.

Pertussis toxin binds target cells through the carbohydrate recognition properties of two subunits, S2 and S3, which share amino acid sequence similarity with the lectin domains of the eukaryotic selectin family. Selectins appear on inflamed endothelial cells and promote rolling of leukocytes by reversibly binding carbohydrates. S2, S3, and synthetic peptides representing their carbohydrate recognition domains competitively inhibited adherence of neutrophils to selectin-coated surfaces and to endothelial cells in vitro. These proteins and peptides also rapidly upregulated the function of the leukocyte integrin CD11b/CD18. These findings implicate mimicry of eukaryotic selectins by prokaryotic adhesive ligands and link the mechanisms underlying leukocyte trafficking to microbial pathogenesis.

Ochrobactrum anthropi bacteremia: report of four cases and short review.

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.