Porphyromonas gingivalis invasion of gingival epithelial cells.

Porphyromonas gingivalis, a periodontal pathogen, can invade primary cultures of gingival epithelial cells. Optimal invasion occurred at a relatively low multiplicity of infection (i.e., 100) and demonstrated saturation at a higher multiplicity of infection. Following the lag phase, during which bacteria invaded poorly, invasion was independent of growth phase. P. gingivalis was capable of replicating within the epithelial cells. Invasion was an active process requiring both bacterial and epithelial cell energy production. Invasion was sensitive to inhibitors of microfilaments and microtubules, demonstrating that epithelial cell cytoskeletal rearrangements are involved in bacterial entry. P. gingivalis, but not epithelial cell, protein synthesis was necessary for invasion. Invasion within the epithelial cells was not blocked by inhibitors of protein kinase activity. Invasion was inhibited by protease inhibitors, suggesting that P. gingivalis proteases may be involved in the invasion process. Low-passage clinical isolates of P. gingivalis invaded with higher efficiency than the type strain. Serum inhibited invasion of the type strain but had no effect on the invasion of a clinical isolate. Invasion of gingival epithelial cells by P. gingivalis may contribute to the pathology of periodontal diseases.     

Invasion of Aortic and Heart Endothelial Cells by Porphyromonas gingivalis

Invasion of host cells is believed to be an important strategy utilized by a number of pathogens, which affords them protection from the host immune system. The connective tissues of the periodontium are extremely well vascularized, which allows invading microorganisms, such as the periodontal pathogen Porphyromonas gingivalis, to readily enter the bloodstream. However, the ability of P. gingivalis to actively invade endothelial cells has not been previously examined. In this study, we demonstrate that P. gingivalis can invade bovine and human endothelial cells as assessed by an antibiotic protection assay and by transmission and scanning electron microscopy. P. gingivalis A7436 was demonstrated to adhere to and to invade fetal bovine heart endothelial cells (FBHEC), bovine aortic endothelial cells (BAEC), and human umbilical vein endothelial cells (HUVEC). Invasion efficiencies of 0.1, 0.2, and 0.3% were obtained with BAEC, HUVEC, and FBHEC, respectively. Invasion of FBHEC and BAEC by P. gingivalis A7436 assessed by electron microscopy revealed the formation of microvillus-like extensions around adherent bacteria followed by the engulfment of the pathogen within vacuoles. Invasion of BAEC by P. gingivalis A7436 was inhibited by cytochalasin D, nocodazole, staurosporine, protease inhibitors, and sodium azide, indicating that cytoskeletal rearrangements, protein phosphorylation, energy metabolism, and P. gingivalis proteases are essential for invasion. In contrast, addition of rifampin, nalidixic acid, and chloramphenicol had little effect on invasion, indicating that bacterial RNA, DNA, and de novo protein synthesis are not required for P. gingivalis invasion of endothelial cells. Likewise de novo protein synthesis by endothelial cells was not required for invasion by P. gingivalis. P. gingivalis 381 was demonstrated to adhere to and to invade BAEC (0.11 and 0.1% efficiency, respectively). However, adherence and invasion of the corresponding fimA mutant DPG3, which lacks the major fimbriae, was not detected. These results indicate that P. gingivalis can actively invade endothelial cells and that fimbriae are required for this process. P. gingivalis invasion of endothelial cells may represent another strategy utilized by this pathogen to thwart the host immune response.     

Uptake pathways of clinical isolates ofProteus mirabilisinto human epithelial cell lines

Proteus mirabilisisolates obtained from urine and faeces showed high invasion levels into several human epithelial cell lines in gentamicin assays. Invasion efficiencies of isolate 102 from a monkey with diarrhoea equalled or even exceeded those ofSalmonella typhistrain Ty2 (6.3 to 13.8% of the inoculum). Vegetative, non-swarmingP. mirabilisinvaded epithelial cells efficiently and were found in endosomes and free in the cytoplasm. Although inhibition of eukaryotic protein synthesis by cycloheximide did not reduce bacterial uptake, inhibition with bacteriostatic antibiotics of bacterial protein-, RNA-, or DNA-synthesis reduced invasion drastically. Involvement of eukaryotic structures and processes in internalization was determined by using various inhibitors in the invasion assay. Uptake ofP. mirabilisisolated from urine into gut (INT407, HCT-8) cells and bladder (T24) cells was dramatically inhibited only by microfilament depolymerization. Internalization of faecal isolate 102 into gut or bladder epithelial cells was inhibited by depolymerization of microfilaments or microtubules. Engulfment of isolate 102 into T24 bladder cells was also reduced by inhibition of receptor-mediated endocytosis. Interference with endosome acidification decreased the number of intracellular bacteria of isolate 102 in all three cell lines. These results suggest thatP. mirabilisisolates from different sources are internalized by epithelial cells by different eukaryotic processes, and that these processes can vary between cell lines.     

A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells.

Isogenic mutants of Porphyromonas gingivalis which differ in the expression of fimbriae were used to examine the contribution of fimbriae in invasion of a human oral epithelial cell line (KB). At a multiplicity of infection of 100, the wild-type P. gingivalis strains 33277, 381, and A7436 exhibited adherence efficiencies of 5.5, 0.11, and 5.0%, respectively, and invasion efficiencies of 0.15, 0.03, and 0.10%, respectively. However, adherence to and invasion of KB cells was not detected with the P. gingivalis fimA mutants, DPG3 and MPG1. Adherence of P. gingivalis wild-type strains to KB cells was completely inhibited by the addition of hyperimmune sera raised to the major fimbriae. Examination by electron microscopy of invasion of epithelial cells by the P. gingivalis wild-type strain 381 revealed microvillus-like extensions around adherent bacteria; this was not observed with P. gingivalis fim mutants. Taken together, these results indicate that the P. gingivalis major fimbriae are required for adherence to and invasion of oral epithelial cells.     

Salmonella enterica serovars gallinarum and pullorum expressing Salmonella enterica serovar typhimurium type 1 fimbriae exhibit increased invasiveness for mammalian cells

Salmonella enterica serovars Gallinarum and Pullorum are S. enterica biotypes that exhibit host specificity for poultry and aquatic birds and are not normally capable of causing disease in mammalian hosts. During their evolution toward host restriction serovars Gallinarum and Pullorum lost their ability to mediate mannose-sensitive hemagglutination (MSHA), a phenotype correlated with adherence to certain cell types. Because adherence is an essential requirement for invasion of cells by bacterial pathogens, we examined whether MHSA type 1 fimbriae would increase the ability of serovars Pullorum and Gallinarum to invade normally restrictive cells. Serovars Gallinarum and Pullorum expressing S. enterica serovar Typhimurium strain LT2 type 1 fimbriae exhibited a 10- to 20-fold increased ability to adhere to and a 20- to 60-fold increased invasion efficiency of the human epithelial HEp-2 cell line. Invasion was accompanied by extensive ruffling of the membranes of the HEp-2 cells. In a murine ligated ileal loop model, a 32% increase in the number of M-cell ruffles was seen when serovar Gallinarum expressed serovar Typhimurium type 1 fimbriae.     

Interaction of Prevotella intermedia Strain 17 Leucine-Rich Repeat Domain Protein AdpF with Eukaryotic Cells Promotes Bacterial Internalization

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.     

Capsule and fimbriae modulate the invasion of Haemophilus influenzae in a human blood-cerebrospinal fluid barrier model

The Gram-negative bacterium Haemophilus influenzae (H. influenzae) can commensally colonize the upper respiratory tract, but also cause life threatening disease including epiglottitis, sepsis and meningitis. The H. influenzae capsule protects the bacteria against both phagocytosis and opsonization. Encapsulated H. influenzae strains are classified into serotypes ranging from a to f dependent on their distinct polysaccharide capsule. Due to the implementation of vaccination the incidence of invasive H. influenzae type b (Hib) infections has strongly decreased and infections with other capsulated types, including H. influenzae type f (Hif), are emerging. The pathogenesis of H. influenzae meningitis is not clarified. To enter the central nervous system (CNS) the bacteria generally have to cross either the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BSCFB). Using a cell culture model of the BCSFB based on human choroid plexus papilloma (HIBCPP) cells and different H. influenzae strains we investigated whether Hib and Hif invade the cells, and if invasion differs between encapsulated vs. capsular-deficient and fimbriated vs. non-fimbriated variants. We find that Hib can adhere to and invade into HIBCPP cells. Invasion occurs in a strongly polar fashion, since the bacteria enter the cells preferentially from the basolateral “blood “side. Fimbriae and capsule attenuate invasion into choroid plexus (CP) epithelial cells, and capsulation can influence the bacterial distribution pattern. Finally, analysis of clinical Hib and Hif isolates confirms the detected invasive properties of H. influenzae. Our data point to roles of capsule and fimbriae during invasion of CP epithelial cells.     

AdpC Is a Prevotella intermedia 17 Leucine-Rich Repeat Internalin-Like Protein

The oral bacterium Prevotella intermedia attaches to and invades gingival epithelial cells, fibroblasts, and endothelial cells. Several genes encoding proteins that mediate both the adhesion and invasion processes are carried on the genome of this bacterium. Here, we characterized one such protein, AdpC, belonging to the leucine-rich repeat (LRR) protein family. Bioinformatics analysis revealed that this protein shares similarity with the Treponema pallidum LRR (LRR_TP) family of proteins and contains six LRRs. Despite the absence of a signal peptide, this protein is localized on the bacterial outer membrane, indicating that it is transported through an atypical secretion mechanism. The recombinant form of this protein (rAdpC) was shown to bind fibrinogen. In addition, the heterologous host strain Escherichia coli BL21 expressing rAdpC (V2846) invaded fibroblast NIH 3T3 cells at a 40-fold-higher frequency than control E. coli BL21 cells expressing a sham P. intermedia 17 protein. Although similar results were obtained by using human umbilical vein endothelial cells (HUVECs), only a 3-fold-increased invasion of V2846 into oral epithelial HN4 cells was observed. Thus, AdpC-mediated invasion is cell specific. This work demonstrated that AdpC is an important invasin protein of P. intermedia 17.Prevotella intermedia, a black-pigmented, Gram-negative, anaerobic bacterium, is associated with periodontal disease. Lopez previously found a high prevalence of Porphyromonas gingivalis and P. intermedia in adult periodontitis lesions (31). Although P. intermedia is also found at healthy sites (30, 44), the profile of degradative enzymes produced by the organism varies depending on the site at which it is present (33). This suggests that under certain conditions, this organism alters its enzyme profile to become more virulent, which promotes the progression of periodontitis. In addition to periodontitis, P. intermedia has also been shown to be associated with endodontic infections such as root canal infection, apical periodontitis, and periapical lesions. In fact, Baumgartner et al. showed previously that 36% of samples from endodontic infections contain P. intermedia (1). Also, P. intermedia has been found in extraoral sites in bacterial tracheitis in children (7) and cancrum oris (also known as noma, an infection that destroys orofacial tissues) lesions (17, 18). Finally, previous studies suggested that chronic infections, including those associated with periodontitis, increase the risk of systemic diseases such as coronary heart disease and preterm delivery of low-birth-weight infants. Haraszthy et al. previously found periodontopathogens, including P. intermedia, in atherosclerotic plaques (22). In addition, Madianos et al. (32) previously demonstrated a significantly higher prevalence of positive fetal IgM to P. intermedia for preterm infants than for full-term infants.P. intermedia invades a variety of nonphagocytic eukaryotic cells (16). The ability to invade these cells affords the bacteria access to a nutritionally rich environment as well as an escape from host immune defenses. The type C fimbriae have been shown to play a major role in the invasion process of epithelial cells, as the anti-type C fimbria antibody inhibited invasion (16). However, we postulated that other proteins mediating the attachment and invasion processes of P. intermedia remain unknown. The availability of the genomic sequence of P. intermedia 17 allowed us to apply genomic approaches to identify and characterize such candidates. Using proteomic approaches, we have identified and characterized the surface protein AdpB, which binds a variety of host extracellular matrix (ECM) components (47). Furthermore, analysis of the P. intermedia 17 genome sequence has revealed the presence of multiple genes encoding proteins with similarity to the leucine-rich repeat (LRR) family of proteins (27). These proteins have been demonstrated to be present in a variety of organisms, including viruses, bacteria, archaea, and eukaryotes, and have been shown to play a role in the immune response, apoptosis, adhesion, invasion, signal transduction, and DNA/RNA processing (5, 27). Thus, we reasoned that the P. intermedia 17 LRR-like proteins play a role in the attachment and invasion of the bacterium into host cells as well. Here, we report one such P. intermedia 17 protein, AdpC, encoded by PI0136 (www.oralgen.lanl.gov), that confers an invasive phenotype to Escherichia coli cells expressing the protein.     

Pseudomonas aeruginosa invasion of and multiplication within corneal epithelial cells in vitro.

Pseudomonas aeruginosa is usually considered an extracellular pathogen. Using assays to determine intracellular survival in the presence of gentamicin, we have previously demonstrated that P. aeruginosa is able to invade corneal cells during infectious keratitis in mice. In vitro, P. aeruginosa was found to enter the following cells: human corneal cells removed by irrigation; epithelial cells in the cornea of rats, mice, and rabbits; and primary corneal epithelial cells cultured from rat and rabbit eyes. The level of invasion was related to the level of adherent or associated bacteria. In general, invasion was more efficient with cultured epithelial cells than with cells tested in situ. Invasion did not occur when assays were performed at 4 degrees C. Cytochalasin D but not colchicine inhibited bacterial invasion, suggesting that bacterial entry was an endocytic process dependent on actin microfilaments but not microtubules. Bacteria that invaded cultured corneal epithelial cells were found to multiply within cells. The ability of P. aeruginosa to invade and multiply within corneal epithelial cells may contribute to the virulence of this organism during infectious keratitis, since intracellular bacteria can evade host immune effectors and antibiotics commonly used to treat infection.     

Effect of type III group B streptococcal capsular polysaccharide on invasion of respiratory epithelial cells.

Group B streptococcal (GBS) capsular polysaccharide is an important virulence factor, and its role in invasion of cultured respiratory epithelial cells was investigated. A type III GBS clinical isolate, COH1, and asialo and unencapsulated isogenic transposon capsule mutants of it were compared in an in vitro invasion assay. The results demonstrated that capsule attenuated the invasion process. Invasion was not affected when the A549 epithelial cells were preincubated with purified type III GBS capsular polysaccharide. Polyclonal type III GBS capsule antibody inhibited invasion by COH1 but did not affect invasion by the capsule mutants. Serotypes Ia, Ib, Ia/c, II, and III all invaded respiratory epithelial cells but demonstrated some strain variation in magnitude of invasion. These observations led us to conclude that type III capsular polysaccharide was not essential for invasion of respiratory epithelial cells by GBS and that bacterial factors other than capsule were responsible for respiratory epithelial cell invasion.     

Differential ability of periodontopathic bacteria to modulate invasion of human gingival epithelial cells by Porphyromonas gingivalis

Periodontitis is a polymicrobial infection caused by selected gram-negative bacteria including Porphyromonas gingivalis. Host cell invasion by P. gingivalis has been proposed as a possible mechanism of pathogenesis in periodontitis. The aim of the present study was to assess the influence of periodontopathogens on P. gingivalis invasion of gingival epithelial cells in polymicrobial infection. P. gingivalis was tested for its ability to invade a human gingival epithelial cell line Ca9-22 in co-infection with periodontopathogens, using an antibiotic protection assay. Among the pathogens tested, only Fusobacterium nucleatum demonstrated the ability to significantly promote P. gingivalis invasion (P < 0.01). This increased invasion was confirmed by confocal scanning laser microscopy utilizing a dual labeling technique. In contrast, co-infection with Aggregatibacter actinomycetemcomitans or Tannerella forsythia attenuated P. gingivalis invasion. The fusobacterial enhancement of host cell invasion was not observed in co-incubation with other periodontopathogens tested. These results suggested that complex synergistic or antagonistic physiologic mechanisms are intimately involved in host cell invasion by P. gingivalis in polymicrobial infection.     

Cronobacter spp. (previously Enterobacter sakazakii) invade and translocate across both cultured human intestinal epithelial cells and human brain microvascular endothelial cells

The mechanism of Cronobacter pathogenesis in neonatal meningitis and potential virulence factors (aside from host cell invasion ability) remain largely unknown. To ascertain whether Cronobacter can invade and transcytose across intestinal epithelial cells, enter into the blood stream and then transcytose across the blood-brain-barrier, we have utilized human intestinal INT407 and Caco-2 cells and brain microvascular endothelial cell (HBMEC) monolayers on Transwell filters as experimental model systems. Our data indicate a wide range of heterogeneity with respect to invasion efficiency among twenty-three Cronobacter isolates screened. For selected isolates, we observed significant levels of transcytosis for Cronobacter sakazakii across tight monolayers of both Caco-2 and HBMEC, mimicking in vivo ability to cross the intestine as well as the blood brain barrier, and at a frequency equivalent to that of a control meningitis-causing Escherichia coli K1 strain. Finally, EM analysis demonstrated intracellular Cronobacter bacteria within host vacuoles in HBMEC, as well as transcytosed bacteria at the basolateral surface. These data reveal that certain Cronobacter isolates can invade and translocate across both cultured human intestinal epithelial cells and HBMEC, thus demonstrating a potential path for neonatal infections of the central nervous system (CNS) following oral ingestion.     

Invasion of HeLa cells by Bordetella bronchiseptica

Invasion, defined as adhesion to, followed by entrance into HeLa cells by Bordetella bronchiseptica was determined by (i) specific staining of intracellular bacteria and (ii) counting of viable intracellular bacteria after killing extracellular bacteria with colistin. It was demonstrated for the first time that B. bronchiseptica, like Bordetella pertussis and Bordetella parapertussis , is able to invade HeLa cells. Comparison of the invasiveness of Bvg⁺ and Bvg^-B. bronchiseptica showed that B. bronchiseptica, in contrast to B. pertussis, invaded HeLa cells in both phases. The number of viable intracellular bacteria isolated after invasion of bacteria in the Bvg^- (flagellated) phase was ten-fold lower than when Bvg⁺ (fimbriated) bacteria were used. Strains which are deficient in the production of either FimX or Fim2 fimbriae were as invasive as wild-type B. bronchiseptica , which indicates that not these fimbriae but probably filamentous haemagglutinin (FHA) is the major adhesin of B. bronchiseptica.     

Oral bacteria modulate invasion and induction of apoptosis in HEp-2 cells by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic bacterial pathogen, causing infections of the respiratory and other organ systems in susceptible hosts. P. aeruginosa infection is initiated by adhesion to and invasion of mucosal epithelial cells. The failure of host defenses to eliminate P. aeruginosa from mucosal surfaces results in P. aeruginosa proliferation, sometimes followed by overt infection and tissue destruction. There is growing evidence that associates poor oral health and respiratory infection. An in vitro model system for bacterial invasion of respiratory epithelial cells was used to investigate the influence of oral bacteria on P. aeruginosa epithelial cell invasion. Oral pathogens including Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter (Actinobacillus) actinomycetemcomitans increased invasion of P. aeruginosa into HEp-2 cells from one- to threefold. In contrast, non-pathogenic oral bacteria such as Actinomyces naeslundii and Streptococcus gordonii showed no significant influence on P. aeruginosa invasion. P. aeruginosa together with oral bacteria stimulated greater cytokine production from HEp-2 cells than did P. aeruginosa alone. P. aeruginosa in combination with periodontal pathogens also increased apoptosis of HEp-2 cells and induced elevated caspase-3 activity. These results suggest that oral bacteria, especially periodontal pathogens, may foster P. aeruginosa invasion into respiratory epithelial cells to enhance host cell cytokine release and apoptosis.     

Differential ability of periodontopathic bacteria to modulate invasion of human gingival epithelial cells by Porphyromonas gingivalis

Periodontitis is a polymicrobial infection caused by selected gram-negative bacteria including Porphyromonas gingivalis. Host cell invasion by P. gingivalis has been proposed as a possible mechanism of pathogenesis in periodontitis. The aim of the present study was to assess the influence of periodontopathogens on P. gingivalis invasion of gingival epithelial cells in polymicrobial infection. P. gingivalis was tested for its ability to invade a human gingival epithelial cell line Ca9-22 in co-infection with periodontopathogens, using an antibiotic protection assay. Among the pathogens tested, only Fusobacterium nucleatum demonstrated the ability to significantly promote P. gingivalis invasion (P < 0.01). This increased invasion was confirmed by confocal scanning laser microscopy utilizing a dual labeling technique. In contrast, co-infection with Aggregatibacter actinomycetemcomitans or Tannerella forsythia attenuated P. gingivalis invasion. The fusobacterial enhancement of host cell invasion was not observed in co-incubation with other periodontopathogens tested. These results suggested that complex synergistic or antagonistic physiologic mechanisms are intimately involved in host cell invasion by P. gingivalis in polymicrobial infection.     

Molecular cloning of epithelial cell invasion determinants from enterotoxigenic Escherichia coli.

Although penetration of the epithelial mucosa has not been identified as a virulence mechanism in enterotoxigenic Escherichia coli (ETEC), we have found that this pathogen is capable of invading human intestinal cell lines. Classical ETEC strain H10407 was most invasive for epithelial cell lines derived from ileocecal and colonic tissues. An ETEC cosmid library was screened for clones that could direct E. coli HB101 to invade cultured human ileocecal epithelial cells (HCT 8 cells). Three invasive recombinant cosmid clones were isolated. These cosmids could direct HB101 invasion at an efficiency that was equal to or greater than that of the parent ETEC strain. The invasion cosmids also allowed for enhanced HCT 8 cell adherence by HB101. Electron micrographs of ETEC and recombinant HB101 strains revealed intracellular bacteria contained within endocytic vacuoles. Restriction endonuclease mapping and hybridization analyses showed that the three ETEC clones represent two separate invasion systems present in the parent ETEC strain and that both systems are chromosomally encoded. The parent ETEC strain and one cloned invasion system did not invade HeLa cells. Interestingly, one cloned invasion system was capable of directing HB101 to invade HeLa cells. Invasion of HCT 8 cells by recombinant HB101 strains and the parent ETEC strain was inhibited by cytochalasin D, indicating that the wild-type and both cloned invasion systems trigger an actin polymerization-dependent uptake process. It is not known whether the invasive phenotype of ETEC is relevant for enterotoxigenic disease. However, the parent ETEC strain, as well as recombinant HB101 strains, was capable of transcytosis through polarized HCT 8 monolayers. This transcytosis suggests that ETEC may cross the gut epithelium in vivo and that this invasion may have a previously unrecognized role in the disease process.     

Enzyme-linked immunosorbent assay for adherence of bacteria to animal cells.

Epithelial cells scraped from human oral mucosa and from pig intestines were immobilized onto the flat bottom surfaces of microtiter plates to study the adherence of various bacterial species to host cells. Bacterial adherence was quantitated either by an enzyme-linked immunosorbent assay technique with specific antibacterial serum as the first antibody followed by peroxidase-conjugated second antibody or by using biotinylated bacteria and avidin-peroxidase as the detecting agent. Unlabeled Escherichia coli and purified E. coli 987P fimbriae inhibited the adherence of biotinylated E. coli to immobilized enterocytes. The adherence of a mannose-sensitive strain of E. coli to immobilized oral epithelial cells was inhibited by mannose derivatives. The adherence of fimbriated E. coli 987P to immobilized enterocytes was approximately four times higher than the adherence of a nonfimbriated variant of the same strain. The adherence of Streptococcus pyogenes to oral cells was detected in the range of 10 to 150 bacteria per cell and was inhibited by lipoteichoic acid and albumin. The data suggest that the putative receptors which bind bacteria on the immobilized cells retain a functional form similar to that of native cells in suspension. The proposed adherence assay is easy to perform, allows the detection of specific adherence of test bacteria, and provides objective quantitation of adherence with a sensitivity of 10 bacteria per cell. Most importantly, the assay allows the testing of many variables in the same day.     

Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells

Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, including Bacteroides forsythus, Campylobacter curvus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatum were also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a "zipping" mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections.     

Functional differences among FimA variants of Porphyromonas gingivalis and their effects on adhesion to and invasion of human epithelial cells.

Fimbriae of Porphyromonas gingivalis, a periodontopathogen, play an important role in its adhesion to and invasion of host cells. The fimA genes encoding fimbrillin (FimA), a subunit protein of fimbriae, have been classified into five types, types I to V, based on nucleotide sequences. We previously reported that P. gingivalis with type II fimA was strongly associated with adult periodontitis. In the present study, we compared the abilities of recombinant FimA (rFimA) types I to V to adhere to and invade human gingival fibroblasts (HGF) and a human epithelial cell line (HEp-2 cells) by using rFimA-conjugated microspheres (rFimA-MS). There were no significant differences in the abilities of the rFimA-MS to adhere to HGF; however, the adhesion of type II rFimA-MS to HEp-2 cells was significantly greater than those of other types of rFimA-MS. We also observed that type II rFimA-MS invaded epithelial cells and accumulated around the nuclei. These adhesion and invasion characteristics were eliminated by the addition of antibodies to type II rFimA and alpha5beta1-integrin. In contrast, Arg-Gly-Asp-Ser peptide and a synthetic peptide of proline-rich protein C had negligible inhibitory effects. Furthermore, P. gingivalis strain HW24D1 with type II fimA adhered to cells and invaded them more than strains with other fimA genotypes. These results suggest that type II FimA can bind to epithelial cells most efficiently through specific host receptors.     

Invasion and intracellular survival of Burkholderia cepacia

Burkholderia cepacia has emerged as an important pulmonary pathogen in immunocompromised patients and in patients with cystic fibrosis (CF). Little is known about the virulence factors and pathogenesis of B. cepacia, although the persistent and sometimes invasive infections caused by B. cepacia suggest that the organism possesses mechanisms for both cellular invasion and evasion of the host immune response. In this study, cultured human cells were used to analyze the invasion and intracellular survival of B. cepacia J2315, a highly transmissible clinical isolate responsible for morbidity and mortality in CF patients. Quantitative invasion and intracellular growth assays demonstrated that B. cepacia J2315 was able to enter, survive, and replicate intracellularly in U937-derived macrophages and A549 pulmonary epithelial cells. Transmission electron microscopy of infected macrophages confirmed the presence of intracellular B. cepacia and showed that intracellular bacteria were contained within membrane-bound vacuoles. An environmental isolate of B. cepacia, strain J2540, was also examined for its ability to invade and survive intracellularly in cultured human cells. J2540 entered cultured macrophages with an invasion frequency similar to that of the clinical strain, but it was less invasive than the clinical strain in epithelial cells. In marked contrast to the clinical strain, the environmental isolate was unable to survive or replicate intracellularly in either cultured macrophages or epithelial cells. Invasion and intracellular survival may play important roles in the ability of virulent strains of B. cepacia to evade the host immune response and cause persistent infections in CF patients.