In a dominant mouse ethylnitrosurea mutagenesis screen for genes regulating erythropoiesis, we identified a pedigree with a novel microcytic hypochromia caused by a V235G missense mutation in Dynamin 2 (Dnm2). Mutations in Dnm2, a GTPase, are highly disease-specific and have been implicated in four forms of human diseases: centronuclear myopathy, Charcot-Marie Tooth neuropathy and, more recently, T-cell leukaemia and Hereditary Spastic Paraplegia, but red cell abnormalities have not been reported to date. The V235G mutation lies within a crucial GTP nucleotide-binding pocket of Dnm2, and resulted in defective GTPase activity and incompatibility with life in the homozygous state. Dnm2 is an essential mediator of clathrin-mediated endocytosis, which is required for the uptake of transferrin (Tf) into red cells for incorporation of haem. Accordingly, we observed significantly reduced Tf uptake by Dnm2+/V235G cells, which led to impaired endosome formation. Despite these deficiencies, surprisingly all iron studies were unchanged, suggesting an unexplained alternative mechanism underlies microcytic anaemia in Dnm2+/V235G mice. This study provides the first in vivo evidence for the requirements of Dnm2 in normal erythropoiesis.

Dynamin I is a highly regulated GTPase enzyme enriched in nerve terminals which mediates vesicle fission during synaptic vesicle endocytosis. One regulatory mechanism involves its interactions with proteins containing Src homology 3 (SH3) domains. At least 30 SH3 domain-containing proteins bind dynamin at its proline-rich domain (PRD). Those that stimulate dynamin activity act by promoting its oligomerisation. We undertook a systematic parallel screening of 13 glutathione-S-transferase (GST)-tagged endocytosis-related SH3 domains on dynamin binding, GTPase activity and oligomerisation. No correlation was found between dynamin binding and their potency to stimulate GTPase activity. There was limited correlation between the extent of their ability to stimulate dynamin activity and the level of oligomerisation, indicating an as yet uncharacterised allosteric coupling of the PRD and G domain. We examined the two variants, dynamin Iab and Ibb, which differ in the alternately splice middle domain α2 helix. They responded differently to the panel of SH3s, with the extent of stimulation between the splice variants varying greatly between the SH3s. This study reveals that SH3 binding can act as a heterotropic allosteric regulator of the G domain via the middle domain α2 helix, suggesting an involvement of this helix in communicating the PRD-mediated allostery. This indicates that SH3 binding both stabilises multiple conformations of the tetrameric building block of dynamin, and promotes assembly of dynamin-SH3 complexes with distinct rates of GTP hydrolysis.

ABSTRACT Lactobacillus rhamnosus HN001 is a probiotic strain reported to increase resistance to epithelium-adherent and -invasive intestinal pathogens in experimental animals. To increase understanding of the relationship between strain HN001 and the bowel, transcription of selected genes in the mucosa of the murine small bowel was measured. Mice previously naive to lactobacilli ( Lactobacillus -free mice) were examined after daily exposure to HN001 in drinking water. Comparisons were made to results from matched Lactobacillus -free mice. Infant and adult mice were investigated to provide a temporal view of gene expression in response to exposure to HN001. Genes sgk1 , angptl4 , and hspa1b , associated with the apoptosis pathway, were selected for investigation by reverse transcription-quantitative PCR on the basis of a preliminary duodenal DNA microarray screen. Normalized to gapdh gene transcription, these three genes were upregulated after 6 to 10 days exposure of adult mice to HN001. Angptl4 was shown by immunofluorescence to be upregulated in duodenal epithelial cells of mucosal samples. Epithelial cell migration was faster in HN001-exposed mice than in the Lactobacillus -free controls. Transcriptional responses in infant mice differed according to bowel region and age. For example, sgk1 was upregulated in duodenal, jejunal, and ileal mucosa of mice less than 25 days old, whereas angptl4 and hspa1b were upregulated at 10 days in the duodenum but downregulated in the jejunal mucosa until mice were 25 days old. Overall, the results provide links between a probiotic strain, mucosal gene expression, and host phenotype, which may be useful in delineating mechanisms of probiotic action.

Early thymocyte progenitor T-cell acute lymphoblastic leukemia (ETP-ALL) is a poor prognosis malignancy that has a distinct genetic basis characterized by activating mutations of the IL-7 signaling pathway. Recurrent mutations spanning Dynamin 2 (Dnm2), a gene encoding a large GTPase required for clathrin-mediated endocytosis, have been identified in ETP-ALL although why these mutations are enriched in ETP-ALL remains obscure. We have utilized the Lmo2 transgenic mouse model to define the role of DNM2 mutations in ETP-ALL. Sequencing of 11 cases of Lmo2-derived T-ALL identified two novel Dnm2 mutations, a frameshift (Serine126fs) and a premature stop (Isoleucine135stop) mutation within the GTPase domain, which confirmed the relevance of this mouse model for studying Dnm2 mutations. We mated Lmo2 mice with mice carrying a germ-line missense mutation of Dnm2 in the GTPase domain (V235G), which impairs GTPase activity. This Dnm2 mutation had two important biologic effects on Lmo2-derived T-ALL: an increased penetrance (90% compared with 50% by 12 months of age; p<0.05) and a more immature phenotype (DN1-3 compared with ISP8). To address the mechanism of these effects, we examined the phenotype and expression profile of early leukemic stem cells harboring the Dnm2 mutation. The major abnormality was an expansion of IL-7 responsive leukemic stem cells with increased proliferation and survival. Intriguingly, the presence of Dnm2 mutations sensitized leukemic cells to killing by a small molecule inhibitor of Dnm2. Together, our data provide the first experimental evidence that Dnm2 mutations are enriched in ETP-ALL by expanding the pool of IL-7 responsive leukemic stem cells. Furthermore, our data supports further testing of Dynamin inhibitors in this subset of poor prognosis T-ALL. Disclosures: No relevant conflicts of interest to declare.

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. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, 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 identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-"docking" protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli.

Abstract 608 Forward-genetic screens have become a powerful method to study the pathogenesis of human disease and gene function. Chemical mutagenesis in mice using the mutagen, N-ethyl N-nitrosourea (ENU), has shown to be highly successful in elucidating novel genes or alleles in a variety of biological pathways, describing new functions of existing genes, and establishing mouse models that accurately recapitulate human disease. Advances in mapping strategies and deep sequencing technologies has dramatically simplified mutation detection, making ENU mutagenesis screens a feasible tool to study specific organ systems. To identify novel alleles regulating erythropoiesis, our laboratory has undertaken a dominant ENU mutagenesis screen. In this screen, the G1 progeny were screened at seven weeks of age for abnormalities in red cell indices (MCV, MCH, and HCT) using an automated hematological analyser. Here, we describe the identification of mice with a missense mutation of the large GTPase Dynamin 2 (DNM2) leading to an amino acid substitution V235G, predicted to lie within the nucleotide binding pocket for GTP. Western blot analysis for DNM2 protein revealed 50% protein levels in heterozygotes, suggesting that the point mutation leads to loss of protein rather than a dominant negative effect. Inherited DNM2 mutations are associated with autosomal dominant Charcot Tooth Myopathy (CTM) and Centronuclear Myopathy (CNM), but no recognised blood disorders. Heterozygous DNM2 V235G displayed hypochromic, microcytic anemia – HGB (15 g/dl compared to 16.5 g/dl in wild type mice), MCV (41.3 fl compared to 45.6 fl in wild type mice) and MCH (12.7 pg compared to 14.5 pg in wild type mice), but no obvious neuropathy or myopathy. Homozygosity was lethal before embryonic (E) day 8.5. DNM2 is an essential component in clathrin-mediated endocytosis, which is required for uptake of transferrin into red cells for incorporation of heme. Accordingly, endocytosis assays for transferrin uptake by FACS and confocal microscopy revealed reduced uptake in heterozygotes, explaining the microcytic hypochromic anemia. Western blot analysis for ferritin demonstrated reduced cellular ferritin, indicating cellular iron deficiency. Thus, this mouse model provides the first in vivo evidence that haplo-insufficiency of DNM2 can lead to iron deficiency anemia. Disclosures: No relevant conflicts of interest to declare.

It is becoming increasingly accepted by consumers that live lactic acid bacteria do exert health benefits when eaten. In addition, it is also becoming recognised that not all probiotic bacteria are equal. It is now no longer just a question of providing sufficient numbers of viable bacteria in a product; industry must also provide proof of efficacy for each strain. In the early 1990s, Fonterra embarked on a programme to develop proprietary probiotic strains, and as a result, commercialised two strains, Bifidobacterium lactis HN019 and Lactobacillus rhamnosus HN001. Over the past decade, Fonterra has developed a significant body of peerreviewed published reports around these strains, including studies showing safety in animal and human trials, protection against pathogens such as Salmonella typhimurium and Escherichia coli O157:H7, modulation of human and animal immune markers at realistic dose rates, and efficacy in human clinical trials. Based on this work, HN019 and HN001 have been applied to several functional foods both by Fonterra (under the DR10 and DR20 brands, respectively) and by third parties (e.g. under the HOWARU brand by Danisco). While the 'gold standard' of proof of efficacy is a phase III clinical trial, ethical considerations as well as expense preclude the use of clinical trials as screening tools for probiotics. Therefore, biomarkers have to be employed to identify strains with probiotic utility, and to define the different positive health benefits of existing probiotic strains. However, as the mechanisms by which most probiotic bacteria exert their health benefits remain unclear, the question of which biomarkers accurately reflect efficacy in vivo remains unresolved. With recent technological advances, and the shift toward probiotics targeted to specific conditions, researchers are beginning to tease out how probiotic bacteria work, and it is this knowledge that will inform biomarker development and improve the ability to offer the market safe and effective probiotic functional foods.

Surface, secreted and transmembrane protein-encoding open reading frames, collectively the secretome, can be identified in bacterial genome sequences using bioinformatics. However, functional analysis of translated secretomes is possible only if many secretome proteins are expressed and purified individually. We have now developed and applied a phage display system for direct selection, identification, expression and purification of bacterial secretome proteins.

Fonterra took a strategic decision in early 1996 to invest in a multi-disciplinary research program to develop its own proprietary probiotic strains. A structured approach based on in vitro and in vivo screening methods was used for the primary screening of potential probiotic candidates. The target health benefit used for selection of strains was immune enhancement. Strains were evaluated using both in vitro immune tests and appropriate in vivo animal models. After screening more than 2000 strains, four potential probiotic strains of food or human origin were selected. Strains were shown to provide protection against disease and mortality in animals challenged with pathogens such as Salmonella typhimurium and Escherichia coli O157:H7. Safety of the strains was tested exhaustively and strains were shown to be safe for human consumption. Four strains were characterised using both classical and modern molecular techniques as Lactobacillus rhamnosus HN001, L. rhamnosus HN067, L. acidophilus HN017 and Bifidobacterium lactis HN019 and were patent protected. Two - L. rhamnosus HN001 and B. lactis HN019 - were trade marked as DR20 and DR10 respectively. The clinical efficacy of selected strains in human subjects has been established for these strains through a number of well-designed clinical trials. Clinical trials have been conducted in number of countries with target populations and the results have been published in peer-reviewed scientific and medical journals. The impact of consumption of the strains on gut micro-ecology in humans has also been established through several dietary intervention studies. One of our current research initiatives is a genomic-based research program on our strains. A draft genome sequence of L. rhamnosus HN001 has allowed us to develop lactobacillus-specific genetic tools, undertake studies on cell surface properties of HN001, and investigate the mechanisms of immune enhancement. This large body of scientific research and efficacy data on strains has provided Fonterra with an ideal platform for commercialisation. Commercialisation of strains has been achieved through two channels. First route to market has been through Fonterra's own consumer products company Fonterra Brands (formerly New Zealand Milk). The second route to commercialisation has been through licensing agreement with Danisco, a major Danish ingredients company. Danisco markets these strains through its premium probiotic brand HOWARU(TM) as HOWARU(TM) Bifido and HOWARU(TM) Rhamnosus.