Lactobacillus casei DN-114 001 Inhibits the Ability of Adherent-Invasive Escherichia coli Isolated 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.     

Modulation of Lactobacillus casei in ileal and fecal samples from healthy volunteers after consumption of a fermented milk containing Lactobacillus casei DN-114 001Rif

Lactobacillus casei DN-114 001 is a probiotic strain able to interact with the immune system and to interfere with gastrointestinal pathogens. The derived strain DN-114 001Rif was studied during its transit through the upper and distal intestine of human volunteers. Seven volunteers participated in the study, which involved intestinal intubation to sample ileal contents and collection of fecal samples, with a wash-out period of 8 days between the 2 steps. The retrieval of the probiotic was analyzed in the ileum every 2 h for 8 h following the ingestion of one dose of the test product and in the feces prior to, during, and after daily consumption of the test product for 8 days. Persistence of the probiotic amplifiable DNA was assessed using temporal temperature gradient gel electrophoresis and real-time PCR. Fluorescent in situ hybridization allowed analysis of the composition of the dominant digestive microbiota. The ingestion of L. casei DN-114 001Rif led to a significant and transient increase of its amplifiable DNA in ileal and fecal samples. This is related to a high stability in the composition of dominant groups of the gut microbiota. Data from ileal samples are scarce and our study confirms the potentiality for interaction between probiotics and the human immune system.     

Lysate of probiotic Lactobacillus casei DN-114 001 ameliorates colitis by strengthening the gut barrier function and changing the gut microenvironment

Background

Probiotic bacteria can be used for the prevention and treatment of human inflammatory diseases including inflammatory bowel diseases (IBD). However, the nature of active components and exact mechanisms of this beneficial effects have not been fully elucidated. Our aim was to investigate if lysate of probiotic bacterium L. casei DN-114 001 (Lc) could decrease the severity of intestinal inflammation in a murine model of IBD.

Methodology/Principal Findings

The preventive effect of oral administration of Lc significantly reduces the severity of acute dextran sulfate sodium (DSS) colitis in BALB/c but not in SCID mice. In order to analyze how this beneficial effect interferes with well-known phases of intestinal inflammation pathogenesis in vivo and in vitro, we evaluated intestinal permeability using the FITC-labeled dextran method and analysed tight junction proteins expression by immunofluorescence and PCR. We also measured CD4⁺FoxP3⁺ regulatory T cells proportion by FACS analysis, microbiota composition by pyrosequencing, and local cytokine production by ELISA. Lc leads to a significant protection against increased intestinal permeability and barrier dysfunction shown by preserved ZO-1 expression. We found that the Lc treatment increases the numbers of CD4⁺FoxP3⁺ regulatory T cells in mesenteric lymph nodes (MLN), decreases production of pro-inflammatory cytokines TNF-α and IFN-γ, and anti-inflammatory IL-10 in Peyer''s patches and large intestine, and changes the gut microbiota composition. Moreover, Lc treatment prevents lipopolysaccharide-induced TNF-α expression in RAW 264.7 cell line by down-regulating the NF-κB signaling pathway.

Conclusion/Significance

Our study provided evidence that even non-living probiotic bacteria can prevent the development of severe forms of intestinal inflammation by strengthening the integrity of intestinal barrier and modulation of gut microenvironment.     

Type 1 pili-mediated adherence of Escherichia coli strain LF82 isolated from Crohn's disease is involved in bacterial invasion of intestinal epithelial cells

We previously characterized the invasive ability of Escherichia coli strain LF82, isolated from an ileal biopsy of a patient with Crohn's disease. In the present study, we performed TnphoA insertion mutagenesis to identify genes involved in LF82 invasion of intestinal epithelial cells. Most of the non-invasive mutants had an insertion mutation within the type 1 pili-encoding operon. Two non-invasive fim mutants, which harboured an insertion within the fimI and fimF genes, still adhered but had lost the ability to induce host cell membrane elongations at the sites of contact with the epithelial cells. Transcomplementation experiments with a fim operon cloned from E. coli K-12 restored both invasive ability and the ability to induce host cell membrane elongations. Expression of the cloned LF82 or K-12 fim operon into the non-invasive laboratory strain JM109 did not confer invasive properties. Thus, these findings showed that: (i) type 1 pili-mediated adherence is involved in LF82-induced perturbation of host cell signalling responsible for membrane elongations; (ii) native shafts are required for type 1 pilus-mediated induction of membrane elongations; (iii) this active phenomenon is a key step in the establishment of the invasive process; and (iv) type 1 pili alone are not sufficient to trigger bacterial internalization.     

Adherent-invasive Escherichia coli isolated from Crohn's disease patients induce granulomas in vitro

Adherent-invasive Escherichia coli (AIEC) have been shown to be highly associated with ileal Crohn's disease (CD). AIEC survive within infected macrophages, residing within the phagolysosomal compartment where they take advantage of the low pH to replicate extensively. We investigated whether, like the tuberculous bacillus which also persists within macrophages, AIEC LF82 induces the formation of granulomas, which are a common histopathological feature of CD. For this purpose, we have taken advantage of an in vitro model of human granulomas that we recently developed, based on blood-derived mononuclear cells. We demonstrated that AIEC LF82 induces aggregation of infected macrophages, fusion of some of them to form multinucleated giant cells and subsequent recruitment of lymphocytes. Light microscopy and scanning electron microscopy analysis of the cell aggregates confirmed their granuloma features. This was further confirmed by histological analysis of granuloma sections. Noteworthy, this phenomenon can be reproduced by soluble protein extracts of AIEC LF82 coated onto beads. Although the cell aggregates not completely mimic natural CD-associated granulomas, they are very similar to early stages of epithelioid granulomas.     

Role of meprins to protect ileal mucosa of Crohn's disease patients from colonization by adherent-invasive E. coli

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to adhere to and invade intestinal epithelial cells (IEC), and to survive within macrophages. The interaction of AIEC with IEC depends on bacterial factors mainly type 1 pili, flagella, and outer membrane proteins. In humans, proteases can act as host defence mechanisms to counteract bacterial colonization. The protease meprin, composed of multimeric complexes of the two subunits alpha and beta, is abundantly expressed in IECs. Decreased levels of this protease correlate with the severity of the inflammation in patients with inflammatory bowel disease. The aim of the present study was to analyze the ability of meprin to modulate the interaction of AIEC with IECs. In patients with ileal CD we observed decreased levels of meprins, in particular that of meprin β. Dose-dependent inhibition of the abilities of AIEC strain LF82 to adhere to and invade intestinal epithelial T84 cells was observed when bacteria were pre-treated with both exogenous meprin α and meprin β. Dose-dependent proteolytic degradation of type 1 pili was observed in the presence of active meprins, but not with heat-inactivated meprins, and pretreatment of AIEC bacteria with meprins impaired their ability to bind mannosylated host receptors and led to decreased secretion of the pro-inflammatory cytokine IL-8 by infected T84 cells. Thus, decreased levels of protective meprins as observed in CD patients may contribute to increased AIEC colonization.     

Type 1, P and S fimbriae, and afimbrial adhesin I are not essential for uropathogenic Escherichia coli to adhere to and invade bladder epithelial cells

Fimbrial (type 1, P, and S) and afimbrial adhesins, the unique virulence traits of uropathogenic Escherichia coli (UPEC), are well recognized for their role in the initial step of uropathogenesis. In this study, we investigated whether these adhesins are dispensable for UPEC in adherence and invasion of uroepithelial cells by using E. coli isolates (n=40) from cystitis patients and T-24 cells, the bladder carcinoma cell line. We found all isolates adherent to T-24 cells within 15 min of infection. In invasion assay, all isolates could invade T-24 cells to a variable degree; 22.5% of them were found highly invasive. About 33% of isolates that do not have any recognized adhesins were as invasive as other isolates. The amplitude of invasiveness was also independent of the adhesins. In conclusion, this study demonstrates that type 1 fimbriae, P fimbriae, S fimbriae, and afimbrial adhesin I are not required for UPEC to adhere to and invade uroepithelial cells.     

Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.     

Immunization of mice with Lactobacillus casei expressing intimin fragments produces antibodies able to inhibit the adhesion of enteropathogenic Escherichia coli to cultivated epithelial cells

Enteropathogenic Escherichia coli (EPEC) are frequently isolated as a cause of infantile diarrhea in developing countries. Its pathogenicity is distinguished by histopathological alterations at the site of infection, known as attaching and effacing (A/E) lesions, in which bacterial virulence factors and host proteins participate. Intimin, a bacterial adhesin expressed by all EPEC described to date, is responsible for the intimate adherence of the bacteria to host cells and is essential for the formation of A/E lesions. Mucosal vaccination may represent an efficacious intervention to prevent EPEC infection and lower morbidity and mortality rates. Strategies for mucosal vaccinations that use lactic acid bacteria for the delivery of heterologous antigens rely on their safety profile and ability to stimulate the immune system. In the present work, we have constructed Lactobacillus casei strains expressing different fragments of intimin beta, a subtype that is frequently expressed by EPEC strains. Mucosal immunization of mice with L. casei expressing intimin fragments induced specific systemic and mucosal antibodies. These antibodies were able to recognize native intimin on the surface of EPEC and to inhibit in vitro EPEC binding to epithelial cells.     

干酪乳杆菌L-乳酸脱氢酶在大肠杆菌中的表达、纯化及酶学性质

L-乳酸脱氢酶(L-lactate dehydrogenase,L-LDH)是发酵生产L-乳酸中催化丙酮酸转化成L-乳酸的关键酶。以干酪乳杆菌G-02(Lactobacillus casei G-02)基因组DNA为模板,克隆得到L-LDH基因(ldhL),经序列分析后将其连接到表达载体pET-28a(+)上,构建成重组质粒pET-ldhL转化到大肠杆菌BL21(DE3)中,实现ldhL基因的表达。30°C加入IPTG诱导表达后,经镍柱亲和层析纯化的重组蛋白样品通过SDS-PAGE分析,约在40 kD处出现显著的特异性条带。对表达的L-LDH生物学特异性研究显示:重组L-LDH的比酶活为1 722 U/mg,最适反应温度为40°C-45°C;果糖-1,6-二磷酸(FBP)为别构激活剂,使最适pH向中性方向偏移(pH为6.6-6.8),Mn2+可拓宽最适酶活pH范围;Mn2+、Ca2+和Mg2+对L-LDH有激活作用,而Zn2+对L-LDH有抑制作用。     

Cloning and sequencing of plasmid pLC494 isolated from human intestinal Lactobacillus casei: construction of an Escherichia coli-Lactobacillus shuttle vector

The complete nucleotide sequence of plasmid pLC494 isolated from Lactobacillus casei L-49 was determined. Plasmid pLC494 is an 8846-bp long circular molecule with a G+C content of 41.5%. Two putative open reading frames, ORF4 (282 amino acids) and ORF5 (169 amino acids), were identified as replication proteins A and B that revealed 100 and 99% similarity, respectively, with the replication proteins of plasmid pLA103 from Lactobacillus acidophilus TK8912. Upstream of ORF4 were the four repeat regions (three perfect 22-bp repeats and one imperfect motif), a putative ribosome binding site, a -10 region, and a -35 region. The shuttle vector pJLE4942 (5318 bp) was constructed using repA from pLC494, a multiple cloning site, ColE1 ori, the ori of gram-negative bacteria from vector pUC19, and the chloramphenicol resistance gene from pJIR418 as a selection marker. Transformation of several lactic acid bacteria with the vector pJLE4942 indicated that this vector might be useful as a genetic tool for the intestinal lactobacilli.     

Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor

Adherence and invasion are thought to be key events in the pathogenesis of non-typeable Haemophilus influenzae (NTHi). The role of NTHi lipooligosaccharide (LOS) in adherence was examined using an LOS-coated polystyrene bead adherence assay. Beads coated with NTHi 2019 LOS adhered significantly more to 16HBE14 human bronchial epithelial cells than beads coated with truncated LOS isolated from an NTHi 2019 pgmB:ermr mutant (P = 0.037). Adherence was inhibited by preincubation of cell monolayers with NTHi 2019 LOS (P = 0.0009), but not by preincubation with NTHi 2019 pgmB:ermr LOS. Competitive inhibition studies with a panel of compounds containing structures found within NTHi LOS suggested that a phosphorylcholine (ChoP) moiety was involved in adherence. Further experiments revealed that mutations affecting the oligosaccharide region of LOS or the incorporation of ChoP therein caused significant decreases in the adherence to and invasion of bronchial cells by NTHi 2019 (P < 0.01). Analysis of infected monolayers by confocal microscopy showed that ChoP+ NTHi bacilli co-localized with the PAF receptor. Pretreatment of bronchial cells with a PAF receptor antagonist inhibited invasion by NTHi 2109 and two other NTHi strains expressing ChoP+ LOS glycoforms exhibiting high reactivity with an anti-ChoP antibody on colony immunoblots. These data suggest that a particular subset of ChoP+ LOS glycoforms could mediate NTHi invasion of bronchial cells by means of interaction with the PAF receptor.     

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.     

Note : Lack of influence of adherent Lactobacillus isolates on the attachment of Escherichia coli to the intestinal epithelial cells of chicken in vitro

Two Lactobacillus isolates, Lact. acidophilus I 26 and Lact. fermentum I 25, were selected, based on their poor aggregation with Escherichia coli and strong ability to adhere to ileal epithelial cells (IEC), to study in vitro interactions with E. coli O1 : K1, O2 : K1 and O78 : K80 in an IEC radioactive-assay under the conditions of exclusion (lactobacilli and IEC, followed by the addition of E. coli ), competition (lactobacilli, IEC and E. coli together) and displacement ( E. coli and IEC, followed by the addition of lactobacilli). The results indicated that Lact. acidophilus I 26 and Lact. fermentum I 25 could not significantly reduce the attachment of E. coli O1 : K1, O2 : K1 and O78 : K80 to IEC under the three conditions tested in vitro , except that the attachment of E. coli O1 : K1 was slightly reduced by Lact. fermentum I 25 in the test for competition.     

Suppression of the ptsH Mutation in Escherichia coli and Salmonella typhimurium by a DNA Fragment from Lactobacillus casei

A DNA fragment from Lactobacillus casei that restores growth to Escherichia coli and Salmonella typhimurium ptsH mutants on glucose and other substrates of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) has been isolated. These mutants lack the HPr protein, a general component of the PTS. Sequencing of the cloned fragment revealed the absence of ptsH homologues. Instead, the complementation ability was located in a 120-bp fragment that contained a sequence homologue to the binding site of the Cra regulator from enteric bacteria. Experiments indicated that the reversion of the ptsH phenotype was due to a titration of the Cra protein, which allowed the constitutive expression of the fructose operon.     

Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7 adhesion to epithelial cells

Adherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface-layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S-layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp-2 and T84) cells were treated with S-layer protein extract alone, infected with E. coli O157:H7, or pretreated with S-layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S-layer protein extracts were characterized by phase-contrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre-treatment of host epithelial cells with S-layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching-effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non-viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.