Generation and selection of anti-flagellin monoclonal antibodies useful for serotyping Salmonella enterica

In developing countries, bacterial acute gastroenteritis continues to be an important cause of morbidity and mortality among young children. Salmonellosis constitutes a major cause of infectious enteritis worldwide, most of them associated to the consumption of contaminated food products. Traditionally, Salmonella has been classified in serovars based on varieties of O and H surface antigens. In the present work we generated and characterized a panel of anti-flagellin monoclonal antibodies (MAbs) in order to select antibodies useful for detecting the H surface antigen. Four different MAbs were obtained by somatic hybridization of splenocytes. We found two MAbs that recognised regions of flagellin conserved among different Salmonella serovars. Other two MAbs recognised structures restricted to Salmonella enterica sv. Typhimurium, being one of them suitable for agglutination tests. Using a diverse panel of S. enterica serovars with different H antigen varieties we confirmed that this MAb agglutinates specifically S. Typhimurium (antigenic formula: 4,12:i:1,2) or other serovars expressing flagellar factor i. In conclusion, we generated a valuable immunochemical tool to be used in simple assays for serotyping of epidemiologically relevant strains. The capacity to characterize specific strains and determine the primary sources of Salmonella contamination generate valuable information of the epidemiology of this microorganism, contributing to the improvement of public health.Fil: Hiriart, Yanina. 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: Serradell, Maria de Los Angeles. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Martinez, Araci. Universidad de la República; UruguayFil: Sampaolesi, Sofia. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gonzalez Maciel, Maria Dolores. 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: Chabalgoity, Jose Alejandro. Universidad de la República; UruguayFil: Yim, Lucia. Universidad de la República; UruguayFil: Algorta, Gabriela. Universidad de la República; UruguayFil: Rumbo, Martín. 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; Argentin

Biophysical Methods for the Elucidation of the S-Layer Proteins/Metal Interaction

Surface-layers (S-layers) are macromolecular paracrystalline arrays of proteins or glycoproteins that can self-assemble into 2-dimensional semi-permeable meshworks to overlay the cell surface of many bacteria and archaea. They usually assemble into lattices with oblique, square or hexagonal symmetry and serve as an interface between the bacterial cell and the environment. Isolated S-layers can recrystallize into two-dimensional regular arrays in suspension or on various surfaces, thus being an appropriate material for several bionanotechnological purposes. Promising applications of S-layers include their use as biotemplates for the capture of metal ions or the synthesis of metal nanoclusters. Considering the use of S-layers as biotemplates for the organization of metal ions or metallic nanoclusters, research on potential of surface layer proteins (SLP) and metals can be understood as an interdisciplinary field, in which different biophysical techniques supply complementary information. In this review, we discuss the SLP as native or engineered “bottom-up” building blocks for metal immobilization structures. We also describe the biophysical techniques used to analyze metal binding properties as well as the information obtained from the investigation of these structures.Fil: Mobili, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigaciones En Criotecnología de Alimentos (i); Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: Serradell, Maria de Los Angeles. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biologicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mayer, Claudine. Instituto Pasteur. Departement de Biologie Structurale Et Chimie; Francia. Centre National de la Recherche Scientifique; Francia. Universite Paris Diderot - Paris 7; FranciaFil: Arluison, Véronique. Commissariat A Energie Atomique; Francia. Laboratoire Jean Perrin; Francia. Universite Paris Diderot - Paris 7; FranciaFil: Gomez Zavaglia, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigaciones En Criotecnología de Alimentos (i); Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentin

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

Administration of kefir-fermented milk protects mice against Giardia intestinalis infection

Giardiasis, caused by the protozoan Giardia intestinalis, is one of the most common intestinal diseases worldwide and constitutes an important problem for the public health systems of various countries. Kefir is a probiotic drink obtained by fermenting milk with ‘kefir grains’, which consist mainly of bacteria and yeasts that coexist in a complex symbiotic association. In this work, we studied the ability of kefir to protect mice from G. intestinalis infection, and characterized the host immune response to this probiotic in the context of the intestinal infection. Six- to 8-week-old C75BL/6 mice were separated into four groups: controls, kefir mice (receiving 1 : 100 dilution of kefir in drinking water for 14 days), Giardia mice (infected orally with 4107 trophozoites of G. intestinalis at day 7) and Giardia–kefir mice (kefir-treated G. intestinalis-infected mice), and killed at 2 or 7 days post-infection. Kefir administration was able to significantly reduce the intensity of Giardia infection at 7 days post-infection. An increase in the percentage of CD4+ T cells at 2 days post-infection was observed in the Peyer’s patches (PP) of mice belonging to the Giardia group compared with the control and kefir groups, while the percentage of CD4+ T cells in PP in the Giardia–kefir group was similar to that of controls. At 2 days post-infection, a reduction in the percentage of B220-positive major histocompatibility complex class II medium cells in PP was observed in infected mice compared with the other groups. At 7 days post-infection, Giardiainfected mice showed a reduction in RcFce-positive cells compared with the control group, suggesting a downregulation of the inflammatory response. However, the percentages of RcFcepositive cells did not differ from controls in the kefir and Giardia–kefir groups. An increase in IgApositive cells was observed in the lamina propria of the kefir group compared with controls at 2 days post-infection. Interestingly, the diminished number of IgA-positive cells registered in the Giardia group at 7 days post-infection was restored by kefir feeding, although the increase in IgApositive cells was no longer observed in the kefir group at that time. No significant differences in CXCL10 expression were registered between groups, in concordance with the absence of inflammation in small-intestinal tissue. Interestingly, a slight reduction in CCL20 expression was observed in the Giardia group, suggesting that G. intestinalis might downregulate its expression as a way of evading the inflammatory immune response. On the other hand, a trend towards an increase in TNF-a expression was observed in the kefir group, while the Giardia–kefir group showed a significant increase in TNF-a expression. Moreover, kefir-receiving mice (kefir and Giardia–kefir groups) showed an increase in the expression of IFN-c, the most relevant Th1 cytokine, at 2 days post-infection. Our results demonstrate that feeding mice with kefir reduces G. intestinalis infection and promotes the activation of different mechanisms of humoral and cellular immunity that are downregulated by parasitic infection, thus contributing to protection.Fil: Correa, Mariana. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigaciones en Criotecnología de Alimentos (i); ArgentinaFil: Golowczyc, Marina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigaciones En Criotecnología de Alimentos (i); Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: de Antoni, Graciela L.. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigaciones en Criotecnología de Alimentos (i); ArgentinaFil: Perez, Pablo Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigaciones En Criotecnología de Alimentos (i); Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: Humen, Martin Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigaciones En Criotecnología de Alimentos (i); Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: Serradell, Maria de Los Angeles. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; Argentin