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Subject: search.filters.subject.Lactobacillus rhamnosus

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    Identification and functional characterisation of a novel surface protein complex of Lactobacillus rhamnosus : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Manawatu Campus, New Zealand
    (Massey University, 2016) Wen, Wesley Xingli
    Proteins are the most diverse structures on bacterial surfaces; hence they are candidates for species- and strain-specific interactions of bacteria with the host, environment and other microorganisms. In probiotic bacteria, some surface and secreted proteins mediate interactions with the host and may consequently contribute to the health-promoting effects. However, a limited fraction of surface-associated proteins from probiotic bacteria have been functionally characterised to date. A secreted protein of Lactobacillus rhamnosus HN001, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like), was previously shown to bind to HN001 cells, however the nature of its ligand on the surface of the cells was unknown. In this study, a series of binding assays first demonstrated that SpcA binds to a cell wall anchored protein of HN001. Next, the SpcA-“docking” protein, named SpcB, was identified using phage display. SpcB is a 3275-residue cell-surface protein that has all the features of large glycosylated serine-rich adhesins/fibrils from Gram-positive bacteria, including the hallmark glycoprotein signal sequence motif KxYKxGKxW and the cell wall anchor motif LPxTG. The spcA and spcB genes are located in a gene cluster, spcBCDA, which is present in 94 out of 100 strains of L. rhamnosus species and some strains of L. casei and L. paracasei whose genome sequences have been determined, but was absent from other Lactobacillus clades. To confirm the role of SpcB as the SpcA anchor and investigate the roles of these two proteins in surface properties of probiotic L. rhamnosus strains HN001 and GG, stable double-crossover mutations of these two genes were constructed. Binding assays to L. rhamnosus mutant cells confirmed dependence on SpcB in both GG and HN001 strains. Comparison of the wild-type and mutant surface properties suggested that SpcB in GG interferes with biofilm formation and aggregation, while it might contribute to the protective effect against TNFa-mediated disruption of the polarised Caco-2 cell monolayer integrity. Deletion of HN001 spcB or spcA had no effect on functions other than the SpcA binding. Our findings indicate that the roles of a surface protein can vary considerably among the strains of a species, requiring functional data to validate the bioinformatics-based hypotheses.
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    The search for Lactobacillus proteins that bind to host targets : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Turitea, New Zealand
    (Massey University, 2014) Erridge, Zoe Amber
    Interactions between microorganisms and host cells in the gastrointestinal tract are crucial to the host’s health. Probiotic bacteria, such as the lactobacilli provide numerous benefits to human health thought to be mediated by bacterial proteins called effectors. Lactobacillus rhamnosus HN001 (L. rhamnosus HN001) is a cheesefermenting isolate with probiotic characteristics and Lactobacillus reuteri 100-23 (L. reuteri 100-23) is a coloniser of the rodent forestomach. Whereas L. rhamnosus HN001 was shown to reduce eczema in children, L. reuteri 100-23 reduces inflammation in mice. The effector proteins for these strains are largely unknown. In this thesis, phage display technology was used to search for proteins that bind specific ligands. Shot-gun genomic phage display library of L. rhamnosus HN001 was affinity screened on fibronectin as bait, leading to enrichment of specific recombinant clones. Analysis of 10 candidate clones, however, determined that these are not genuine binders, but may have been selected due to a potential growth advantage during amplification steps of the library. The L. reuteri 100-23 genomic shot-gun phage display library was subjected to two affinity screens on two baits: fibronectin and murine stomach tissue. The aim of the screen on the murine stomach tissue was to identify keratin-binding proteins, as this strain naturally colonises the murine keratinous forestomach. Whereas no enrichment was detected in the screen on fibronectin as a bait, a strong enrichment of a phagemid displaying a short peptide, IGINS, derived from a cell-surface protease of L. reuteri 100-23 was identified. Identifying and characterising probiotic bacterial proteins that positively influence health will lead to a greater understanding of gastrointestinal tract interactions. Ultimately, this aids development of probiotic use as therapeutic agents.
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    Safety studies on probiotic strains Lactobacillus rhamnosus HN001, Lactobacillus acidophilus HN017, and Bifidobacterium lactis HN019 : a thesis submitted for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
    (Massey University, 2001) Zhou, Joseph Shengli
    Lactic acid bacteria (LAB) have been consumed in foods by human beings for several centuries without any obvious adverse effects. But the safety of consumption of these organisms, especially novel strains, which are added to foods as probiotics, has been questioned recently due to occasionally reported infections implicated with some particular LAB strains. Evaluation of the safety or potential toxicity of probiotic candidate strains, especially novel strains for which no prior safety data exist, is highly recommended. The LAB strains Lactobacillus rhamnosus HN001 (DR20TM), Lb, acidophilus HN017 and Bifidobacterium lactis HN019 (DR10TM) are three newly identified probiotic organisms with immune-enhancing properties. Their safety/potential toxicity was investigated in this study through a series of both in vitro and in vivo experiments. The mucus layer coating the surface of the gastrointestinal tract plays an important role in the gut mucosal defence system. Platelet activation and /or aggregation is a critical factor in the pathogenesis of infective endocarditis (IE). In the first part of this study, the potential pathogenicity of LAB strains was examined by in vitro mucin degradation (HN001, HN017, and HN019) and platelet aggregation (HN001 and HN019) assays. Following incubation with hog gastric mucin (HGM) in a minimal medium, the mucin degradation activity of test strains was determined via changes in the carbohydrate and protein concentration of the culture media and molecular weight changes of mucin glycoproteins (SDS-polyacrylamide gel electrophoresis, SDS-PAGE). The mucinolytic activity of test strains was also measured in an agarose petri dish assay. The results from these experiments suggested that HN001, HN017 and HN019 had no ability to degrade HGM in vitro. Flow cytometry analysis using platelet specific monoclonal antibodies demonstrated an inability of the test strains HN001 and HN019 to induce or enhance human platelet aggregation. These experiments indicated that the test strains are unlikely to degrade the mucin layer of the gastrointestinal mucosal surface or participate in the pathogenesis of endocarditis. Resistance of LAB strains to commonly used antibiotics has caused safety concerns regarding the genetic stability of these resistance properties. The antibiotic susceptibility and plasmid profiles of test organisms were investigated in another series of experiments. The susceptibility of the test strains to 18 antibiotics in common clinical use was examined by disk diffusion method. No extraordinary antimicrobial resistance was detected among the test strains (HN001, HN017, HN019, and HN067), and there were several antibiotics that efficiently suppressed the growth of test bacterial cells. A plasmid screening experiment demonstrated that all LAB strains examined were plasmid-free, this was verified by Southern blotting and DNA hybridisation techniques. These results indicate that the probiotic organisms tested here do not express or carry plasmid-associated antibiotic resistance, so their antibiotic resistance attributes are unlikely to disseminate to other clinically significant strains. To investigate the oral toxicity of test strains (HN001, HN017, and HN019), conventional BALB/c mice were inoculated with a high dose (10 11cfu/mouse/day) of the test probiotic LAB strains for 8 consecutive days. The feed and water intake, body weight gain, and general health status, of the mice were monitored. The potential translocation of inoculated LAB strains and gut mucosal histological changes following feeding were also investigated. Random amplified polymorphic DNA (RAPD) fingerprinting techniques were used for bacterial identification. Results showed that the test LAB strains had no adverse effects on the parameters observed; no viable bacteria were recovered from blood or tissue samples (mesenteric lymph nodes, liver, and spleen). These results suggest that the test strains had no acute toxicity and had no potential to result in infection in normal mice at the high dose applied in this study. To observe the consequences of longer-term consumption of test LAB strains, groups of BALB/c mice were orally administered with test LAB strains (HN001, HN017 and HN019) at doses of 5 x 10 7, 10 9 or 5 x 10 10 cfu/mouse/day for 4 weeks. In addition to the indicators observed in the acute toxicity study, the animals' haematological parameters; total and differential leucocyte counts; and blood biochemistry (plasma total protein, albumin, cholesterol, and glucose) were also investigated. Similar results to those of the acute toxicity study were obtained, i.e. 4 weeks consumption of HN001, HN017, and HN019 had no significant effects on the animals' general health status, haematology, blood biochemistry, or gut mucosal histological parameters. No dose-related effects were detected for any of the observed indicators. Translocation of test LAB strains was not observed. These results suggest that longer-term consumption of test strains is unlikely to cause any obvious health problems in host animals. In the final stage of this study, the potentially detrimental effects of HN001 and HN019 on hosts with sub-optimal immune functions were tested. To characterise the potential infectivity of test strains in immune deficient hosts, a group of adult male BALB/c mice pre-treated with dexamethasone (200µg/mouse/48 hrs) were fed with freshly cultured living HN001 or HN019 at doses of 1.5 ~ 2.5 x 10 7 cfu/mouse/day for 7 days; similar safety indicators to those outlined above were monitored. Results showed that no significant changes were noted in any of the safety parameters measured. No translocation of dietary LAB or systemic infection was detected. These findings suggest that HN001 and HN019 are well tolerated in immunocompromised mice without any significant safety concerns. To investigate the effects of consumption of test LAB strains in hosts with a preexisting immunological dysfuction, a group of female CBA/CaH mice (6 to 8 weeks) with experimentally induced autoimmune thyroiditis (EAT) were fed with freshly prepared probiotic preparations (HN001 4.2 x 10 8 cfu/mouse/day; HN019 2.16 x 10 8 cfu/mouse/day) for 5 to 8 weeks. Probiotic feeding was commenced one week prior to the immunization with auto antigens (MTg, mouse thyroglobulin). Antibody titres and spleen cell proliferative responses to the autoimmune inducing antigens (MTg) were determined via in vitro immunoassays. Lymphocyte (or mononuclear leucocyte) infiltration into thyroid tissue was also examined. Results showed that HN001 or HN019 feeding did not exacerbate spleen cell proliferative responses to MTg or lymphocyte infiltrations in thyroid tissues. These results indicate that feeding of HN001 or HN019 had no adverse effect on the induction or progress of autoimmune responses in CBA/CaH mice. Overall, the combined results from these studies suggest that the probiotic LAB strains HN001, HN017, and HN019 are non-pathogenic for experimental animals and are likely to be safe for human consumption.