Intestine-407 Cells Research Articles

P719 Identification of differentially expressed genes between AIEC and non-AIEC clinical isolates during in vitro cell infection

Abstract Background Adherent invasive Escherichia coli (AIEC) have been related to Crohn’s disease. Phenotypic assays of adhesion and invasion of intestinal epithelial cells and intra-macrophage replication are used to identify AIEC. No molecular tools are still available for AIEC identification. We hypothesise that differential gene expression may drive to the AIEC phenotype. To test this hypothesis we sequenced the genes expressed by AIEC and non-AIEC strains during infection, and we investigated differential expressed genes (DEGs) suitability as molecular markers. Methods Comparative transcriptomics was performed between two AIEC/non-AIEC strain pairs, belonging to the phylogroups B1 and D, during Intestine-407 cells infection. Each strain pair displayed identical pulsotypes and similar genomes. Supernatant fractions of the infected cell cultures (SN) and eukaryotic cells containing adhered and/or intracellular bacteria (A/I) were considered separately. DEGs obtained were quantified by RT-qPCR in the same samples to confirm the results, and in a strain collection of 13 AIEC and 23 non-AIEC to investigate their suitability as molecular markers. Binary logistical regression was performed to identify DEGs whose quantification could be used as AIEC biomarker. Results Comparative transcriptomics revealed 67 DEGs between the two phenotypes in the strain pairs (19 over-expressed in AIEC, and 48 under-expressed), 51 of which (82.26%) were corroborated by RT-qPCR. When explored in the whole strain collection, 29 DEGs were found to be differentially expressed between AIEC and non-AIEC phenotypes (p < 0.042), and 42 genes between SN and A/I fractions (p < 0.049). Notably, six DEGs detected in the strain collection were implicated in a metabolic pathway that could be involved in acid resistance, and eight in the synthesis of two virulence determinants. Finally, binary logistical regression revealed three DEGs able to predict the AIEC phenotype with an accuracy of ≥ 85%. Conclusion No previous comparative transcriptomic studies have been performed using AIEC-infected cell cultures. Our research revealed three genes, whose quantification presents higher sensitivities and specificities than other molecular markers previously suggested. Moreover, we have identified functionally related genes that can be involved in AIEC pathogenicity. These results open the door to further research to confirm the suitability of these genes as AIEC biomarkers and to investigate new therapeutic targets against AIEC colonisation.

Amino Acid Substitutions and Differential Gene Expression of Outer Membrane Proteins in Adherent-Invasive Escherichia coli.

Variations in the sequence and/or the expression of outer membrane proteins (OMPs) may modulate bacterial virulence. OmpA and OmpC have been involved in the interaction of adherent-invasive Escherichia coli (AIEC) strain LF82 with intestinal epithelial cells (IECs). Scarce data exist about OMPs sequence variants in a collection of AIEC strains, and no study of OMPs expression during infection exists. We aimed to determine whether particular mutations or differential expression of OMPs are associated with AIEC virulence. The ompA, ompC, and ompF genes in 14 AIEC and 30 non-AIEC strains were sequenced by Sanger method, and the protein expression profile was analyzed by urea-SDS-PAGE. Gene expression was determined during in vitro bacterial infection of intestine-407 cells by RT-qPCR. The distribution of amino acid substitutions in OmpA-A200V, OmpC-S89N, V220I, and W231D associated with pathotype and specific changes (OmpA-A200V, OmpC-V220I, D232A, OmpF-E51V, and M60K) correlated with adhesion and/or invasion indices but no particular variants were found specific of AIEC. OMPs protein levels did not differ according to pathotype when growing in Mueller-Hinton broth. Interestingly, higher OMPs gene expression levels were reported in non-AIEC growing in association with cells compared with those non-AIEC strains growing in the supernatants of infected cultures (p < 0.028), whereas in AIEC strains ompA expression was the only increased when growing in association with cells (p = 0.032), but they did not significantly alter ompC and ompF expression under this condition (p > 0.146). Despite no particular OMPs sequence variants have been found as a common and distinctive trait in AIEC, some mutations could facilitate a better interaction with the host. Moreover, the different behavior between pathotypes regarding OMPs gene expression at different stages of infection could be related with the virulence of the strains.

Complete genome sequence of Crohn's disease-associated adherent-invasive E. coli strain LF82.

BackgroundIleal lesions of Crohn's disease (CD) patients are abnormally colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells and macrophages.Principal FindingsWe report here the complete genome sequence of E. coli LF82, the reference strain of adherent-invasive E. coli associated with ileal Crohn's disease. The LF82 genome of 4,881,487 bp total size contains a circular chromosome with a size of 4,773,108 bp and a plasmid of 108,379 bp. The analysis of predicted coding sequences (CDSs) within the LF82 flexible genome indicated that this genome is close to the avian pathogenic strain APEC_01, meningitis-associated strain S88 and urinary-isolated strain UTI89 with regards to flexible genome and single nucleotide polymorphisms in various virulence factors. Interestingly, we observed that strains LF82 and UTI89 adhered at a similar level to Intestine-407 cells and that like LF82, APEC_01 and UTI89 were highly invasive. However, A1EC strain LF82 had an intermediate killer phenotype compared to APEC-01 and UTI89 and the LF82 genome does not harbour most of specific virulence genes from ExPEC. LF82 genome has evolved from those of ExPEC B2 strains by the acquisition of Salmonella and Yersinia isolated or clustered genes or CDSs located on pLF82 plasmid and at various loci on the chromosome.ConclusionLF82 genome analysis indicated that a number of genes, gene clusters and pathoadaptative mutations which have been acquired may play a role in virulence of AIEC strain LF82.

Abnormally expressed ER stress response chaperone Gp96 in CD favours adherent-invasive Escherichia coli invasion

Background and aimsCrohn's disease (CD) ileal lesions are colonised by pathogenic adherent-invasive Escherichia coli (AIEC) producing outer membrane vesicles (OMVs) that contribute to the bacterial invasion process. In addition, increased...

Molecular diversity of Escherichia coli in the human gut: New ecological evidence supporting the role of adherent-invasive E. coli (AIEC) in Crohnʼs disease

Escherichia coli, particularly the adherent-invasive E. coli (AIEC) pathovar, has been increasingly implicated in the ethiopathogenesis of Crohn's disease (CD). We describe the richness, abundance, diversity, and pathogenic features of E. coli and AIEC strains that colonize the intestinal mucosa. Approximately 100 E. coli colonies per biopsy from 20 CD patients (18 biopsies from colon and 23 from ileum) and 28 healthy controls (C) (25, colon; 27, ileum) were isolated. Repetitive extragenic palindrome-polymerase chain reaction (Rep-PCR) and pulsed field gel electrophoresis (PFGE) were used to analyze the clonality of isolates. For AIEC identification, adhesion and invasion assays were performed over Intestine-407 cells, and the capacity to survive and replicate intracellularly was determined over macrophages J774. The serotypes, phylotypes, and genotypes (19 virulence genes) of strains were also investigated. Mucosa-associated E. coli richness (E. coli subtypes/patient: C = 2.0 +/- 1.0; CD = 2.1 +/- 1.3) and diversity (Shannon Index: H'(C): 2.1 +/- 0.6; H'(CD): 2.5 +/- 0.8) were similar between CD and C, but higher E. coli counts were characteristic of CD patients (P = 0.010), particularly those with Crohn's ileitis (P = 0.001). Host-specific pulsotypes shared virulence features of ExPEC at similar frequencies between CD and C, except for iucD, which was more prevalent in E. coli from controls (C: 75%, CD: 40%, P = 0.027). In contrast, greater AIEC prevalence (% subjects with AIEC: CD = 51.9%; C = 16.7%; P = 0.003), abundance (% AIEC/E. coli: CD = 3.8 +/- 5.0%; C = 1.5 +/- 3.8%; P = 0.039), and richness (number of AIEC subtypes: CD = 0.8 +/- 1.4; C = 0.2 +/- 0.4; P = 0.015) of E. coli strains belonging to the AIEC pathovar was observed for CD patients. AIEC subtypes showed a high variability of seropathotypes and pulsotypes, although the B2 phylogroup was the most prevalent (AIEC: 64%, non-AIEC: 38%, P = 0.044). New data about ecological parameters of AIEC reinforces the implication of AIEC in CD.

OmpC and the σE regulatory pathway are involved in adhesion and invasion of the Crohn's disease‐associated Escherichia coli strain LF82

Ileal lesions of 36.4% of patients with Crohn's disease (CD), an inflammatory bowel disease in humans, are colonized by pathogenic adherent-invasive Escherichia coli (AIEC), and high levels of antibodies directed against E. coli OmpC are present in 37-55% of CD patients. We therefore investigated the expression of OmpC and its role in the interaction of CD-associated adherent-invasive E. coli strain LF82 with intestinal epithelial cells. High osmolarity induced a significant increase in the ability of LF82 bacteria to interact with Intestine-407 cells, which correlates with increased OmpC expression. Deletion of ompC gene markedly decreased the adhesion and invasion levels of the corresponding mutant. A LF82-DeltaompR mutant impaired in OmpC and OmpF expression, showed decreased adhesion and invasion, and unlike a K-12-negative OmpR mutant did not express flagella and type 1 pili. Interestingly, the wild-type phenotype was restored when OmpC or OmpF expression was induced in the LF82-DeltaompR mutant. Overexpression of RpoE in the LF82-DeltaompR isogenic mutant restored a full wild-type phenotype without restoring OmpC expression. Increased expression of RpoE was observed in wild-type strain LF82 at high osmolarity. Hence, the role of OmpC in the AIEC LF82 adhesion and invasion is indirect and involves the sigma(E) regulatory pathway.

Hypoxia induced by benign intestinal epithelial cells is associated with cyclooxygenase-2 expression in stromal cells through AP-1-dependent pathway

Cyclooxygenase-2 (COX-2) plays important roles in tumor development. Especially in the early-stage colorectal tumors, COX-2 expression is often observed in the tumor stroma. However, the mechanism regulating such stromal expression of COX-2 remains unknown. In the present study, we simulated the indirect interaction between epithelial cells and stromal cells in the process of colorectal tumor development using an in vitro co-culture model in which NIH3T3 fibroblasts were co-cultured with 'sparsely' or 'densely' populated intestinal epithelial cells, Intestine-407 as a model of premalignant or benign intestinal epithelial cells, and DLD-1 and Caco-2 as models of malignant epithelial cells. COX-2 expression in NIH3T3 fibroblasts was upregulated when co-cultured with the 'dense' epithelial cells regardless of their character. Interestingly, there was pericellular hypoxia in the vicinity of NIH3T3 fibroblasts when co-cultured with 'dense' epithelial cells, and the recovery of the partial pressure of oxygen level resulted in the reduction of enhanced COX-2 expression only in NIH3T3 fibroblasts co-cultured with 'dense' Intestine-407 cells. Furthermore, COX-2 expression was also reduced by the inhibition of transcription factor AP-1. Thus, pericellular hypoxia of the stromal cells caused by densely populated epithelial cells may be one of the potent COX-2 enhancers before completion of malignant transformation during intestinal tumor development.

Lactobacillus caseiDN-114 001 Inhibits the Ability of Adherent-InvasiveEscherichia coliIsolated from Crohn's Disease Patients To Adhere to and To Invade Intestinal Epithelial Cells

Ileal lesions in 36.4% of patients with Crohn's disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli isolated from Crohn's disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in coincubation and 43% to 62% in preincubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli or to a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn's disease.

Clostridium botulinum type A progenitor toxin binds to Intestine-407 cells via N-acetyllactosamine moiety

Botulism is a highly fatal disease caused by the botulinum progenitor toxin. In this study, the role of oligosaccharides for the binding of botulinum type A progenitor toxin (type A PTX) to human intestinal cells was investigated. The binding of type A PTX to Intestine-407 cells was inhibited by the addition of N-acetyllactosamine, lactose, and galactose. Treatment of Intestine-407 cells with neuraminidase led to a significant increase in the binding of type A PTX, while further digestion of cell surface oligosaccharides by β-galactosidase and β- N-acetylhexosaminidase decreased the binding. These results indicate that the N-acetyllactosamine moiety is responsible for the binding of type A PTX. These findings were further confirmed by a binding assay using synthesized oligosaccharides. Interestingly, sialylation or fucosylation of oligosaccharides inhibited the binding of type A PTX. These data suggest that the type A PTX binds to intestinal cells via cell surface N-acetyllactosamine moiety.

Strong Decrease in Invasive Ability and Outer Membrane Vesicle Release in Crohn's Disease-Associated Adherent-Invasive Escherichia coli Strain LF82 with the yfgL Gene Deleted

Adherent-invasive Escherichia coli strain LF82 recovered from a chronic lesion of a patient with Crohn's disease is able to invade cultured intestinal epithelial cells. Three mutants with impaired ability to invade epithelial cells had the Tn5phoA transposon inserted in the yfgL gene encoding the YfgL lipoprotein. A yfgL- negative isogenic mutant showed a marked decrease both in its ability to invade Intestine-407 cells and in the amount of the outer membrane proteins OmpA and OmpC in the culture supernatant, as shown by analysis of the culture supernatant protein contents by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Transcomplementation of the LF82-DeltayfgL isogenic mutant with the cloned yfgL gene restored invasion ability and outer membrane protein release in the culture supernatant. The outer membrane proteins in the culture supernatant of strain LF82 resulted from the formation of vesicles. This was shown by Western blot analysis of periplasmic and outer membrane fraction markers typically found in outer membrane vesicles and by transmission electron microscopic analysis of ultracentrifuged cell-free LF82 supernatant pellets, indicating the presence of vesicles with a bilayered structure surrounding a central electron-dense core. Thus, deletion of the yfgL gene in strain LF82 resulted in a decreased ability to invade intestinal epithelial cells and a decreased release of outer membrane vesicles.

High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn’s disease

Background & Aims: Adherent-invasive Escherichia coli (AIEC) pathovar has been identified in the intestinal mucosa of patients with Crohn’s disease (CD). AIEC reference strain LF82 is able to adhere to intestinal epithelial cells, to invade epithelial cells via a mechanism involving actin polymerization and microtubules, and to survive and replicate within macrophages. This study was performed to assess the prevalence of AIEC associated with intestinal mucosa of patients with CD, ulcerative colitis (UC), and of controls. Methods: A search for E. coli strains was performed with ileal specimens of 63 patients with CD and 16 controls without inflammatory bowel disease (IBD), and with colonic specimens of 27 patients with CD, 8 patients with UC, and 102 controls. The abilities of E. coli strains to invade epithelial cells and to survive and replicate within macrophages were assessed using the gentamicin protection assay. Bacterial uptake by epithelial cells was analyzed using cytoskeletal inhibitors. Bacterial adhesion was quantified with Caco-2 and Intestine-407 cells. The presence of known E. coli virulence genes was assessed by polymerase chain reaction and DNA hybridization. Results: In ileal specimens, AIEC strains were found in 21.7% of CD chronic lesions vs. in 6.2% of controls. In neoterminal ileal specimens, AIEC strains were found in 36.4% of CD early lesions (P = 0.034 vs. controls) and 22.2% of healthy mucosa of CD patients. In colonic specimens, AIEC strains were found in 3.7% of CD patients, 0% of UC patients, and 1.9% of controls. Conclusions: AIEC strains are associated specifically with ileal mucosa in CD.

Involvement of lipoprotein NlpI in the virulence of adherent invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease.

Escherichia coli strain LF82 recovered from a chronic lesion of a patient with Crohn's disease (CD) is able to adhere to and invade cultured intestinal epithelial cells and to replicate within macrophages. One mutant selected for its impaired ability to invade epithelial cells had an insertion of a Tn phoA transposon within the nlpI gene encoding the lipoprotein NlpI. A NlpI-negative isogenic mutant showed a 35-fold decrease in its ability to adhere to and a 45-fold decrease in its ability to invade Intestine-407 cells, but its ability to survive and to replicate within macrophages was similar to that of wild-type strain LF82. In addition, this mutant did not express flagella and synthesized very small amounts of type 1 pili. Downregulation of type 1 pili in the NlpI-negative mutant resulted from a preferential switch toward the OFF position of the invertible DNA element located upstream of the fim operon. The FimB and FimE recombinases act in concert to control the switch, and a large decrease in fimB and fimE mRNA levels was observed. The absence of flagellar structures correlated with a drastic 19-fold decrease in the fliC mRNA level, regardless of the FlhD(2)C(2) transcriptional regulator and of the sigma(28) transcription factor. The key role of NlpI in virulence is independent of type 1 pili and motility, since induced type 1 pilus expression and/or forced contact between bacteria and intestinal epithelial cells did not restore the ability of the NlpI mutant to adhere to and to invade intestinal epithelial cells.

Medium- and Long-Chain Fatty Acids Differentially Modulate Interleukin-8 Secretion in Human Fetal Intestinal Epithelial Cells

The primary therapeutic effects of enteral nutrition in patients with Crohn's disease have been reported previously. Although the quantity and type of fat in enteral nutrition are considered to be important, it is unclear how fat modulates mucosal inflammatory responses in the intestine. In the present study, we evaluated the effects of medium-chain and long-chain fatty acids (MCFA and LCFA) on interleukin (IL)-8 secretion in a fetal intestinal epithelial cell line, intestine-407 cells. IL-8 expression was evaluated at the protein and mRNA levels. The activation of nuclear factor-kappaB was assessed with an electrophoretic gel mobility shift assay. The addition of oleic acid (LCFA) micelles, but not octanoic acid (MCFA) micelles, weakly but significantly enhanced basal IL-8 secretion in the intestine-407 cells. The addition of MCFA (5 mmol/L) induced a 40% increase in IL-1beta-induced IL-8 secretion and a 35% increase in tumor necrosis factor (TNF)-alpha-induced IL-8 secretion, respectively. The addition of LCFA (5 mmol/L) induced a 140% increase in IL-1beta-induced IL-8 secretion and a 110% increase in TNF-alpha-induced IL-8 secretion, respectively. These responses were also observed at the mRNA levels. The electrophoretic gel mobility shift assay indicated that both MCFA and LCFA enhanced IL-1beta- and TNF-alpha-induced nuclear factor-kappaB activation. We demonstrated the proinflammatory activities of MCFA and especially LCFA. It is likely that medium-chain triglycerides may be more suitable than long-chain triglycerides as an energy source in enteral diets in the treatment of patients with Crohn's disease.

Invasive ability of an Escherichia coli strain isolated from the ileal mucosa of a patient with Crohn's disease.

Crohn's disease (CD) is an inflammatory bowel disease in which Escherichia coli strains have been suspected of being involved. We demonstrated previously that ileal lesions of CD are colonized by E. coli strains able to adhere to intestinal Caco-2 cells but devoid of the virulence genes so far described in the pathogenic E. coli strains involved in gastrointestinal infections. In the present study we compared the invasive ability of one of these strains isolated from an ileal biopsy of a patient with CD, strain LF82, with that of reference enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteraggregative (EAggEC), enterohemorrhagic (EHEC), and diffusely adhering (DAEC) E. coli strains. Gentamicin protection assays showed that E. coli LF82 was able to efficiently invade HEp-2 cells. Its invasive level was not significantly different from that of EIEC and EPEC strains (P > 0.5) but significantly higher than that of ETEC (P < 0.03), EHEC (P < 0. 005), EAggEC (P < 0.004) and DAEC (P < 0.02) strains. Strain LF82 also demonstrated efficient ability to invade intestinal epithelial cultured Caco-2, Intestine-407, and HCT-8 cells. Electron microscopy examination of infected HEp-2 cells revealed the presence of numerous intracellular bacteria located in vacuoles or free in the host cell cytoplasm. In addition, the interaction of strain LF82 with epithelial cells was associated with the elongation of microvillar extensions that extruded from the host cell membranes and engulfed the bacteria. This internalization mechanism strongly resembles Salmonella- or Shigella-induced macropinocytosis. The use of cytochalasin D and colchicine showed that the uptake of strain LF82 by HEp-2 cells was mediated by both an actin microfilament-dependent mechanism and microtubule involvement. In addition, strain LF82 survived for at least 24 h in HEp-2 and Intestine-407 cells and efficiently replicated intracellularly in HEp-2 cells. PCR and hybridization experiments did not reveal the presence of any of the genetic determinants encoding EIEC, EPEC, or ETEC proteins involved in bacterial invasion. Thus, these findings show that LF82, which colonized the ileal mucosa of a patient with CD, is a true invasive E. coli strain and suggest the existence of a new potentially pathogenic group of E. coli, which we propose be designated adherent-invasive E. coli.

Novel cytotoxicity test based on menadione-catalyzed H2O2 productivity for food safety evaluation.

Menadione-catalyzed H(2)O(2) production by viable cells was proportional to viable cell number, and the assay of this H(2)O(2) production was applied to the cytotoxicity test of 17 substances which were used for international validation of fixed-dose procedure as an alternative to the classical LD(50) test. The cytotoxicity of substances tested was observed 4 h after the incubation with animal cells, and the viability was determined in 10 min according to menadione-catalyzed H(2)O(2) production assay. IC(50) of each substance required for 50% inhibition of menadione-catalyzed H(2)O(2) production was similar among HepG2, HuH-6KK, HUVE, Vero, Intestine407, NIH/3T3 and Neuro-2a cells. Twelve substances, 3 substances and 2 substances showed the difference of one, two and three orders in the magnitude between LD(50) and IC(50), respectively. These results show that menadione-catalyzed H(2)O(2) production assay is useful for the rapid detection of toxic compounds having the basal cytotoxicity common to various cells, but is unfit for the detection of organ-specific toxic compounds.

Studies on the relationship between adhesive activity and haemagglutination by Helicobacter pylori.

The adhesion of Helicobacter pylori to gastric carcinoma cells (MKN45, KatoIII and MKN28) and Intestine-407 cells was tested by flow cytometric analysis. The mean adhesion rates of H. pylori strains to MKN45, KatoIII and Intestine-407 cells were 90.5, 42.7 and 15.1%, respectively. There was no statistical correlation between the adhesion rates to MKN45 cells and haemagglutination (HA) activity of H. pylori strains, although H. pylori strains with high HA activity with human type O erythrocytes tended to adhere effectively to MKN45 cells. No correlation between adhesion and production of vacuolating toxin was observed.

The central variable V2 region of the CS31A major subunit is involved in the receptor-binding domain.

CS31A is a K88-related capsule-like surface protein that mediates Escherichia coli and Klebsiella pneumoniae adhesion to the human Caco-2 and Intestine-407 cell lines. In this study, we demonstrate that ClpG, the major subunit of CS31A, contains the adhesive domain of the polymerized structure. We mapped this domain within the ClpG protein by performing adhesion inhibition experiments with Intestine-407 cells with nine synthetic peptides (CLP1 to CLP9) covering the dominant antigenic regions of ClpG and with the corresponding rabbit antipeptide antibodies. The peptides CLP1 (amino acid positions in parentheses) (5-18), CLP2 (44-56), CLP3 (82-96), CLP7 (174-190), CLP8 (185-199), and CLP9 (235-249) and corresponding antipeptide antibodies targeting parts of the N- and C-terminal regions of ClpG had no effect on the adhesion of the TCFF15 recombinant strain expressing CS31A. Only the CLP5 (132-146) peptide, corresponding to the central part of the protein, and relevant antibodies inhibited bacterial adhesion to intestinal epithelial cells. Anti-CLP4 (97-109) and anti-CLP6 (148-162) antibodies targeting regions surrounding the CLP5 sequence also inhibited bacterial adhesion. Site-directed mutagenesis experiments inducing changes in the amino acid sequence of the ClpG protein corresponding to the CLP5 peptide resulted in the expression of nonadhesive CS31A antigen. These findings indicate that the ClpG receptor-binding domain is located in the central variable V2 region.

Aggregative adherence of Klebsiella pneumoniae to human intestine-407 cells

Aggregative adhesion of Klebsiella pneumoniae LM3 to Intestine-407 (Int-407) cells was studied. Adhesive capacities were affected by the bacterial growth phase (with a maximum of adherence obtained during the exponential phase), temperature, multiplicity of infection, and length of incubation with Int-407 cells. Adhesion occurred through a cytochalasin D-sensitive process and was greatly reduced after treatment of Int-407 with cycloheximide, indicating that aggregative adhesion requires active participation of Int-407 cells. Transmission electron microscopy revealed that adherent bacteria were surrounded by a capsule-like material, apparently involved in both bacterium-Int-407 cell and bacterium-bacterium adherence. Examination with a scanning electron microscope showed interactions of intestinal cell microvilli with bacteria and formation in 3 h of a fibrous network within and around the bacterial clusters. We speculate that aggregative adhesion of K. pneumoniae mediated by a capsule-like extracellular material might explain the persistence of these strains inside the host gastrointestinal tract.