Adherence and colonization properties of Lactobacillus rhamnosus TB1, a broiler chicken isolate

AIMS: Selected lactic acid bacteria (LAB) isolated from intestinal tract of chicken have been studied in order to investigate their ability to adhere in vitro to Basement Membrane Matrigel (BMM). A selected strain showing a good adherence in BMM test was used for in vivo colonization assays. METHODS AND RESULTS: In vitro assessment of adhesion of broiler chicken isolates was performed using BMM assay. Among LAB strains tested, Lactobacillus rhamnosus TB1 showed a good adherence that was comparable to the one of an Escherichia coli EPEC strain used as positive control. For in vivo colonization assays this strain was fluorescently stained with the carboxyfluorescein diacetate succinimidyl ester (cFDA-SE) thus allowing its detection in different layers of intestinal tract after inoculation in broiler chicken. Further, stained L. rhamnosus were found with a highest value in rectum, jejunum and ileum both 3 and 24 h after administration. CONCLUSIONS: BMM assay is a quick method to test in vitro adhesion properties of bacterial strains and cFDA-SE-stained bacteria may be considered as an alternative method to test in vivo adhesion and colonization properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus rhamnosus TB1 was therefore showed to be able to adhere strongly in vitro to BMM and in vivo to intestinal epithelial cells of chicken and may be considered as a potential probiotic for chicken.     

Some factors affecting the adherence of probiotic Propionibacterium acidipropionici CRL 1198 to intestinal epithelial cells

Adhesion to the intestinal mucosa is generally considered an important property of probiotic microorganisms and has been related to many of their health benefits. This study investigated some factors that could affect or be involved in the adherence of Propionibacterium acidipropionici CRL 1198, a dairy strain with suggested probiotic effects and high adherence in vitro and in vivo to intestinal epithelial cells. In vitro adhesion of propionibacteria was decreased by gastric digestion but not affected by bile and pancreatic enzymes. Adherence was also decreased by pretreatment of bacterial cells with protease, sodium metaperiodate, and trichloroacetic acid, revealing that different features of the cell surface, like protein factors, carbohydrates, and teichoic acids, are involved in the process. Adherence to intestinal epithelial cells was enhanced by calcium and was dependent on other divalent cations. Adhesion to intestinal mucus was also demonstrated. The results should explain the metabolic effects in the host previously obtained with this strain and support the potential of Propionibacterium for development of new probiotics.     

Contribution of plasmid-encoded peptidase S8 (PrtP) to adhesion and transit in the gut of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IBB477 strain

The ability of Lactococcus lactis to adhere to the intestinal mucosa can potentially prolong the contact with the host, and therefore favour its persistence in the gut. In the present study, the contribution of plasmid-encoded factors to the adhesive and transit properties of the L. lactis subsp. cremoris IBB477 strain was investigated. Plasmid-cured derivatives as well as deletion mutants were obtained and analysed. Adhesion tests were performed using non-coated polystyrene plates, plates coated with mucin or fibronectin and mucus-secreting HT29-MTX intestinal epithelial cells. The results indicate that two plasmids, pIBB477a and b, are involved in adhesion of the IBB477 strain. One of the genes localised on plasmid pIBB477b (AJ89_14230), which encodes cell wall-associated peptidase S8 (PrtP), mediates adhesion of the IBB477 strain to bare, mucin- and fibronectin-coated polystyrene, as well as to HT29-MTX cells. Interactions between bacteria and mucus secreted by HT29-MTX cells were further investigated by fluorescent staining and confocal microscopy. Confocal images showed that IBB477 forms dense clusters embedded in secreted mucus. Finally, the ability of IBB477 strain and its ΔprtP deletion mutant to colonise the gastrointestinal tract of conventional C57Bl/6?mice was determined. Both strains were present in the gut for up to 72 h. In summary, adhesion and persistence of IBB477 were analysed by in vitro and in vivo approaches, respectively. Our studies revealed that plasmidic genes encoding cell surface proteins are more involved in the adhesion of IBB477 strain than in the ability to confer a selective advantage in the gut.     

Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany

AIMS: To verify the presence of Escherichia coli Nissle 1917 as a natural isolate in swine and to characterize in vitro probiotic properties as well as in vivo persistence in a feeding experiment. METHODS AND RESULTS: During studies on the intestinal microflora of pigs, we isolated E. coli Nissle 1917 sporadically from a pig population over a period of 1 year. The identity of the isolates as E. coli Nissle 1917 was verified by serotyping, Nissle-specific PCR, macrorestriction analysis (pulsed field gel electrophoresis) and the determination of in vitro probiotic properties in invasion and adhesion assays using a porcine intestinal epithelial cell line. Both the E. coli isolates and the E. coli Nissle 1917 strain showed strong reductions in adhesion of porcine enteropathogenic E. coli and invasion of Salmonella typhimurium with epithelial cells in vitro, with a probiotic effect. Screening of five epidemiologically unlinked swine farms and two wild boar groups showed one farm positive for E. coli Nissle 1917. A feeding experiment with four piglets showed viable E. coli Nissle 1917 in the intestine of three animals. CONCLUSIONS: The results of this study suggest that the E. coli Nissle 1917 strain is already partially established in swine herds, but the colonization of individual animals is variable. SIGNIFICANCE AND IMPACT OF THE STUDY: We report natural, long-term colonization and transmission of the probiotic E. coli Nissle 1917 strain in a swine herd, characterized individual persistence and colonization properties in swine and established an in vitro porcine intestinal epithelial cell model of probiotic action. The results of this study would have implications in the use of this strain as a probiotic in swine and contribute to a better understanding of the individual nature of intestinal bacterial persistence and establishment.     

Effect of fructooligosaccharide metabolism on chicken colonization by an extra-intestinal pathogenic Escherichia coli strain

Extra-intestinal pathogenic Escherichia coli (ExPEC) strains cause many diseases in humans and animals. While remaining asymptomatic, they can colonize the intestine for subsequent extra-intestinal infection and dissemination in the environment. We have previously identified the fos locus, a gene cluster within a pathogenicity island of the avian ExPEC strain BEN2908, involved in the metabolism of short-chain fructooligosaccharides (scFOS). It is assumed that these sugars are metabolized by the probiotic bacteria of the microbiota present in the intestine, leading to a decrease in the pathogenic bacterial population. However, we have previously shown that scFOS metabolism helps BEN2908 to colonize the intestine, its reservoir. As the fos locus is located on a pathogenicity island, one aim of this study was to investigate a possible role of this locus in the virulence of the strain for chicken. We thus analysed fos gene expression in extracts of target organs of avian colibacillosis and performed a virulence assay in chickens. Moreover, in order to understand the involvement of the fos locus in intestinal colonization, we monitored the expression of fos genes and their implication in the growth ability of the strain in intestinal extracts of chicken. We also performed intestinal colonization assays in axenic and Specific Pathogen-Free (SPF) chickens. We demonstrated that the fos locus is not involved in the virulence of BEN2908 for chickens and is strongly involved in axenic chicken cecal colonization both in vitro and in vivo. However, even if the presence of a microbiota does not inhibit the growth advantage of BEN2908 in ceca in vitro, overall, growth of the strain is not favoured in the ceca of SPF chickens. These findings indicate that scFOS metabolism by an ExPEC strain can contribute to its fitness in ceca but this benefit is fully dependent on the bacteria present in the microbiota.     

Survival of lactococci during passage through mouse digestive tract

One of the important properties of probiotics is the ability to survive in the intestine. There have been few studies on the probiotic property of lactococci, since they are formally not considered to be natural inhabitants of the intestine. To evaluate lactococci as probiotic bacteria, we investigated their ability to survive during gastric transit by in vitro and in vivo tests. When exposed to an in vitro simulated gastrointestinal environment, such as low pH and bile, only Lactococcus lactis subsp. lactis bv. diacetylactis N7 showed a moderate survival rate among the four strains tested. The tested strains were orally administered to mice, and intestinal passage of the ingested strains was monitored by two methods: antibiotics and PCR. Viable cells of strain N7 were recovered from feces within 24-48 h after administration but not at 72 h. Lactococcus lactis subsp. cremoris ATCC 19257, which had a poor survival rate in vitro test, was also detected at 12 h but not at 24 h. These results indicate that lactococci can reach the mouse intestine alive, but not colonize it. If administered daily, viable strain N7 may exist continuously in the intestine. The effect of strain N7 on intestinal microbial balance and on animal health will be the subject of a further study.     

不同宿主源弯曲菌黏附侵袭肠上皮细胞及在鸡肠道内定殖的比较

【背景】弯曲菌(Campylobacter)是重要的人畜共患病原菌,可在多种动物肠道定殖,但不同宿主源弯曲菌对肠上皮细胞的黏附侵袭特征及在鸡肠道内的定殖能力并不明确。【目的】探究不同宿主源弯曲菌对不同宿主肠上皮细胞黏附侵袭及在鸡肠道内定殖能力的差异性。【方法】利用 5株来自不同宿主源弯曲菌,包括人源、鸡源、鸭源和牛源空肠弯曲菌(Campylobacter jejuni)及猪源结肠弯曲菌(Campylobacter coli),在对菌株PCR鉴定、运动力及生物膜形成能力测定的基础上,分别测定各菌株对人源肠上皮细胞Caco-2、猪源肠上皮细胞IPEC-J2和大鼠源肠上皮细胞IEC-6的黏附能力,通过庆大霉素保护试验测定菌株对肠上皮细胞的侵袭能力,比较黏附量和侵袭量的差异;将5株弯曲菌分别口服攻毒鸡,于攻毒后不同日龄(different days post inoculation,DPI)采集肠道样品测定弯曲菌的菌落数,比较不同弯曲菌在鸡肠道内定殖的差异。【结果】人源弯曲菌运动力显著高于其他4株动物源弯曲菌,而牛源和猪源弯曲菌生物膜形成能力显著高于其他菌株。黏附侵袭测定结果显示,人源弯曲菌对Caco-2细胞的黏附能力显著高于动物源弯曲菌,但侵袭能力显著低于动物源弯曲菌;鸭源和牛源弯曲菌对IPEC-J2细胞的黏附能力显著低于其他菌株,而且鸭源弯曲菌的侵袭能力显著低于其他菌株;不同菌株对IEC-6细胞的黏附能力无显著差异,但鸡源弯曲菌侵袭能力显著低于其他菌株。不同弯曲菌口服攻毒鸡后1、3和6d动物源弯曲菌定殖水平显著高于人源,在攻毒后10d和15d仅牛源弯曲菌显著高于人源,于攻毒后15d所有菌株达到约8-10Log₁₀(CFU/g)的稳定定殖水平。【结论】来源于不同宿主的弯曲菌对不同宿主肠上皮细胞均具有黏附侵袭能力,同时可在鸡肠道内稳定定殖,提示弯曲菌在不同动物间传播和适应性定殖的特征,对开展弯曲菌针对性防控措施具有一定的借鉴意义。     

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.     

补体攻膜复合体致肾小球脏层上皮细胞粘附性改变研究

为探讨膜性肾病中补体攻膜复合体(MAC)介导蛋白尿机制,本研究制作了MAC致肾小球脏层上皮细胞(GVEC)亚溶破模型。通过对细胞局部粘附及相关蛋白的观察发现,MAC亚溶破致伤GVEC后,其粘附性发生改变。其机理与ECM分泌失调、膜硫酸化物质及整合素减少、细胞骨架重排有关。这些改变导致体内GVEC脱附,足突退缩融合,从而参与膜性肾病的蛋白尿产生。     

Salmonella Typhimurium SPI-1 genes promote intestinal but not tonsillar colonization in pigs

Salmonella Pathogenicity Island 1 (SPI-1) genes are indispensable for virulence of Salmonella Typhimurium in several animal species. The role of SPI-1 in the pathogenesis of Salmonella Typhimurium infections of pigs, however, is not well described. The interactions of a porcine Salmonella Typhimurium field strain and its isogenic mutants with disruptions in the SPI-1 genes hilA, sipA and sipB with porcine intestinal epithelial cells were characterized in vitro and in a ligated intestinal loop model in pigs. HilA and SipB were essential in the invasion of porcine intestinal epithelial cells in vitro. A sipA mutant was impaired for invasion using a polarized cell line, but fully invasive in a non-polarized cell line. All SPI-1 mutants induced a significant decrease in influx of neutrophils in the porcine intestinal loop model compared with the wild type strain. Pigs were orally inoculated with 10(8) colony forming units of both the wild type Salmonella Typhimurium strain and its isogenic sipB::kan mutant strain. The sipB mutant strain was significantly impaired to invade the intestinal, but not the tonsillar tissue, one day after inoculation and was unable to efficiently colonize the intestines and the GALT, but not the tonsils, 3 days after inoculation. This study shows that SPI-1 plays a crucial role in the invasion and colonization of the porcine gut and in the induction of influx of neutrophils towards the intestinal lumen, but not in the colonization of the tonsils.     

Starvation of marine flounder, squid and laboratory mice and its effect on the intestinal microbiota

Abstract Starvation processes of microorganisms in natural ecosystems were studied, both in vivo and in vitro, using marine animals (flounder and squid) and laboratory mice. In flounder, starvation resulted in the mixed intestinal microbiota maintaining its viability but decreasing its cell volume. It was also observed that during starvation there was an increased liability for adhesion of th microbiota to both the oil-water interface in the hexadecane water separation technique, and to Sepharose beads with either exposed hydrophobic or cationic charge groups. The population also exhibited the capacity to respond immediately to the addition of nutrients. When the flounder microbiota was starved in vitro the ratio of bacteria cultured on high nutrient: low nutrient media decreased with time of starvation. A similar effect on the intestinal microbiota of squid was observed in vivo when the animals were starved. The in vivo starvation of the mouse also produced a decrease in the mean bacterial cell volume which was concurrrent with a promotion of coliform bacteria. A coliform isolate exhibited similar starvation survival characteristics in vitro. From the data obtained from the flounder, squid and mice, it was concluded that components of the large intestinal microbiota exhibited the starvation survival characteristics previously reported for laboratory studies of planktonic bacteria, when exposed to energy- or nutrient-limited conditions.     

Aggregation Factor as an Inhibitor of Bacterial Binding to Gut Mucosa

Modern research in the area of probiotics is largely devoted to discovering factors that promote the adherence of probiotic candidates to host mucosal surfaces. The aim of the present study was to test the role of aggregation factor (AggL) and mucin-binding protein (MbpL) from Lactococcus sp. in adhesion to gastrointestinal mucosa. In vitro, ex vivo, and in vivo experiments in rats were used to assess the adhesive potential of these two proteins expressed in heterologous host Lactobacillus salivarius BGHO1. Although there was no influence of MbpL protein expression on BGHO1 adhesion to gut mucosa, expression of AggL had a negative effect on BGHO1 binding to ileal and colonic rat mucosa, as well as to human HT29-MTX cells and porcine gastric mucin in vitro. Because AggL did not decrease the adhesion of bacteria to intestinal fragments in ex vivo tests, where peristaltic simulation conditions were missing, we propose that intestinal motility could be a crucial force for eliminating aggregation-factor-bearing bacteria. Bacterial strains expressing aggregation factor could facilitate the removal of pathogens through the coaggregation mechanism, thus balancing gut microbial ecosystems in people affected by intestinal bacteria overgrowth.     

Identification of a Surface Protein from <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> JCM1081 That Adheres to Porcine Gastric Mucin and Human Enterocyte-Like HT-29 Cells

Adhesion of lactobacilli to the host gastrointestinal (GI) tract is considered an important factor in health-promoting effects. However, studies addressing the molecular mechanisms of the adhesion of lactobacilli to the host GI tract have not yet been performed. The aim of this work was to identify Lactobacillus reuteri surface molecules mediating adhesion to intestinal epithelial cells and mucins. Nine strains of lactobacilli were tested for their ability to adhere to human enterocyte-like HT-29 cells. The cell surface proteins involved in the adhesion of Lactobacillus to HT-29 cells and gastric mucin were extracted. The active fractions were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with horseradish peroxidase-labeled mucin and NHS-Biotin-labeled HT-29 cells. Furthermore, tandem mass spectrometry analysis was performed to identify the surface protein that participates in adhesion. It was shown that the ability of lactobacilli to adhere to HT-29 cells in vitro varied considerably among different strains. The most adhesive strain was the chicken intestinal tract isolate Lactobacillus reuteri JCM1081 (495.07 +/- 80.03 bacterial cells/100 HT-29 cells). The adhesion of L. reuteri JCM1081 to HT-29 cells appeared to be mediated by a cell surface protein, with an approximate molecular mass of 29 kDa. The peptides generated from the 29-kDa protein significantly matched the Lr0793 protein sequence of L. reuteri strain ATCC55730 (~71.1% identity) and displayed significant sequence similarity to the putative ATP-binding cassette transporter protein CnBP.     

Some probiotic properties of chicken lactobacilli

The beneficial effect of lactobacilli has been attributed to their ability to colonize human and animal gastrointestinal tracts. In this work, adhesion assays with three lactobacillus strains and intestinal fragments obtained from chickens were assessed. Lactobacillus animalis and L. fermentum were able to adhere to three kinds of epithelial cells (crop, small and large intestines) with predominance to small intestine. Among the strains considered, L. fermentum subsp. cellobiosus showed the lowest and L. animalis the highest adhesion ability. Scanning electron microphotographs showing L. animalis and L. fermentum adhering to intestinal cells were obtained. The characterization of L. animalis adhesion indicated that lectin-like structure of this strain has glucose/mannose as specific sugars of binding. However, a calcium requirement was not observed. The adhesion of L. fermentum was reduced by addition of sialic acid or mannose (P < 0.01). These carbohydrates can be involved in the interaction between adhesin and epithelial surface. In this case, the dependence on bivalent cations was demonstrated. Lactobacillus fermentum was effective in reducing the attachment of Salmonella pullorum by 77%, while L. animalis was able to inhibit (90%, 88%, and 78%) the adhesion of S. pullorum, S. enteritidis, and S. gallinarum to host-specific epithelial fragments respectively. Our results from this in vitro model suggest that these lactobacilli are able to block the binding sites for Salmonella adhesion.     

SopB effector protein of Salmonella Typhimurium is translocated in mesenteric lymph nodes during murine salmonellosis

Salmonella Typhimurium harbors two Salmonella pathogenicity islands (SPIs), each encoding a type three secretion system for virulence proteins. Although there is increasing evidence of postinvasion roles for SPI-1, it has been generally accepted that SPI-1 genes are downregulated following the invasion process. Here, we analyzed the expression and translocation of SopB in vitro, in cell culture and in vivo. To this end, a sopB-FLAG-tagged strain of Salmonella Typhimurium was obtained by epitope tagging. Tagged proteins were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with anti-FLAG antibodies. SopB expression was observed in vitro under cultured conditions that mimic the intestinal niche and different intracellular environments. In agreement, bacteria isolated from infected monolayers expressed and translocated SopB for at least 24?h postinoculation. For in vivo experiments, BALB/c mice were inoculated intraperitoneally with the tagged strain of Salmonella Typhimurium. Infecting bacteria and infected cells were recovered from mesenteric lymph nodes. Our results showed that SopB continues to be synthesized in vivo during 5 days after inoculation. Interestingly, translocation of SopB was detected in the cytosol of cells isolated from lymph nodes 1 day after infection. Altogether, these findings indicate that the expression and translocation of SopB during Salmonella infection is not constrained to the initial host-bacteria encounter in the intestinal environment as defined previously.     

Green fluorescent protein for detection of the probiotic microorganism Escherichia coli strain Nissle 1917 (EcN) in vivo

Probiotic microorganisms are defined as viable nutritional agents conferring benefit to the health of the human host. Especially, Escherichia coli strain Nissle 1917 (EcN) was shown to be equally effective as mesalazine in the maintenance of remission in ulcerative colitis (UC). Presumably, the therapeutic effect of EcN is linked to the presence of the strain in the region of interest; however, it remains difficult to follow the orally administered strain on its passage through the complex microbial environment of the intestine in vivo, inhabited dominantly by various E. coli strains, using traditional culturing methods. In this study we transformed EcN and a wild-type E. coli from a laboratory rat (EcR) with a plasmid carrying a gfp gene (pUC-gfp) to obtain EcN- and EcR-GFP to allow in vivo detection without alteration of strain-specific characteristics. Analysis of different strain-specific characteristics included the measurement of stimulation of IL-8 secretion and adhesion in vitro using the epithelial cell line HT-29. The kinetics of intestinal distribution in mice and colonization properties in rats following oral administration was studied in vivo. Detectability of the strain in histologic specimens was analysed using fluorescence microscopy and immunohistochemistry. The identity of fluorescent E. coli strains isolated from stool samples, Peyer's patches (PP) and mesenteric lymph nodes (MLN) was determined by REP-PCR. We were able to demonstrate that EcN and EcN-GFP do not differ in stimulation of IL-8 secretion or adhesion to HT-29 cells. In vivo, EcN-GFP colonies were readily detectable by fluorescence microscopy in luminal samples and also by immunohistochemistry in histological sections allowing analysis of the kinetics of the intestinal passage following oral administration. Translocation of fluorescent and non-fluorescent bacteria into PP and MLN was noted at 6 h post oral administration. EcN-GFP was detectable initially for 14 days in faecal samples of rats, while EcR-GFP was detectable throughout the whole experiment (45 days). Challenge with ampicillin at day 45 demonstrated continuing presence of EcN-GFP in small numbers by reappearing fluorescent colonies. The plasmid was not stable in vivo since non-fluorescent EcN colonies were detected also in faecal samples by REP-PCR. In summary, transformation of EcN to obtain EcN-GFP in our study had no detectable influence on the probiotic microorganism regarding adhesion on and induction of IL-8 secretion of HT-29 cells and allows the detection in mixed microbial environments in vivo but the stability of EcN-GFP in vivo is limited.     

Deletion of peb4 gene impairs cell adhesion and biofilm formation in Campylobacter jejuni

Campylobacter jejuni is a microaerophilic bacterium that causes diarrhea in humans. The first step in establishing an infection is adherence to a host cell, which involves two major cell-binding proteins, Peb1A (CBF1) and Peb4 (CBF2). Because the functional role of Peb4 on the cell adhesion remains unclear compared with that of Peb1A, a C. jejuni peb4 deletion mutant was constructed and cell adherence and ability to colonize mouse intestine were studied. The result showed that adherence of the peb4 mutant strain to INT407 cells was 1-2% that of the wild-type strain. Mouse challenge experiments showed a reduced level and duration of intestinal colonization by the mutant compared with the wild-type strain. In addition, fewer peb4 mutant cells than wild-type cells responded to stress by forming a biofilm. Proteomic analysis revealed that the expression levels of proteins involved in various adhesion, transport, and motility functions, which are required for biofilm formation by the pathogen, were lower in the peb4 mutant than in the wild-type strain. A Peb4 homolog has prolyl cis/trans-isomerase activity, suggesting that the loss of this activity in the mutant strain may be responsible for the repression of these proteins.     

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.     

Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM

Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.     

Functional Analysis of Putative Adhesion Factors in Lactobacillus acidophilus NCFM

Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in Lactobacillus acidophilus that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in L. acidophilus NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.