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Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 215-245). / Microfiche. / xii, 245 leaves, bound ill. (some col.) 29 cm

Lactobacilli are intestinal bacteria with known immunomodulatory competence. Numerous strains of this genus have been implicated in both the prevention and treatment of intestinal inflammation as well as in maintenance of immunological homeostasis. The frequent inclusion of lactobacilli in probiotic products attests to this ability. These lactic acid bacteria colonise the murine forestomach and burgeon in other environments similarly rich in carbohydrate-containing substrates. Accordingly, lactobacilli may utilise fermentable carbohydrates to synthesise exopolysaccharides (EPS). These polymers are secreted into the cellular milieu and, while the ecological function of EPS is yet to be defined, evidence points towards a protective role. This function may include bacterial protection from immunological attack, via EPS recognition by immune cells, resulting in modulation of immunological activity. Dendritic cells (DC) are potent antigen presenting cells, providing an essential link between the innate and adaptive arms of the intestinal immune system. DC efficiently sample intestinal antigens and present peptides to cognate naive CD4⁺ T cells in secondary lymphatic tissue. Under the influence of secreted cytokines, DC direct the differentiation of naive CD4⁺ T cells and therefore, instruct the resultant immune response. Anti-inflammatory Th2 and regulatory T cells can down-regulate the destructive Th1 pro-inflammatory effects associated with inflammatory bowel disease (IBD). As such, bioactives with the aptitude to direct DC activity and T cell differentiation have the potential to prevent or reduce intestinal inflammation. Therefore, this study aimed to determine whether heat-killed EPS-producing strains of lactobacilli, and their secreted EPS, exert an immunomodulatory effect on the murine gut which may down-regulate the immune reactions associated with IBD. Lactobacilli were screened for their ability to produce EPS when grown in the presence of glucose, sucrose or lactose. Heat-killed EPS-producing strains were then used to stimulate bone marrow-derived DC (BMDC) and the resultant cytokine profile was analysed. Nine Lactobacillus strains were found to produce EPS when grown in the presence of sucrose. Of these, L. reuteri 100-23 and L. johnsonii 100-33 exhibited potential anti-inflammatory effects. Therefore, these strains, as well as L. johnsonii 100-5 and L. johnsonii #21, with relatively weak BMDC stimulatory effect, were selected for further investigation. EPS of the potentially anti-inflammatory strain L. reuteri 100-23 was analysed. This sample contained approximately 85% carbohydrate and was composed of a (2[to]6)-β-fructofuranan (levan) and a mannan. The fructan, with an estimated molecular weight of 7 kDa, comprised at least 50% of the EPS, while the mannan made up at least 22%. The mannan component was likely linked to a protein and may have originated from the culture medium. The immunostimulatory capacity of heat-killed Lactobacillus bacterial cells and their EPS was determined in vitro. Firstly, the effect of lactobacilli and EPS on BMDC cytokine secretion, particularly levels of anti-inflammatory IL-10 and pro-inflammatory IL-12, as well as the expression of cell surface activation markers, was determined. L. reuteri 100-23 stimulated relatively high IL-10 secretion but low IL-12, while L. johnsonii 100-33-stimulated BMDC produced elevated levels of both IL-10 and IL-12. All bacterial cells up-regulated co-stimulatory molecules CD40 and CD80 on BMDC. The effect of these stimulated BMDC on T cell proliferation and cytokine production was then assessed, employing the ovalbuminDO11.10 T cell model. L. reuteri 100-23-stimulated BMDC down-regulated T cell production of the proliferation-stimulating cytokine, IL-2, up-regulated regulatory TGF-β secretion, but did not affect pro-inflammatory IFN-γ levels. The EPS of all strains did not stimulate significant BMDC cytokine production and failed to alter BMDC activation marker expression. However, BMDC stimulated with L. reuteri 100-23 and L. johnsonii 100-33 EPS significantly enhanced T cell IL-2 secretion, but did not alter TGF-β or IFN-γ levels. The effect of in vivo L. reuteri 100-23 and EPS intestinal stimulation on the reactivity of immune cells was subsequently investigated. Mesenteric lymph node (MLN) cells and splenic T cells from reconstituted Lactobacillus-free mice fed stimulant or PBS on two occasions were co-cultured with stimulated or unstimulated donor CD11c⁺ splenic DC ex vivo. Cellular proliferation as well as TGF-β and IFN-γ secretion was analysed, and IL-10 neutralisation assays were carried out to ascertain the involvement of this cytokine. Primary exposure of MLN cells to L. reuteri 100-23 resulted in suppressed cell proliferation in the presence of enhanced TGF-β levels, which may have also involved IL-10. Primed splenic T cells exhibited increased proliferation in the presence of elevated TGF-β levels following re-exposure to L. reuteri 100-23, and IL-10 may be involved in limiting this proliferation. L. reuteri 100-23 EPS did not alter MLN cell proliferation, possibly due to the suppressive activity of IL-10, but did enhance that of naive and primed splenic T cells. The effect of ingested L. reuteri 100-23 and EPS on intestinal sIgA concentration was assessed by quantifying IgA levels in the faecal supernatant of RLF mice previously ingesting L. reuteri 100-23 and EPS. L. reuteri 100-23 EPS-fed female mice exhibited significantly elevated levels of sIgA, while heat-killed bacteria did not affect antibody levels. The present study demonstrated that oral administration of heat-killed L. reuteri 100-23 and EPS exerts immunomodulatory effects on the murine intestine. These bioactives may promote a suppressive environment by conditioning DC to secrete a cytokine profile conducive to regulatory T cell induction and memory generation. Additionally, mucosal protection may be favoured by the stimulation of elevated sIgA levels. Therefore, a therapeutic composite is possibly obtained to preserve the intestinal barrier by defending against pathogen-induced injury and buffering inflammatory events. In these ways, L. reuteri 100-23 and EPS may confer long-lasting protection against, and down-regulate the immune reactions associated with, IBD.

The genes coding for the expression of a ropy exopolysaccharide responsible for commercially desirable textural and rhealogical traits in fermented milk products by a natural lactococcal ropy isolate were sought. Using a transposon mutagenesis vector, pGh9:ISS1, three mutants lacking expression of the ropy exopolysaccharide were isolated. One of the mutants was chosen for further characterization. Using a Southern hybridization analysis, the interrupted gene was localized to the chromosome. The non-ropy mutant was further characterized and shown to be unable to produce ropy exopolysaccharide in fermented milk. A 2006 by fragment of the interrupted gene was sequenced. The DNA sequence over a short region showed homology to sugar transfer enzymes found in exopolysaccharide biosynthesis pathways. The DNA sequence was translated into its predicted amino acid sequences and two partial open reading frames of 236 and 338 amino acid residues in length were identified. These open reading frames were found to exhibit identity to glycosyltransferases present in exopolysaccharide biosynthesis pathways in other bacteria. / Graduation date: 1999

Paenibacillus velaei, sp. nov. is a soil bacterium capable of producing an unusually large amount of exopolysaccharide (EPS). The EPS contains glucose, mannose, galactose and fucose in a molar ratio of 4:2:1:1. The molecular weight of the EPS is higher than 2x106. The viscosity of 1% EPS is 1300 cP when measured at a shear rate of 1 sec-1. Physiological parameters for optimal production of the EPS were studied and it was found that 1.4 g dry weight per 1 l of medium was produced when the bacteria were grown at 30EC and the pH adjusted at 7± 0.2 in a medium containing glucose as the carbon source. Growing the bacteria on different carbon sources did not alter the quantity or the composition of the EPS produced. No toxicity effects were observed in mice or rats when EPS was administered in amounts ranging from 20 to 200 mg per kg body weight. The data obtained from physical, chemical and biological properties suggest that the EPS may be employed in several industrial and environmental applications. It is an excellent emulsifier, it holds 100 times its own weight in water, it is not toxic, and it can be used to remove mercury, cadmium and lead from aqueous solutions.

Lactococcus lactis subsp. cremoris Ropy 352 and L. lactis subsp. cremoris Hollandicus produce an exopolysaccharide (EPS) that imparts commercially desirable textural and rheological properties to fermented milk products. This ropy phenotype is expressed under specific environmental conditions. A mucoid EPS phenotype, also expressed under specific environmental conditions, but not involved in the fermentation of ropy milk was identified. The two EPS phenotypes can be expressed individually or concurrently. Genetic regulators involved in expression of the EPS phenotypes were sought. DNA probes and polyclonal antiserum specific to two regulators of EPS in Escherichia coli, Lon protease and RcsA protein, were used to probe ropy and non-ropy strains of L. lactis. The two ropy strains of L. lactis subsp. cremoris, Ropy 352 and Hollandicus, expressed significantly less of the Lon protein than non-ropy strains. Southern and Western blot analysis was extended to a number of Gram negative and Gram positive bacteria. All of the Gram negative bacteria probed contained DNA sequences that hybridized to the Ion and rcsA gene probes, and all of these bacteria has at least one protein that reacted with antiserum to E. coli Lon and RcsA proteins. Two of the Gram positive bacteria contained DNA sequences that hybridized to the E. coli rcsA probe. None of the other Gram positive organisms contained DNA sequences that hybridized to the rcsA or the Ion probes. However, all the Gram positive bacteria contained one high molecular weight protein that reacted with Lon antiserum. In addition, Streptococcus salivarius expressed a protein that reacted with RcsA antiserum. In the course of this study, a second RcsA protein was identified in E. coll. The two RcsA proteins are expressed from one rcsA gene. One RcsA protein is not the proteolytic product of the other RcsA protein. Limited peptide digest profiles of each RcsA protein reveals almost identical peptides indicating the two proteins share a high degree of homology but are not identical. Ferguson plot analysis strongly suggests that the two RcsA proteins differ by size not by charge. Neither RcsA protein can be detected in cells mutant for Ion and rcsB. / Graduation date: 1997

Thesis (Ph. D.)--Victoria University (Melbourne, Vic.), 2009. / Includes bibliographical references.

Submitted by Programa de Pós-graduação em Biotecnologia (mebiotec.ufba@gmail.com) on 2017-04-06T13:04:01Z No. of bitstreams: 1 Dissertação Final - Jamille Almeida.pdf: 1302682 bytes, checksum: 996b2da859b03a0f196518660811cde6 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-06-29T15:06:56Z (GMT) No. of bitstreams: 1 Dissertação Final - Jamille Almeida.pdf: 1302682 bytes, checksum: 996b2da859b03a0f196518660811cde6 (MD5) / Made available in DSpace on 2017-06-29T15:06:56Z (GMT). No. of bitstreams: 1 Dissertação Final - Jamille Almeida.pdf: 1302682 bytes, checksum: 996b2da859b03a0f196518660811cde6 (MD5) / CAPES / Os polissacarídeos microbianos estão sendo muito utilizados atualmente por causa das vantagens em relação aos provenientes de outras fontes. Muitos são sintetizados por bactérias pertencentes à família Sphingomonadaceae como gelana, ramsana, welana, diutana, entre outras. Apesar da quantidade de polissacarídeos existentes, a descoberta de novos polissacarídeos microbianos é importante, tendo em vista a sua vasta aplicabilidade industrial, como espessantes, emulsificantes, estabilizantes e quelantes. Além disso, há a possibilidade de propriedades mais vantajosas e maior produção bacteriana. Este trabalho teve como objetivo selecionar linhagens bacterianas nativas de ambiente marinho produtoras de exopolissacarídeos e caracterizá-los. Neste contexto, a otimização da composição dos meios de cultivo e condições de processo podem modificar a produção, com possibilidade de aplicação industrial. Quatro bactérias foram selecionadas a partir da Coleção de Cultura Microbiana do Instituto de Ciências da Saúde pela resistência ao meio ágar nutriente contendo o antibiótico estreptomicina nas concentrações 100 e 200 μg.mL-1, sendo posteriormente identificadas por análise molecular como pertencentes aos gêneros Sphingomonas sp., Sphingobium sp. e Bacillus sp. A produção dos polímeros sintetizados por essas bactérias foi realizada em meio de cultivo, com alteração da fonte de carbono (sacarose ou glicerina bruta). A quantidade dos exopolissacarídeos sintetizados pelas bactérias pertencentes aos gêneros Sphingomonas sp. e Bacillus sp foi de 0,2 g.L-1 independente da fonte de carbono utilizada. O polímero produzido por Sphingobium sp. foi de 0,1 g.L-1 no meio contendo sacarose e 0,2 g.L-1 no meio com glicerina bruta. A CCMICS SB 22 não produziu exopolissacarídeo no meio contendo sacarose, enquanto que com a glicerina bruta foi de 0,2 g.L-1. As viscosidades dos exopolissacarídeos produzidos pelas quatro linhagens estudadas não apresentaram diferença entre si. A massa molecular do exopolissacarídeo produzido por Sphingobium sp. foi de 1,13 x 103 Daltons. Os outros polímeros não tiveram a massa molecular determinada por não apresentarem solubilidade em água. / The microbial polysaccharides are currently used being much because of advantages over from other sources. Most of those which are being studied are synthesized by bacteria of Sphingomonadaceae family, like gelan, rhamsan, welan, diutan, among others. Despite the amount of existing polysaccharides, the discovery of new polysaccharides is important, in view of its wide industrial applicability, such as thickeners, emulsifiers, stabilizers, and binders. Furthermore, there is the possibility of further advantageous properties and increased bacterial production. This work aimed to select native bacterial strains of exopolysaccharides-producing marine environment and characterize them. In this context, optimization of the composition of culture media and process conditions may change the production, with the possibility of industrial application. Four bacteria were selected from the Microbial Culture Collection of Sciences Institute of Health the resistance to the nutrient agar containing the antibiotic streptomycin in concentrations 100 and 200 μg.mL-1, subsequently identified by molecular analysis as belonging to the Sphingomonas sp., Sphingobium sp. and Bacillus sp. genres. The production of polymers synthesized by those bacteria was held in the culture medium, by changing the carbon source (sucrose or crude glycerin). The quantity of synthesized exopolysaccharides by the bacteria belonging to the Sphingomonas sp. and Bacillus sp genres was 0,2 g.L-1 regardless of the carbon source used. The polymer produced by Sphingobium sp. was 0,1 g.L-1 in the medium containing sucrose and 0,2 g.L-1 in the medium with crude glycerin. The CCMICS SB 22 produced no exopolysaccharide in the medium containing sucrose, while with crude glycerin was 0,2 g.L-1. The viscosities of exopolysaccharides produced by the four strains studied did not differ among themselves. The molecular mass of the exopolysaccharide produced by Sphingobium sp. was 1,13 x 10³ Daltons. The others polymers did not have the molecular mass determined for not showing solubility in water.

Thesis (Ph.D.)--Victoria University (Melbourne, Vic.), 2009.