Upregulation of Immune checkpoint PD-L1 in Colon cancer cell lines and activation of T cells by Leuconostoc mesenteroides.

Globally colorectal cancer ranks as the third most widespread disease and the third leading cause of cancer-associated mortality. Immunotherapy treatments like PD-L1 blockade have been used to inhibit the PD-L1 legend, which boosts the activity of cytotoxic T lymphocytes. Recently, studies suggest that some probiotics could potentially enhance the effectiveness of immunotherapy treatments for cancer patients. We found that in Caco-2 and HT-29 cells, the live Leuconostoc mesenteroides treatment resulted an increase in the PD-L1 expression and this treatment stimulated interferon-gamma (IFN-γ) production in Jurkat T-cells. Due to the well-established ability of IFN-γ to enhance PD-L1 expression, the combination of IFN-γ and L. mesenteroides was used in colon cancer cell lines and a resulting remarkable increase of over tenfold in PD-L1 expression was obtained. Interestingly, when L. mesenteroides and IFN-γ are present, the blockage of PD-L1 using PD-L1 antibodies not only improved the viability of Jurkat T-cells but also significantly boosted the levels of IFN-γ and IL-2, the T-cells activation marker cytokines. In addition to upregulating PD-L1, L. mesenteroides also activated Toll-like receptors (TLRs) and NOD-like receptors (NODs) pathways, specifically through TLR2 and NOD2, while also exerting a suppressive effect on autophagy in colon cancer cell lines. In conclusion, our findings demonstrate a significant upregulation of PD-L1 expression in colon cancer cells upon co-culturing with L. mesenteroides. Moreover, the presence of PD-L1 antibodies during co-culturing activates Jurkat T cells. The observed enhancement in PD-L1 expression may be attributed to the inhibition of the Autophagy pathway or activation of the hippo pathway. KEY POINTS: Co-culturing L. mesenteroides increases PD-L1 gene and protein transaction in colon cancer. L. mesenteroides existing enhances T cells viability and activity. GPCR41/42 is a possible link between L. mesenteroides, YAP-1 and PD-L1.

Escherichia coli K12: An evolving opportunistic commensal gut microbe distorts barrier integrity in human intestinal cells.

Commensal enteric microbes under specific conditions viz. immunocompromised system, altered microbiota or uncompetitive niche induce their otherwise dormant pathogenic phenotype to distort host cellular functioning. Here we investigate how under in vitro environment established by using Caco-2 cells, commensal gut microbe E. coli K12 (ATCC 14849) disrupt intestinal epithelial barrier function. Caco-2 cells exposed to E. coli showed the time dependent significant (P < 0.01) decrease in transepithelial electrical resistance (TEER) and concomitantly increased phenol red flux across cell monolayer in contrast to non infected control cells. E. coli infected intestinal cells were observed with suppressed (p < 0.05) mRNA levels of ZO-1, Claudin-1, Occludin and Cingulin-1 in contrast to significantly (p < 0.05) higher PIgR and hbd-2 mRNA fold changes. Immunofluorescent and electron micrographs revealed the disrupted distribution and localisation of specific tight junction proteins (Zo-1 and Claudin-1) and actin filament in E. coli infected Caco-2 cells that ultimately resulted in deformed cellular morphology. Taken together, E. coli K12 under compromised in vitro milieu disrupted the intestinal barrier functions by decreasing the expression of important tight junction genes along with the altered distribution of associated proteins that increased the intestinal permeability as reflected by phenol red flux and TEER values.

Virulence, attachment and invasion of Caco-2 cells by multidrug-resistant bacteria isolated from wild animals.

Wild animals may be considered important reservoirs for bacterial pathogens and, consequently, possible sources of infection for humans. In this study, selected multidrug-resistant bacteria (Acinetobacter spp., Aeromonas salmonicida, Klebsiella pneumoniae, Pseudomonas fluorescens and Shewanella putrefaciens) isolated from wild animals were characterized on their ability to attach and invade/internalize human colonic carcinoma (Caco-2) cells. In addition, the viability of these bacteria to survive under simulated human gastrointestinal tract conditions as well as the production of virulence factors (homoserine lactones signal molecules, gelatinases, proteases, siderophores and biofilm formation) were studied. The results suggests that all the bacteria presented the capacity to attach and internalize into Caco-2 cells. A. salmonicida and P. fluorescens exhibited the highest ability to internalize. These bacteria were also found to be the highest proteases producers. A. salmonicida and K. pneumoniae survived under simulated human gastrointestinal conditions. These were the bacteria with the highest capacity to produce biofilms. K. pneumoniae was the only bacterium producing siderophores. Taken together, the present results reinforce the need for the "One Health" initiative, underscoring the environment and the animals as important reservoirs of infectious determinants.

Protective effect of the riboflavin-overproducing strain Lactobacillus plantarum CRL2130 on intestinal mucositis in mice.

OBJECTIVES: Intestinal mucositis (IM) is a local inflammatory response that causes alterations of the intestinal structure that in turn affect nutrient absorption and a side effect that is commonly associated with cancer treatments. Lactobacillus plantarum CRL2130 is a riboflavin-overproducing strain that has previously been shown to provide antiinflammatory properties. The objective of this study was to evaluate the effects of this riboflavin-producing strain in a chemically induced murine mucositis model. METHODS: Mucositis was induced by daily injections of 5-fluororacil (5-FU) after which mice were either given L. plantarum CRL2130, CRL725 (strain from which CRL2130 was derived that does not overproduce riboflavin), or commercial riboflavin twice daily during 6 d of chemotherapy agent injections. The effect of the strains and riboflavin was also evaluated in vitro using Caco-2 intestinal cancer cell cultures to determine if they interfere with 5-FU's anticancer activity. RESULTS: The administration of L. plantarum CRL2130 significantly attenuated the pathologic changes induced by 5-FU in mice such as body weight loss, diarrhea, shortening of villus height, increases in proinflammatory cytokine concentrations, and elevated production of interleukin 10. In vitro assays using Caco-2 cells showed that the effectiveness of 5-FU was not affected by L. plantarum CRL2130 and that this strain exerted an inhibitory mechanism against oxidative stress. CONCLUSIONS: These results indicate that the riboflavin-overproducing strain L. plantarum CRL2130 could be useful to prevent mucositis during cancer treatments and would not affect the primary treatment.

Effect of Pseudomonas graminis strain CPA-7 on the ability of Listeria monocytogenes and Salmonella enterica subsp. enterica to colonize Caco-2 cells after pre-incubation on fresh-cut pear.

To further gain insight into the mechanism by which the biopreservative bacterium Pseudomonas graminis CPA-7 develops its antimicrobial activity, we have examined the effect that the prior interaction stablished by this bacterium and two foodborne pathogens on fresh-cut pear, has on their capacity to colonize human epithelial cells (Caco-2 cell line) which is crucial for establishing infection. CPA-7 inhibited the growth of L. monocytogenes and S. enterica subsp. enterica ser. Enteritidis by 5.5 and 3.1 log10, respectively, after 7d of interaction at 10°C. Furthermore, CPA-7 attenuated the adherence of S. enterica to Caco-2 cells by 0.8 log10 regardless of the pre-adaptation on the fruit. Conversely, the adhesiveness of L. monocytogenes was not influenced by the interaction with the antagonist but it was reduced by 0.5 log10 after incubation on the food matrix. Pathogen-antagonist-food matrix interaction was associated to a significant reduction of the relative invasiveness of both pathogens, by 1.3 log10 in the case of L. monocytogenes and to an undetectable level (below 5CFU/g fruit) for S. enterica. CPA-7 can adhere to and internalize into intestinal epithelium which enables it for competition. Its adherence positively correlates to the multiplicity of infection (MOI) with respect to Caco-2 cells, increasing by 0.6 log10 in an MOI range of 0.1:1 to 100:1. For the same levels of inoculum, internalized cells could only be detected after 7d of pre-adaptation in the fruit (pH4.5-5.0). However, the combination of gastrointestinal digestion and habituation on the fruit resulted in a significant reduction of CPA-7 populations (by 2 log10 more after 7d of incubation than on inoculation day) as well as in the decrease of its adhesiveness (by 0.8 log10) and invasiveness (to undetectable levels).

Supplemental invasion of Salmonella from the perspective of Salmonella enterica serovars Kentucky and Typhimurium.

BACKGROUND: Critical to the development of Salmonellosis in humans is the interaction of the bacterium with the epithelial lining of the gastrointestinal tract. Traditional scientific reasoning held type III secretion system (T3SS) as the virulence factor responsible for bacterial invasion. In this study, field-isolated Salmonella enterica serovar Kentucky and a known human pathogen Salmonella enterica serovar Typhimurium were mutated and evaluated for the invasion of human colorectal adenocarcinoma epithelial cells. RESULTS: S. enterica serovar Kentucky was shown to actively invade a eukaryotic monolayer, though at a rate that was significantly lower than Typhimurium. Additionally, strains mutated for T3SS formation were less invasive than the wild-type strains, but the decrease in invasion was not significant in Kentucky. CONCLUSIONS: Strains mutated for T3SS formation were able to initiate invasion of the eukaryotic monolayer to varying degrees based on strain, In the case of Kentucky, the mutated strain initiated invasion at a level that was not significantly different from the wild-type strain. A different result was observed for Typhimurium as the mutation significantly lowered the rate of invasion in comparison to the wild-type strain.

Iron-induced virulence of Salmonella enterica serovar typhimurium at the intestinal epithelial interface can be suppressed by carvacrol.

Oral iron therapy can increase the abundance of bacterial pathogens, e.g., Salmonella spp., in the large intestine of African children. Carvacrol is a natural compound with antimicrobial activity against various intestinal bacterial pathogens, among which is the highly prevalent Salmonella enterica serovar Typhimurium. This study aimed to explore a presumed interaction between carvacrol and bacterial iron handling and to assess the potential of carvacrol in preventing the increase of bacterial pathogenicity during high iron availability. S. Typhimurium was cultured with increasing concentrations of iron and carvacrol to study the effects of these combined interventions on growth, adhesion to intestinal epithelial cells, and iron uptake/influx in both bacterial and epithelial cells. In addition, the ability of carvacrol to remove iron from the high-affinity ligand transferrin and an Fe-dye complex was examined. Carvacrol retarded growth of S. Typhimurium at all iron conditions. Furthermore, iron-induced epithelial adhesion was effectively reduced by carvacrol at high iron concentrations. The reduction of growth and virulence by carvacrol was not paralleled by a change in iron uptake or influx into S. Typhimurium. In contrast, bioavailability of iron for epithelial cells was moderately decreased under these conditions. Further, carvacrol was shown to lack the properties of an iron binding molecule; however, it was able to weaken iron-ligand interactions by which it may possibly interfere with bacterial virulence. In conclusion, our in vitro data suggest that carvacrol has the potential to serve as a novel dietary supplement to prevent pathogenic overgrowth and colonization in the large intestine during oral iron therapy.

Entrapment of intracytosolic bacteria by septin cage-like structures.

Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.

Listeria monocytogenes uses Listeria adhesion protein (LAP) to promote bacterial transepithelial translocation and induces expression of LAP receptor Hsp60.

Listeria monocytogenes interaction with the intestinal epithelium is a key step in the infection process. We demonstrated that Listeria adhesion protein (LAP) promotes adhesion to intestinal epithelial cells and facilitates extraintestinal dissemination in vivo. The LAP receptor is a stress response protein, Hsp60, but the precise role for the LAP-Hsp60 interaction during Listeria infection is unknown. Here we investigated the influence of physiological stressors and Listeria infection on host Hsp60 expression and LAP-mediated bacterial adhesion, invasion, and transepithelial translocation in an enterocyte-like Caco-2 cell model. Stressors such as heat (41°C), tumor necrosis factor alpha (TNF-α) (100 U), and L. monocytogenes infection (10(4) to 10(6) CFU/ml) significantly (P < 0.05) increased plasma membrane and intracellular Hsp60 levels in Caco-2 cells and consequently enhanced LAP-mediated L. monocytogenes adhesion but not invasion of Caco-2 cells. In transepithelial translocation experiments, the wild type (WT) exhibited 2.7-fold more translocation through Caco-2 monolayers than a lap mutant, suggesting that LAP is involved in transepithelial translocation, potentially via a paracellular route. Short hairpin RNA (shRNA) suppression of Hsp60 in Caco-2 cells reduced WT adhesion and translocation 4.5- and 3-fold, respectively, while adhesion remained unchanged for the lap mutant. Conversely, overexpression of Hsp60 in Caco-2 cells enhanced WT adhesion and transepithelial translocation, but not those of the lap mutant. Furthermore, initial infection with a low dosage (10(6) CFU/ml) of L. monocytogenes increased plasma membrane and intracellular expression of Hsp60 significantly, which rendered Caco-2 cells more susceptible to subsequent LAP-mediated adhesion and translocation. These data provide insight into the role of LAP as a virulence factor during intestinal epithelial infection and pose new questions regarding the dynamics between the host stress response and pathogen infection.

Transcriptional profiling of Clostridium difficile and Caco-2 cells during infection.

Clostridium difficile is well recognized as the most common infectious cause of nosocomial diarrhea. The incidence and severity of C. difficile infection (CDI) is increasing worldwide. Here, we evaluated simultaneously the transcriptional changes in the human colorectal epithelial Caco-2 cells and in C. difficile after infection. A total of 271 transcripts in Caco-2 cells and 207 transcripts in C. difficile were significantly differentially expressed at 1 time point during CDI. We used the gene ontology annotations and protein-protein network interactions to underline a framework of target molecules that could potentially play a key role during CDI. These genes included those associated with cellular metabolism, transcription, transport, cell communication, and signal transduction. Our data identified certain key factors that have previously been reported to be involved in CDI, as well as novel determinants that may participate in a complex mechanism underlying the host response to infection, bacterial adaptation, and pathogenesis.

Sialic acid is required for nonspecific adherence of Salmonella enterica ssp. enterica serovar Typhi on Caco-2 cells.

To initiate infection, bacteria must adhere to and colonize host tissues. Specific and nonspecific mechanisms participate in the adherence process. Salmonella enterica ssp. enterica serovar Typhi (S. Typhi) must first adhere to the intestinal epithelium to invade and disseminate throughout the host. In this study, the role of colonic epithelial cell surface sialic acid in the adherence of S. Typhi was defined. Neuraminidase treatment of colonic Caco-2 cells removed 27-58% of surface sialic acid. Thus desialylation diminished the adherence of S. Typhi by 41%. Sialic acid treatment of S. Typhi had no effect on their adherence to neuraminidase-treated or control cells. These results indicate that sialic acid on the surface of colonic cells enhances S. Typhi adherence. These findings may suggest novel therapeutic strategies for S. Typhi infections.

A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy.

Bacteria-mediated transfer of plasmid DNA to mammalian cells (bactofection) has been shown to have significant potential as an approach to express heterologous proteins in various cell types. This is achieved through entry of the entire bacterium into cells, followed by release of plasmid DNA. In a murine model, we show that Listeria monocytogenes can invade and spread in tumors, and establish the use of Listeria to deliver genes to tumors in vivo. A novel approach to vector lysis and release of plasmid DNA through antibiotic administration was developed. Ampicillin administration facilitated both plasmid transfer and safety control of vector. To further improve on the gene delivery system, we selected a Listeria monocytogenes derivative that is more sensitive to ampicillin, and less pathogenic than the wild-type strain. Incorporation of a eukaryotic-transcribed lysin cassette in the plasmid further increased bacterial lysis. Successful gene delivery of firefly luciferase to growing tumors in murine models and to patient breast tumor samples ex vivo was achieved. The model described encompasses a three-phase treatment regimen, involving (1) intratumoral administration of vector followed by a period of vector spread, (2) systemic ampicillin administration to induce vector lysis and plasmid transfer, and (3) systemic administration of combined moxifloxacin and ampicillin to eliminate systemic vector. For the first time, our results reveal the potential of Listeria monocytogenes for in vivo gene delivery.

Osmoregulated periplasmic glucans synthesis gene family of Shigella flexneri.

UNLABELLED: Osmoregulated periplasmic glucans (OPGs) of food- and water-borne enteropathogen Shigella flexneri were characterized. OPGs were composed of 100% glucose with 2-linked glucose as the most abundant residue with terminal glucose, 2-linked and 2,6-linked glucose also present in high quantities. Most dominant backbone polymer chain length was seven glucose residues. Individual genes from the opg gene family comprising of a bicistronic operon opgGH, opgB, opgC and opgD were mutagenized to study their effect on OPGs synthesis, growth in hypo-osmotic media and ability to invade HeLa cells. Mutation in opgG and opgH abolished OPGs biosynthesis, and mutants experienced longer lag time to initiate growth in hypo-osmotic media. Longer lag times to initiate growth in hypo-osmotic media were also observed for opgC and opgD mutants but not for opgB mutant. All opg mutants were able to infect HeLa cells, and abolition of OPGs synthesis did not affect actin polymerization or plaque formation. Ability to synthesize OPGs was beneficial to bacteria in order to initiate growth under low osmolarity conditions, in vitro mammalian cell invasion assays, however, could not discriminate whether OPGs were required for basic aspect of Shigella virulence. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00203-009-0538-z) contains supplementary material, which is available to authorized users.

Predicting virulence of Aeromonas isolates based on changes in transcription of c-jun and c-fos in human tissue culture cells.

AIMS: To screen for the virulence potential of Aeromonas isolates based on the change in regulation of c-jun and c-fos in the human intestinal tissue culture cell line Caco-2. METHODS AND RESULTS: Aeromonas cells were added to Caco-2 cells at a ratio of approx. 1 : 1. After 1-, 2- and 3-h incubation at 37 degrees C, mRNA was extracted from the cells and gene expression of two host genes, c-jun and c-fos, quantified. Aeromonas isolates which were pathogenic in the neonatal mouse model demonstrated up-regulation of c-jun and c-fos compared to avirulent isolates. CONCLUSIONS: Human cell culture results showed that c-jun and c-fos were predictive of Aeromonas virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: An Aeromonas relative virulence scale is proposed for use in the testing of Aeromonas drinking water isolates.

Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells.

AIMS: This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and to clarify the mechanism of action. METHODS AND RESULTS: Tempe was prepared at controlled laboratory scale using Rhizopus microsporus var. microsporus as the inoculum. Extracts of raw, soaked and cooked soya beans reduced ETEC adhesion to brush border cells by 40%. Tempe extracts reduced adhesion by 80% or more. ETEC adhesion to Caco-2 cells reduced by 50% in the presence of tempe extracts. ETEC K88 bacteria were found to interact with soya bean extracts, and this may contribute to the observed decrease of ETEC adhesion to intestinal epithelial cells. CONCLUSIONS: Fermented soya beans (tempe) reduce the adhesion of ETEC to intestinal epithelial cells of pig and human origin. This reduced adhesion is caused by an interaction between ETEC K88 bacteria and soya bean compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: The results strengthen previous observations on the anti-diarrhoeal effect of tempe. This effect indicates that soya-derived compounds may reduce adhesion of ETEC to intestinal cells in pigs as well as in humans and prevent against diarrhoeal diseases.

High potential of adhesion to abiotic and biotic materials in fish aquaculture facility by Vibrio alginolyticus strains.

AIMS: The ability of Vibrio alginolyticus strains isolated from Sparus aurata and Dicentrarchus labrax nursery to adhere to epithelial cell lines (Hep-2 and Caco-2), fish mucus and their ability to form a biofilm on different surfaces (glass, polystyrene, polyethylene and polyvinyl-chloride) was investigated in this study. METHODS AND RESULTS: The extracellular products were rich in enzymes and the strains were haemolytic on Wagatsuma agar and possessed several hydrolytic exoenzymes such as proteases, DNase and lipases. Most strains tested were multiresistant to the 17 antibiotics tested including those used in the farm to treat vibriosis. CONCLUSIONS: These bacteria were able to form a biofilm on all the surfaces tested and the cell density was the highest on the PVC surface followed by that on the glass slides, polystyrene and the polyethylene surface. More than 50% of the tested strains were adhesive to the epithelial cell lines (Hep-2 and Caco-2). SIGNIFICANCE AND IMPACT OF THE STUDY: These properties allow these bacteria to survive, proliferate and persist in all stages of fish rearing nursery even after seawater treatment with UV light.

Contributions of six lineage-specific internalin-like genes to invasion efficiency of Listeria monocytogenes.

Listeria monocytogenes strains are divided into at least three lineages, which seem to differ in virulence. Internalins are surface-attached or secreted proteins that encode leucine-rich repeats, and L. monocytogenes encodes species-specific as well as lineage-specific internalin and internalin-like genes. Internalins A and B have previously been shown to be critical for L. monocytogenes host cell invasion. Transcription of selected internalins is regulated by the virulence gene regulator PrfA and/or the stress-responsive alternative sigma factor sigma(B). We hypothesized that lineage-specific internalin-like genes may contribute to differential virulence and niche adaptation of the L. monocytogenes lineages. Initial quantitative real time, reverse transcriptase PCR (RT-PCR) showed that the six selected lineage-specific internalin-like genes were transcribed in cells grown at 16 degrees and 37 degrees C. Lineage-specific internalin-like gene, lineage II (lsiIIX) showed significantly higher transcript levels in log-phase cells grown at 37 degrees C as compared to 16 degrees C. The gene lsiIA was preceded by a putative sigma(B)-dependent promoter and showed sigma(B)-dependent transcription. None of the null mutants in lineage-specific internalin-like genes differed from their respective parent strain in ability to invade either human intestinal epithelial or hepatocyte-like cell lines. All three mutants in lineage I-specific internalin-like genes exhibited the same growth condition-dependent invasion phenotype as their parent strain ( approximately 1.5 log higher invasion efficiency when grown at 30 degrees C without aeration versus 37 degrees C with aeration). Despite structural similarities to internalins with known roles in host cell attachment and invasion, none of the six lineage-specific internalin-like genes characterized here appear to contribute to invasion. Combined with the observation that some nonpathogenic Listeria species also carry internalin genes, our findings suggest a broad role of Listeria internalins, not limited to attachment and invasion of human cells. Due to the wide host range of L. monocytogenes and the fact that transcription of internalin-like genes can differ considerably depending on growth condition, elucidating the function of different internalins and internalin-like genes will remain a challenge.

Evaluation of the probiotic potential of Lactobacillus paracasei KW3110 based on in vitro tests and oral administration tests in healthy adults.

Lactobacillus paracasei KW3110 is a strain which has been reported to possess a strong antiallergic activity. Since many lactic acid bacteria have shown to be useful as the probiotics, the possibility of this strain for use as a probiotic was studied. An in vitro test showed that this strain is strong enough to survive in the gastric juice. In addition, this strain showed exceptionally strong adherence to human intestinal epithelial cells, Caco-2 and HT29 compared to normal L. paracasei strains. Human oral administration testing of L. paracasei KW3110 showed that this strain survived in the human gut and increased the number of bifidobacteria and lactobacilli in the fecal samples of the subjects. In addition, KW 3110 was detected in 50% of the subjects up to 1 week after the end of the administration test. These results showed that L. paracasei KW3110 has a strong ability to survive in and colonize the human gut and improve the human intestinal microflora.

Implication of an outer surface lipoprotein in adhesion of Bifidobacterium bifidum to Caco-2 cells.

We found that the human intestinal isolate Bifidobacterium bifidum MIMBb75 strongly adhered to Caco-2 cells. Proteinase K and lithium chloride treatments showed that proteins play a key role in MIMBb75 adhesion to Caco-2 cells. By studying the cell wall-associated proteins, we identified a surface protein, which we labeled BopA. We purified the protein chromatographically and found that it functioned as an adhesion promoter on Caco-2 cells. In silico analysis of the gene coding for this protein and globomycin experiments showed that BopA is a cysteine-anchored lipoprotein expressed as a precursor polypeptide. A database search indicated that BopA appears to function biologically as an oligopeptide/tripeptide-solute-binding protein in the ABC transport system. We discovered a protein corresponding to BopA and its gene in eight other highly adherent B. bifidum strains. Finally, we found that B. bifidum MIMBb75 and BopA affected the production of interleukin-8 in Caco-2 epithelial cells. BopA is the first protein described to date to be directly involved in the adhesion of bifidobacteria to Caco-2 cells and to show immunomodulatory activity.

Subtractive screening for probiotic properties of lactobacillus species from the human gastrointestinal tract in the search for new probiotics.

In the search for new probiotics, 61 Lactobacillus spp. isolates, belonging to 12 species and isolated as dominant lactic acid bacteria from the feces of healthy humans, were subjected to a subtractive system of in vitro analyses, which included desirable and undesirable traits. Twenty-four isolates were able to grow in 2% bovine bile, of which 13 grew in acidified broth at pH 3.5 in acidified cysteine-containing MRS broth. Intrinsic resistance to certain antimicrobial agents (cefoxitin, metronidazole, vancomycin) was observed in most isolates, but atypical resistances to erythromycin, clindamycin, or tetracycline were also found in 5 strains. Undesirable traits such as alpha-chymotrypsin or N-acetyl-beta-glucosaminidase activities were not detected, but low beta-glucuronidase and moderate beta-glucosidase activities were recorded in 2 strains. Two Lactobacillus gasseri and 2 Lactobacillus paracasei selected strains inhibited several intestinal pathogens in an agar spot test, including strains of Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus. They also adhered to human Caco-2 and HT-29 epithelial cells in a manner comparable to Lactobacillus rhamnosus strain GG, and were unable to degrade pig gastric mucin in a plate assay. Together, these results suggest these 4 strains to be good probiotic candidates, concluding that the subtractive screening devised in this work could be a valuable tool in large-scale surveys for probiotics.