Phage T5 straight tail fiber is a multifunctional protein acting as a tape measure and carrying fusogenic and muralytic activities

We report a bioinformatic and functional characterization of Pb2, a 121-kDa multimeric protein that forms phage T5 straight fiber and is implicated in DNA transfer into the host. Pb2 was predicted to consist of three domains. Region I (residues 1-1030) was mainly organized in coiled coil and shared features of tape measure proteins. Region II (residues 1030-1076) contained two alpha-helical transmembrane segments. Region III (residues 1135-1148) included a metallopeptidase motif. A truncated version of Pb2 (Pb2-Cterm, residues 964-1148) was expressed and purified. Pb2-Cterm shared common features with fusogenic membrane polypeptides. It formed oligomeric structures and inserted into liposomes triggering their fusion. Pb2-Cterm caused beta-galactosidase release from Escherichia coli cells and in vitro peptidoglycan hydrolysis. Based on these multifunctional properties, we propose that binding of phage T5 to its receptor triggers large conformational changes in Pb2. The coiled coil region would serve as a sensor for triggering the opening of the head-tail connector. The C-terminal region would gain access to the host envelope, permitting the local degradation of the peptidoglycan and the formation of the DNA pore by fusion of the two membranes

Association of levels of antibodies from patients with inflammatory bowel disease with extracellular proteins of food and probiotic bacteria

Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals (HC, n=50), Crohn's disease patients (CD, n=37), and ulcerative colitis patients (UC, n=15). Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH) were significantly higher in the IBD group (P<0.002; n=52). The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS- 377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria. © 2014 Arancha Hevia et al.Borja Sánchez and Arancha Hevia were recipients of a Ramón y Cajal postdoctoral contract and a FPI Grant, respectively, from the Spanish Ministry of Economy and Competitiveness. Research in our group is supported by Grants AGL2010-14952 and RM2010-00012-00-00 from the Spanish Plan Nacional de I+D.Peer Reviewe

Enterococcus durans EP1 a promising anti-inflammatory probiotic able to stimulate sIgA and to increase Faecalibacterium prausnitzii abundance

Enterococcus species, principally Enterococcus faecium are used as probiotics since a long time with preference in animal applications but safety considerations were updated and also new uses as probiotics can be envisaged. Fifteen Enterococcus strains isolated from different foods were identified and analyzed for virulence factors and antibiotic resistance. Three Enterococcus durans strains were selected to study their immunomodulatory properties on PBMC and Caco2 cells. Two strains presented a profile toward a mild inflammatory Th1 response considering TNF-α/IL-10 and IL-1β/IL-10 cytokines ratios. The third strain EP1, presented an anti-inflammatory potential and was selected for in vivo studies. In mice, the strain was well tolerated and did not cause any adverse effects. EP1 administration increased the amount of IgA+ cells in mesenteric lymph node (MLN) after 7 days of administration. In fecal samples, the IgA content increased gradually and significantly from day 7 to day 21 in treated group. Additionally, IL-17, IL-6, IL-1β, IFN-γ, and CXCL1 gene expression significantly decreased on day 21 in Peyer's patches and IL-17 decreased in MLN. Mice treated with the probiotic showed significant lower mRNA levels of pro-inflammatory cytokines and mucins in the ileum at day 7 while their expression was normalized at day 21. Colonic expression of il-1β, il6, and mucins remain diminished at day 21. Ileum and colon explants from treated mice stimulated in vitro with LPS showed a significant reduction in IL-6 and an increase in IL-10 secretion suggesting an in vivo protective effect of the probiotic treatment against a proinflammatory stimulus. Interestingly, analysis of feces microbiota demonstrated that EP1 administration increase the amount of Faecalibacterium prausnitzii, a butyrate-producing bacteria, which is known for its anti-inflammatory effects. In conclusion, we demonstrated that EP1 strain is a strong sIgA inducer and possess mucosal anti-inflammatory properties. This strain also modulates gut microbiota increasing Faecalibacterium prausnitzii, a functionally important bacterium. Thus, E. durans EP1 is not only a good candidate to increases F. prausnitzii in some cases of dysbiosis but can also be interesting in gut inflammatory disorders therapy.Facultad de Ciencias Exacta

Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast-ovarian cancer susceptibility locus

A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P=9.2 × 10-20), ER-negative BC (P=1.1 × 10-13), BRCA1-associated BC (P=7.7 × 10-16) and triple negative BC (P-diff=2 × 10-5). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P=2 × 10-3) and ABHD8 (P<2 × 10-3). Chromosome conformation capture identifies interactions between four candidate SNPs and ABHD8, and luciferase assays indicate six risk alleles increased transactivation of the ADHD8 promoter. Targeted deletion of a region containing risk SNP rs56069439 in a putative enhancer induces ANKLE1 downregulation; and mRNA stability assays indicate functional effects for an ANKLE1 3′-UTR SNP. Altogether, these data suggest that multiple SNPs at 19p13 regulate ABHD8 and perhaps ANKLE1 expression, and indicate common mechanisms underlying breast and ovarian cancer risk

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer

Abstract: Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM−/− patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors

Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast-ovarian cancer susceptibility locus

A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P = 9.2 x 10(-20)), ER-negative BC (P = 1.1 x 10(-13)), BRCA1-associated BC (P = 7.7 x 10(-16)) and triple negative BC (P-diff = 2 x 10(-5)). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P = 2 x 10(-3)) and ABHD8 (PPeer reviewe

Secreted compounds of the probiotic Bacillus clausii O/C inhibit the cytotoxic effects induced by Clostridium difficile and Bacillus cereus toxins

Although the use of probiotics based on Bacillus strains to fight off intestinal pathogens and antibiotic-associated diarrhea is widespread, the mechanisms involved in producing their beneficial effects remain unclear. Here, we studied the ability of compounds secreted by the probiotic Bacillus clausii strain O/C to counteract the cytotoxic effects induced by toxins of two pathogens, Clostridium difficile and Bacillus cereus, by evaluating eukaryotic cell viability and expression of selected genes. Coincubation of C. difficile and B. cereus toxic culture supernatants with the B. clausii supernatant completely prevented the damage induced by toxins in Vero and Caco-2 cells. The hemolytic effect of B. cereus was also avoided by the probiotic supernatant. Moreover, in these cells, the expression of rhoB, encoding a Rho GTPase target for C. difficile toxins, was normalized when C. difficile supernatant was pretreated using the B. clausii supernatant. All of the beneficial effects observed with the probiotic were abolished by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Suspecting the involvement of a secreted protease in this protective effect, a protease was purified from the B. clausii supernatant and identified as a serine protease (M-protease; GenBank accession number Q99405). Experiments on Vero cells demonstrated the antitoxic activity of the purified protease against pathogen supernatants. This is the first report showing the capacity of a protease secreted by probiotic bacteria to inhibit the cytotoxic effects of toxinogenic C. difficile and B. cereus strains. This extracellular compound could be responsible, at least in part, for the protective effects observed for this human probiotic in antibiotic-associated diarrhea

Impact of Kefir Derived Lactobacillus kefiri on the Mucosal Immune Response and Gut Microbiota

The evaluation of the impact of probiotics on host health could help to understand how they can be used in the prevention of diseases. On the basis of our previous studies and in vitro assays on PBMC and Caco-2 ccl20:luc reporter system presented in this work, the strain Lactobacillus kefiri CIDCA 8348 was selected and administrated to healthy Swiss mice daily for 21 days. The probiotic treatment increased IgA in feces and reduced expression of proinflammatory mediators in Peyer Patches and mesenteric lymph nodes, where it also increased IL-10. In ileum IL-10, CXCL-1 and mucin 6 genes were upregulated; meanwhile in colon mucin 4 was induced whereas IFN-gamma, GM-CSF, and IL-1beta genes were downregulated. Moreover, ileum and colon explants showed the anti-inflammatory effect of L. kefiri since the LPS-induced increment of IL-6 and GM-CSF levels in control mice was significantly attenuated in L. kefiri treated mice. Regarding fecal microbiota, DGGE profiles allowed differentiation of experimental groups in two separated clusters. Quantitative PCR analysis of different bacterial groups revealed only significant changes in Lactobacillus population. In conclusion, L. kefiri is a good candidate to be used in gut inflammatory disorders

Enterococcus durans EP1 a Promising Anti-inflammatory Probiotic Able to Stimulate slgA and to Increase Faecalibacterium prausnitzii Abundance

Enterococcus species, principally Enterococcus faecium are used as probiotics since a long time with preference in animal applications but safety considerations were updated and also new uses as probiotics can be envisaged. Fifteen Enterococcus strains isolated from different foods were identified and analyzed for virulence factors and antibiotic resistance. Three Enterococcus durans strains were selected to study their immunomodulatory properties on PBMC and Caco2 cells. Two strains presented a profile toward a mild inflammatory Th1 response considering TNF-alpha/IL-10 and IL-1 beta/IL-10 cytokines ratios. The third strain EP1, presented an anti-inflammatory potential and was selected for in vivo studies. In mice, the strain was well tolerated and did not cause any adverse effects. EP1 administration increased the amount of IgA+ cells in mesenteric lymph node (MLN) after 7 days of administration. In fecal samples, the IgA content increased gradually and significantly from day 7 to day 21 in treated group. Additionally, IL-17, IL-6, IL-1 beta, IFN-gamma, and CXCL1 gene expression significantly decreased on day 21 in Peyer's patches and IL-17 decreased in MLN. Mice treated with the probiotic showed significant lower mRNA levels of pro-inflammatory cytokines and mucins in the ileum at day 7 while their expression was normalized at day 21. Colonic expression of il-1 beta, il6, and mucins remain diminished at day 21. Ileum and colon explants from treated mice stimulated in vitro with LPS showed a significant reduction in IL-6 and an increase in IL-10 secretion suggesting an in vivo protective effect of the probiotic treatment against a proinflammatory stimulus. Interestingly, analysis of feces microbiota demonstrated that EP1 administration increase the amount of Faecalibacterium prausnitzii, a butyrate-producing bacteria, which is known for its anti-inflammatory effects. In conclusion, we demonstrated that EP1 strain is a strong sIgA inducer and possess mucosal anti-inflammatory properties. This strain also modulates gut microbiota increasing Faecalibacterium prausnitzii, a functionally important bacterium. Thus, E. durans EP1 is not only a good candidate to increases F. prausnitzii in some cases of dysbiosis but can also be interesting in gut inflammatory disorders therapy

Safety and potential beneficial properties of Enterococcus strains isolated from kefir

Kefir is a fermented milk containing microorganisms showing several properties beneficial to health. In the present work the safety aspects and beneficial properties of kefir-isolated Enterococcus strains were investigated for the first time. Enterococcus strains were identified as Enterococcus durans using phenotypical and molecular characterisation methods. Neither resistance to clinically important antibiotics, nor genes encoding for eight different virulence factors were found. Interestingly, selected E. durans strains were able to inhibit different Gram+ and Gram− pathogens. These strains were able to survive simulated gastrointestinal conditions and showed a similar power of adhesion to mucins. Of note, the E. durans strains possessed anti-inflammatory properties as demonstrated by a significant inhibition of flagellin-induced response on Caco-2 cells. Our results indicate that the presence of E. durans in kefir does not represent a threat to consumers' health, and shows their potential both functionality and as probiotics.Fil: Carasi, Paula. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jacquot, Claudine. Universite de Bordeaux; FranciaFil: Romanin, David Emmanuel. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigaciones del Sistema Inmune; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Elie, Anne Marie. Universite de Bordeaux; FranciaFil: de Antoni, Graciela Liliana. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas; ArgentinaFil: Urdaci, María C.. Universite de Bordeaux; FranciaFil: Serradell, María de los Ángeles. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin