Effects of Escherichia coli and Porphyromonas gingivalis lipopolysaccharide on pregnancy outcome in the golden hamster.

This report describes the effects of two gram-negative bacterial endotoxin (lipopolysaccharide [LPS]) preparations on hamster pregnancy outcome variables. Single intravenous challenges with Escherichia coli and Porphyromonas gingivalis LPS on day 8 of pregnancy produced dose-dependent effects on fetal weight malformation and fetal resorption with E. coli LPS having potent embryolethal effects. Premating maternal exposure to P. gingivalis produced embryolethal effects similar to those of E. coli. These data suggest that maternal exposure to P. gingivalis LPS prior to and during pregnancy can induce deleterious effects on the developing fetus.     

Interactions of Escherichia coli and Proteus mirabilis with mouse mononuclear phagocytes

Five strains of enterobacteria (three of Escherichia coli and two of Proteus mirabilis) were studied to assess and compare their phagocytic uptake and intracellular killing by mouse macrophages. Each strain was injected intraperitoneally into separate groups of mice and peritoneal exudate cells were harvested after 3 min for phagocytosis to occur in vivo. Acridine orange staining showed that there were approximately 10-fold fewer intracellular P. mirabilis than E. coli cells. The average numbers of viable intracellular bacteria per leucocyte were 0.03 and 0.02 for P. mirabilis strains M13 and H1, respectively, and 0.48, 0.45, and 0.28 for E. coli strains M14, A-D M5 and H40. Thus, both P. mirabilis strains were ingested less readily than any of the three E. coli strains (p less than 0.01). The rates of in-vitro intracellular killing were similar for all five strains of bacteria. The intracellular killing constants (Kk) for the three mouse isolates were 0.017, 0.016 and 0.020 min for E. coli M14 and A-D M5, and P. mirabilis M13, respectively; the Kks for the two human isolates were 0.026 and 0.029/min for E. coli H40 and P. mirabilis H1, respectively. The Kks for all five strains were not significantly different. Assuming that the numbers of viable intracellular bacteria at the beginning of the assay represented 100% viability, 6-17% of the intracellular bacteria remained viable after 2 h, reflecting log10 3.9-5.6 bacteria (6-8) x 10(6) peritoneal exudate cells. Intravenous injection of these five strains into separate groups of mice demonstrated that the P. mirabilis strains were more virulent than the E. coli strains. Injection of each P. mirabilis strain was associated with ruffled fur and death, whereas mice given any of the three E. coli strains remained visibly healthy and none died. Consistent with these observations, quantitation of viable bacteria in the liver and spleen showed that greater numbers of P. mirabilis M13 than of E. coli M14 or A-D M5 persisted in these organs; similarly greater numbers of P. mirabilis H1 than of E. coli H40 persisted in the liver and spleen. Because the rates of intracellular killing of these five strains were similar, the relative virulence of both strains of P. mirabilis appeared to be associated with decreased phagocytic uptake rather than differences in intracellular survival.     

Helicobacter pylori and Porphyromonas gingivalis lipopolysaccharides are poorly transferred to recombinant soluble CD14.

Helicobacter pylori and Porphyromonas gingivalis are gram-negative bacteria associated with chronic inflammatory diseases. These bacteria possess lipopolysaccharides (LPSs) that are able to activate human monocytes to produce tumor necrosis factor alpha but fail to activate human endothelial cells to express E-selectin. With Escherichia coli LPS, tumor necrosis factor alpha activation requires membrane-bound CD14 and E-selectin expression requires soluble CD14 (sCD14). Therefore, the ability of H. pylori and P. gingivalis LPSs to transfer to and bind sCD14 was examined by using immobilized recombinant sCD14 and human serum or recombinant LPS-binding protein (LBP). H. pylori and P. gingivalis LPSs were transferred to sCD14 when serum or LBP was present. However, the transfer of these LPSs to CD14 in serum was significantly slower than the transfer of E. coli LPS. Quantitation of the transfer rates by Michaelis-Menten kinetics yielded K(m) values of 6 and 0.1 nM for H. pylori and E. coli LPSs, respectively. The amount of P. gingivalis LPS required to obtain half-maximum binding to CD14 was approximately 10-fold greater than the amount of E. coli LPS required. The slower transfer rates displayed by these LPSs can be explained by the poor binding to LBP observed in direct binding assays. These results are consistent with the proportionately lower ability of these LPSs to activate monocytes compared with E. coli LPS. However, the ability of H. pylori and P. gingivalis LPSs to bind LBP and transfer to sCD14 demonstrates that the lack of endothelial cell CD14-dependent cell activation by these LPSs occurs distal to sCD14 binding.     

The atherogenic bacterium Porphyromonas gingivalis evades circulating phagocytes by adhering to erythrocytes

A relationship between periodontitis and coronary heart disease has been investigated intensively. A pathogenic role for the oral bacterium Porphyromonas gingivalis has been suggested for both diseases. We examined whether complement activation by P. gingivalis strain ATCC 33277 allows the bacterium to adhere to human red blood cells (RBCs) and thereby evade attack by circulating phagocytes. On incubation with normal human serum, the P. gingivalis strain efficiently fixed complement component 3 (C3). Incubation of bacteria with washed whole blood cells suspended in autologous serum resulted in a dose- and time-dependent adherence to RBCs. The adherence required functionally intact complement receptor 1 (CR1; also called CD35) on the RBCs and significantly inhibited the uptake of P. gingivalis by neutrophils and B cells within 1 min of incubation (by 64% and 51%, respectively) and that by monocytes after between 15 min and 30 min of incubation (by 66% and 53%, respectively). The attachment of C3b/iC3b to bacterium-bearing RBCs decreased progressively after 15 min, indicating that conversion of C3 fragments into C3dg occurred, decreasing the affinity for CR1 on RBCs. We propose that P. gingivalis exploits RBCs as a transport vehicle, rendering it inaccessible to attack by phagocytes, and by doing so plays a role in the development of systemic diseases.     

Cloning of two distinct hemolysin genes from Porphyromonas (Bacteroides) gingivalis in Escherichia coli

Hemolysin is considered a potent virulence factor in a large number of Gram-positive and Gram-negative bacterial pathogens. The hemolysin produced by the oral pathogen Porphyromonas gingivalis functions to provide the cell with its required heme-containing molecules for growth in the periodontal pocket. Two distinct P. gingivalis genes, each of which confers a hemolytic phenotype in Escherichia coli, were isolated by screening genomic DNA libraries of P. gingivalis on sheep blood agar plates. The results obtained from physical maps and Southern blots indicated a considerable degree of divergence in the nucleotide sequences of these two genes. Maxicell analyses of the recombinant plasmids in E. coli suggested that plasmid pPGH5 encoded a polypeptide of molecular weight 48 kDa, while an 18-kDa polypeptide was obtained with pPGH1 and pPGH7. When E. coli harboring these hemolysin genes was subjected to iron starvation, the levels of hemolysin activity increased. Biochemical characterization of hemolytic activities indicated that the activity of both hemolysins was inhibited by Mg²⁺ and Ca²⁺; but not by EDTA. Elevated levels of hemolytic activity were obtained from the E. coli recombinant strains in the presence of glutathione, DTT and 2-mercaptoethanol. Cholesterol inhibited the activity.     

Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide

Porphyromonas gingivalis lipopolysaccharide (LPS) (strain W50) interacts with Toll‐like receptor 2 (TLR‐2) leading to cytokine expression and inflammation, and thereby plays a key role in the pathogenesis of periodontal disease. The aims of this study were to investigate gene expression of key regulatory mediators of innate immune responses in a human monocytic cell line (THP‐1) to P. gingivalis LPS and to compare these results with those obtained using the TLR‐4 ligand, Escherichia coli LPS. Custom‐made Taqman low‐density arrays were used for expression profiling of 45 different cytokine‐related genes. Both types of LPS highly up‐regulated interleukin (IL)‐1α and IL‐1β, IL‐18 receptor (IL‐18R), IL‐18R accessory protein and IL‐1 family (IL‐1F)9. Expression levels of IL‐1F6, IL‐1F7 and caspase‐1 were unaltered by either LPS. Genes for tumour necrosis factor‐α, IL‐6, leukaemia inhibitory factor and IL‐32 were also highly induced by both LPS. For a subset of genes, including CXC chemokine ligand 5 (CXCL5), expression was induced only by E. coli LPS or was up‐regulated more highly by E. coli compared with P. gingivalis LPS in THP‐1 monocytes. A similar expression pattern was also observed in dendritic cells. Analysis of signalling pathways which lead to CXCL5 expression indicated that the mechanisms underpinning the differential responses did not involve the recruitment of different adaptor proteins by TLR‐2 and TLR‐4, and therefore occur downstream of the receptor–adaptor complex. We conclude that differences in signalling pathways activated by TLR‐2 and TLR‐4 ligands lead to differential innate immune responses which may be important in polymicrobial diseases such as periodontal disease.     

Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at toll-like receptor 4 in human endothelial cells

E. coli lipopolysaccharide (LPS) induces cytokine and adhesion molecule expression via the toll-like receptor 4 (TLR4) signaling complex in human endothelial cells. In the present study, we investigated the mechanism by which Porphyromonas gingivalis LPS antagonizes E. coli LPS-dependent activation of human endothelial cells. P. gingivalis LPS at 1 micro g/ml inhibited both E. coli LPS (10 ng/ml) and Mycobacterium tuberculosis heat shock protein (HSP) 60.1 (10 micro g/ml) stimulation of E-selectin mRNA expression in human umbilical vein endothelial cells (HUVEC) without inhibiting interleukin-1 beta (IL-1beta) stimulation. P. gingivalis LPS (1 micro g/ml) also blocked both E. coli LPS-dependent and M. tuberculosis HSP60.1-dependent but not IL-1beta-dependent activation of NF-kappaB in human microvascular endothelial (HMEC-1) cells, consistent with antagonism occurring upstream from the TLR/IL-1 receptor adaptor protein, MyD88. Surprisingly, P. gingivalis LPS weakly but significantly activated NF-kappaB in HMEC-1 cells in the absence of E. coli LPS, and the P. gingivalis LPS-dependent agonism was blocked by transient expression of a dominant negative murine TLR4. Pretreatment of HUVECs with P. gingivalis LPS did not influence the ability of E. coli LPS to stimulate E-selectin mRNA expression. Taken together, these data provide the first evidence that P. gingivalis LPS-dependent antagonism of E. coli LPS in human endothelial cells likely involves the ability of P. gingivalis LPS to directly compete with E. coli LPS at the TLR4 signaling complex.     

Effects of mycobacteria on regulation of apoptosis in mononuclear phagocytes.

Since apoptosis is observed in tuberculous granulomata, we investigated the molecular mechanisms underlying the apoptotic pathway in an in vitro model of mycobacterial infection of mononuclear phagocytes. We postulated that Mycobacterium tuberculosis could trigger the apoptotic pathway in macrophages, resulting in death of the microorganism by modulating the expression of bcl-2, bax, bcl-xL, and bcl-xS. We found that the mRNA of bcl-2, an inhibitor of apoptosis, was downregulated in peripheral blood monocytes (PBM) between 2 and 6 h following infection with M. bovis BCG or induction with heat-killed M. tuberculosis H37Ra. Western analysis showed a downregulation of the Bcl-2 protein, with a half-life of 24 h. At the same time points, there was no change in the expression of Bax or Bcl-xS, inducers of apoptosis, but Bcl-xL, another inhibitor of apoptosis, was minimally upregulated by BCG. To determine if apoptosis could be a mechanism for growth inhibition in vivo, we obtained alveolar macrophages by bronchoalveolar lavage from involved sites in patients with active pulmonary tuberculosis. Using the TUNEL (terminal deoxynucleotidyltransferase mediated nick end labeling) technique, we observed significantly more apoptosis in involved segments of five tuberculosis patients (14.8 +/- 1.9%) than in those of normal controls (<1%, P = 0.02) or in uninvolved segments (4.3 +/- 0.9%, P < 0.05). We conclude that apoptosis of mononuclear phagocytes induced by M. tuberculosis occurs in vivo and that in an in vitro model of mycobacterial infection, apoptosis may be mediated by downregulation of Bcl-2.     

Effects of Escherichia coli and E. coli lipopolysaccharides on the function of human ureteral epithelial cells cultured in serum-free medium.

Escherichia coli is the microorganism most commonly isolated from human urinary tract infections. Earlier studies by others have shown that bacterial attachment and production of toxins (e.g., lipopolysaccharides [LPS]) enhance recruitment of leukocytes to the infection site and mucosal inflammation. The mechanisms by which these changes occur have not been completely defined. In the present study, epithelial cell cultures isolated from the human ureter (UT cells) (A. Elgavish, J. J. Wille, F. Rahemtulla, and L. Debro, Am. J. Physiol. 261:C916-C926, 1991; J. J. Wille, J. Park, and A. Elgavish, J. Cell. Physiol. 150:52-58, 1992) served as a model system with which to explore the mechanisms of action of Escherichia coli and E. coli LPS in UT cells. E. coli adhered to UT cells and inhibited carrier-mediated sulfate uptake to half of that in untreated UT cells, suggesting that the intracellular pool of sulfate available for sulfation may be lower in infected cells and may lead to the production of undersulfated glycoconjugates. Incubation of UT cells with E. coli LPS inhibited carrier-mediated sulfate uptake to an extent similar to that caused by whole E. coli, indicating that the effect of E. coli on sulfate uptake may be mediated by LPS. LPS caused an increase in Na+ content in rapidly proliferating UT cells but not in quiescent cells. We postulated that this change in the intracellular ionic environment or changes coupled to it (e.g., pH or Ca2+ levels) may serve as a transducing signal. This possibility was supported by the fact that LPS stimulated clustering of ICAM-1 on the cell surface of rapidly proliferating but not quiescent UT cells. This study suggests that, in vivo, LPS stimulation of ICAM-1 clustering on the surface of the urothelium may allow more effective binding of leukocytes. This may be the mechanism underlying earlier findings in vivo indicating a role for LPS in the recruitment of leukocytes to the urinary tract as a host defense mechanism following urinary tract infection.     

Cloning, expression, and sequencing of a protease gene (tpr) from Porphyromonas gingivalis W83 in Escherichia coli.

Porphyromonas gingivalis is a highly proteolytic organism which metabolizes small peptides and amino acids. Indirect evidence suggests that the proteases produced by this microorganism constitute an important virulence factor. In this study, a gene bank of P. gingivalis W83 DNA was constructed by cloning 0.5- to 20-kb HindIII-cut DNA fragments into Escherichia coli DH5 alpha by using the plasmid vector pUC19. A clone expressing a protease from P. gingivalis was isolated on LB agar containing 1% skim milk. The clone contained a 3.0-kb insert that coded for a protease with an apparent molecular mass of 64 kDa. Sequencing part of the 3.0-kb DNA fragment revealed an open reading frame encoding a protein of 482 amino acids with a molecular mass of 62.5 kDa. Putative promoter and termination elements flanking the open reading frame were identified. The activity expressed in E. coli was extensively characterized by using various substrates and protease inhibitors, and the results suggest that it is possibly a thiol protease.     

Influence of adhesins on the interaction of Escherichia coli with human phagocytes.

The fitness between bacterial adhesins and target cell receptors, determining bacterial adherence to epithelial cells in urinary tract infections, was shown to influence also the interaction with human polymorphonuclear leukocytes (PMNL). Two sets of homogenic strains, constructed to express either, both, or none of the globotetraosylceramide-sensitive (GS) adhesins specific for globoseries glycolipid receptors or the mannose-sensitive (MS) adhesins inhibited by alpha-methyl mannoside were compared regarding charge, hydrophobicity, and binding to PMNL. The mutants of a hydrophilic pyelonephritis strain required MS adhesins for binding to and activation of the PMNL. Removal of the MS adhesins from the mutant carrying both MS and GS adhesins abolished chemiluminescence and binding. A pronounced chemiluminescence reaction was induced by the hydrophobic strain without GS or MS adhesins . Transformants of this strain expressing the MS adhesin bound to and activated the PMNL. Poor binding and activation were found with mutants and transformants carrying only the GS adhesins . The improved reactivity after coating of the PMNL with the appropriate receptor glycolipid supported the previously reported absence of globoseries glycolipids in those cells as the reason for the refractoriness to bacteria with GS adhesins . The mechanism of binding, which improves epithelial cell adhesion, may prevent binding to PMNL, thus improving the survival of Escherichia coli in the kidney.     

Effects of temperature stress on expression of fimbriae and superoxide dismutase by Porphyromonas gingivalis.

We examined the biosynthesis of fimbriae and superoxide dismutase (SOD) produced by the periodontopathic bacterium Porphyromonas gingivalis in response to elevated temperature. P. gingivalis 2561, grown at 37 degrees C to mid-logarithmic phase, was subsequently incubated at 39, 41, and 43 degrees C, respectively, to stationary phase. There was no difference in the growth of cells at 37 and 39 degrees C. However, at 39 degrees C there was a 54% reduction in the amount of fimbrillin (fimbriae) as well as decreased expression of mRNA for fimA. On the other hand, under the same conditions, a more than twofold increase in the amount of SOD activity, as well as in the levels of SOD mRNA, was observed. Moreover, cells cultured for 20 h at 39 degrees C showed an 86% decrease of fimbrillin protein and a threefold increase in SOD activity. These observations suggest that P. gingivalis may undergo alterations in its virulence and susceptibility to host immune responses as a result of the elevated temperatures found in inflamed periodontal pockets.     

Expression of a functional Porphyromonas gingivalis fimbrillin polypeptide in Escherichia coli: purification, physicochemical and immunochemical characterization, and binding characteristics.

Fimbriae have been reported to play an important role in the adherence of Porphyromonas gingivalis to oral surfaces and possibly in triggering host responses. A structural subunit of the fimbriae, fimbrillin, has been shown to be important in binding of the bacterium to saliva-coated oral surfaces. In the present study, a coding region of the fimbrillin gene from P. gingivalis 2561 was amplified by the polymerase chain reaction and cloned into the pET-11d vector. The recombinant plasmid was transformed into Escherichia coli BL21, and protein expression was induced with isopropyl-beta-D-thiogalactopyranoside. The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and gel filtration chromatography. The purified recombinant fimbrillin polypeptide, r-fim 10-337, corresponded to amino acid residues 10 to 337 of the deduced amino acid sequence of fimbrillin. In immunoblot analysis, r-fim 10-337 reacted with antibodies to fimbrillin purified from P. gingivalis, as well as with antibodies to synthetic peptides corresponding to the amino acid sequence of fimbrillin. The apparent molecular mass of r-fim 10-337 was estimated to be 41 kDa on sodium dodecyl sulfate-polyacrylamide gels. The r-fim 10-337 polypeptide was capable of inhibiting the binding of P. gingivalis 2561 to saliva-coated hydroxyapatite beads. These results suggest that the fimbrillin subunit polypeptide plays an important role in binding of P. gingivalis cells to saliva-coated surfaces. We describe here the successful expression and purification of a functionally and immunologically reactive recombinant P. gingivalis fimbrillin subunit from E. coli.     

Plasmodium and mononuclear phagocytes

Plasmodium, the causative agent of malaria, initially multiplies inside liver cells and then in successive cycles inside erythrocytes, causing the symptoms of the disease. In this review, we discuss interactions between the extracellular and intracellular forms of the Plasmodium parasite and innate immune cells in the mammalian host, with a special emphasis on mononuclear phagocytes. We overview here what is known about the innate immune cells that interact with parasites, mechanisms used by the parasite to evade them, and the protective or detrimental contribution of these interactions on parasite progression through its life cycle and pathology in the host.     

Porphyromonas gingivalis lipopolysaccharides induce maturation of dendritic cells with CD14+CD16+ phenotype

Primary immune responses are initiated by dendritic cells (DC) that inform naive T helper cells about invading pathogens. DC undergo sequential events leading to irreversible maturation upon bacterial stimulation. To investigate the responses of DC during periodontal infection, we studied the effects of LPS from Porphyromonas gingivalis on DC. DC generated from human peripheral monocytes by culture with IL-4 and GM-CSF were incubated with P. gingivalis LPS (Pg LPS) or Escherichia coli LPS (Ec LPS). Flow cytometry and real-time quantitative RT-PCR analysis revealed that Pg LPS, but not Ec LPS, preferentially up-regulated CD14 and CD16 expression at protein and mRNA levels. Furthermore, Pg LPS preferentially induced the secretion of soluble CD14. CD1a, HLA-DR and CD54 were highly expressed on DC stimulated with both kinds of LPS; however, CD40, CD80, CD83 and CD86 expression on Pg LPS-stimulated DC was lower than on Ec LPS-stimulated DC. With regard to IL-6, IL-8, IL-10, IL-12 and RANTES production from DC and allogeneic T cell proliferation, Pg LPS was a weaker stimulator than Ec LPS. These results suggested that Pg LPS triggers maturation of DC with unique characteristics, which exhibited weak immunostimulatory activity and may contribute to induction of chronic inflammation at the site of periodontal infection.     

Age and prevalence of Porphyromonas gingivalis in children.

The acquisition of Porphyromonas gingivalis was examined in a cross-sectional study of 198 subjects from 0 to 18 years of age using a PCR-based assay. P. gingivalis was detected in the oral cavities of 37% of subjects and at similar frequencies among subjects of all ages. These data indicate that P. gingivalis may be acquired in the first days of life.     

Effects of cholera exotoxin on Fc receptor activity of lymphoid cells and mononuclear phagocytes.

The effect of cholera exotoxin and aminophylline on Fc receptors in a murine lymphoid-cell line and in rabbit pulmonary alveolar macrophages has been investigated. Although both agents elevated intracellular cyclic AMP levels in macrophages and lymphoid cells, the effects on Fc receptor expression were distinct. Cholera toxin at 10 microng/ml reversibly inhibited Fc-receptor activity in the murine lymphoid cell line. In contrast, cholera toxin at 10 microng/ml or 0-01 microng/ml was ineffective in altering pulmonary alveolar macrophage receptor expression. Fc receptor activity on the macrophage was reduced by 20-30 per cent following incubation with aminophylline, (10(-3)M) from 0-6 h. There was no direct correlation between Fc-receptor activity and cyclic AMP levels in the cells studied. The differential susceptibility of these lymphoid and phagocytic cell populations to cholera toxin and also toward aminophylline suggests that there may be fundamental differences in topography on the membrane surface, or in the intracellular regulation of Fc receptors between lymphoid and phagocytic cells.     

Escherichia coli lipopolysaccharides diminish and enhance cell function of human polymorphonuclear leukocytes.

The effects of the lipopolysaccharide (LPS) of Escherichia coli J5 and 0111B4 on the function of human polymorphonuclear leukocytes (PMN) were tested. E. coli J5 is a UDP-galactose-4-epimerase-deficient mutant of E. coli 0111B4, and its LPS, therefore, contains mainly lipid A, as it lacks the polysaccharide side chains. PMN which had been incubated with J5 LPS showed decreased phagocytic, chemotactic, and metabolic activities as compared with control PMN. In contrast, incubation of PMN with 0111B4 LPS had no effect or even an enhancing effect on PMN function. When lipid A and the polysaccharide fraction were isolated from 0111B4 LPS, it was shown that lipid A had the same deleterious effect on PMN function as did J5 LPS and that the LPS fraction had no effect. When PMN were incubated with J5 LPS or lipid A, it could be shown that these structures were able to induce PMN to generate superoxide and chemiluminescence. 0111B4 LPS and the polysaccharide component were able to generate a metabolic burst by the PMN to a lesser extent. The induced defects in PMN function by J5 LPS could be prevented when polymyxin B or an oxygen-radical scavenger was present. We hypothesize that the lipid A portion of LPS is toxic for PMN due to the induction of toxic oxygen species by the PMN. These toxic oxygen species destroy the phagocytic, chemotactic, and metabolic activities of the PMN.     

Kinetics of mononuclear phagocytes after thymectomy.

Some kinetic parameters of mononuclear phagocytes have been studied. Wistar rats were injected with 3H-thymidine. Samples of peripheral blood and peritoneal macrophages were obtained at 12, 14, 24, 36, 48, 60, and 84 hours respectively after injection. The labelling index and the mean grain count were determined. The turnover of monocytes was slowed down in the thymectomized animals. It is assumed that thymectomy had changed some biological qualities of mononuclear phagocytes and/or the humoral relations connected with the function and the regulation of mononuclear phagocytes.