Type IV Effector Proteins Involved in the Medicago-Sinorhizobium Symbiosis.

In this study, we investigated genetic elements of the type IV secretion system (T4SS) found in Sinorhizobium spp. and the role they play in symbiosis. Sinorhizobium meliloti and S. medicae each contain a putative T4SS similar to that used by Agrobacterium tumefaciens during pathogenesis. The Cre reporter assay for translocation system was used to validate potential effector proteins. Both S. meliloti and S. medicae contained the effector protein TfeA, which was translocated into the host plant. Sequence analysis revealed the presence of a nod box involved in transcriptional activation of symbiosis-related genes, upstream of the transcriptional regulator (virG) in the Sinorhizobium T4SS. Replicate quantitative reverse transcription-polymerase chain reaction analyses indicated that luteolin, released by roots and seeds of Medicago truncatula, upregulated transcription of tfeA and virG. Mutations in the T4SS apparatus or tfeA alone resulted in reduced numbers of nodules formed on M. truncatula genotypes. In addition, S. meliloti KH46c, which contains a deletion in the T4SS, was less competitive for nodule formation when coinoculated with an equal number of cells of the wild-type strain. To our knowledge, TfeA is the first T4SS effector protein identified in Sinorhizobium spp. Our results indicate that Sinorhizobium i) uses a T4SS during initiation of symbiosis with Medicago spp., and ii) alters Medicago cells in planta during symbiosis. This study also offers additional bioinformatic evidence that several different rhizobial species may use the T4SS in symbiosis with other legumes.

The effects of Gremlin1 on human umbilical cord blood hematopoietic progenitors.

Bone morphogenetic proteins (BMPs) support malignant hematopoiesis in CML. Conversely, the multi-functional BMP antagonist Gremlin1 supports self-renewing cancer stem cells of other malignancies. Inhibition of BMP signaling in CML, or of Gremlin1 in solid tumors, may therefore have therapeutic potential. However, since BMPs regulate hematopoietic stem cell (HSC) decisions in the stem cell niche, it is necessary to determine how Gremlin1 influences normal HSC. We examined the effects of Gremlin1 on long-term culture-initiating cells (LTC-IC) and transplantable hematopoietic stem cells (SCID-repopulating cells: SRC) in human umbilical cord blood. Gremlin1 inhibited BMP signaling, downregulated BMP-6 and cyclin E2 expression and upregulated hairy and enhancer of split-1 (HES-1; a Notch transcriptional target) and Hedgehog interacting protein-1 (HHIP-1; an inhibitor of Hedgehog signaling). The functional effects of Gremlin1 on SRC, i.e. skewing of their myelopoietic:lymphopoietic potential towards B lymphopoiesis without affecting long-term engraftment potential, were entirely consistent with changes in gene expression induced by Gremlin1. Since both BMPs and Gremlin1 are secreted by osteoblasts in vivo, our studies provide potential insights into the molecular regulation of hematopoiesis in the stem cell niche. These results also suggest that Gremlin1 (and possibly its mimetics that may be developed for therapeutic use) may not adversely affect normal human hematopoietic stem cell survival, though they may reduce their myelopoietic potential.

Characterization of an immunodeficient mouse model of mucopolysaccharidosis type I suitable for preclinical testing of human stem cell and gene therapy.

Mucopolysaccharidosis type I (MPS-I or Hurler syndrome) is an inherited deficiency of the lysosomal glycosaminoglycan (GAG)-degrading enzyme alpha-l-iduronidase (IDUA) in which GAG accumulation causes progressive multi-system dysfunction and death. Early allogeneic hematopoietic stem cell transplantation (HSCT) ameliorates clinical features and extends life but is not available to all patients, and inadequately corrects its most devastating features including mental retardation and skeletal deformities. To test novel therapies, we characterized an immunodeficient MPS-I mouse model less likely to develop immune reactions to transplanted human or gene-corrected cells or secreted IDUA. In the liver, spleen, heart, lung, kidney and brain of NOD/SCID/MPS-I mice IDUA was undetectable, and reduced to half in heterozygotes. MPS-I mice developed marked GAG accumulation (3-38-fold) in these organs. Neuropathological examination showed GM(3) ganglioside accumulation in the striatum, cerebral peduncles, cerebellum and ventral brainstem of MPS-I mice. Urinary GAG excretion (6.5-fold higher in MPS-I mice) provided a non-invasive and reliable method suitable for serially following the biochemical efficacy of therapeutic interventions. We identified and validated using rigorous biostatistical methods, a highly reproducible method for evaluating sensorimotor function and motor skills development. This Rotarod test revealed marked abnormalities in sensorimotor integration involving the cerebellum, striatum, proprioceptive pathways, motor cortex, and in acquisition of motor coordination. NOD/SCID/MPS-I mice exhibit many of the clinical, skeletal, pathological and behavioral abnormalities of human MPS-I, and provide an extremely suitable animal model for assessing the systemic and neurological effects of human stem cell transplantation and gene therapeutic approaches, using the above techniques to measure efficacy.

Draft Genome Sequences of Four Novel Thermal- and Alkaline-Tolerant Egyptian Rhizobium Strains Nodulating Berseem Clover.

Four Rhizobium strains were isolated from berseem clover in Egypt. The symbiotically effective, salt-tolerant, strain Rhiz950 was identified as new species, Rhizobium aegypticaum sv. trifolii (USDA 7124(T)). The other three thermal- and pH-tolerant strains were identified as Rhizobium bangladeshense sv. trifolii, the type strain is USDA 7125(T).

Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes.

The formation of symbiotic nitrogen-fixing nodules on the roots and/or stem of leguminous plants involves a complex signal exchange between both partners. Since many microorganisms are present in the soil, legumes and rhizobia must recognize and initiate communication with each other to establish symbioses. This results in the formation of nodules. Rhizobia within nodules exchange fixed nitrogen for carbon from the legume. Symbiotic relationships can become non-beneficial if one partner ceases to provide support to the other. As a result, complex signal exchange mechanisms have evolved to ensure continued, beneficial symbioses. Proper recognition and signal exchange is also the basis for host specificity. Nodule formation always provides a fitness benefit to rhizobia, but does not always provide a fitness benefit to legumes. Therefore, legumes have evolved a mechanism to regulate the number of nodules that are formed, this is called autoregulation of nodulation. Sequencing of many different rhizobia have revealed the presence of several secretion systems - and the Type III, Type IV, and Type VI secretion systems are known to be used by pathogens to transport effector proteins. These secretion systems are also known to have an effect on host specificity and are a determinant of overall nodule number on legumes. This review focuses on signal exchange between rhizobia and legumes, particularly focusing on the role of secretion systems involved in nodule formation and host specificity.

Intracerebroventricular transplantation of human bone marrow-derived multipotent progenitor cells in an immunodeficient mouse model of mucopolysaccharidosis type I (MPS-I).

Mucopolysaccharidosis type I (MPS-I; Hurler syndrome) is an inborn error of metabolism caused by lack of the functional lysosomal glycosaminoglycan (GAG)-degrading enzyme α-L-iduronidase (IDUA). Without treatment, the resulting GAG accumulation causes multisystem dysfunction and death within the first decade. Current treatments include allogeneic hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy. HSCT ameliorates clinical features and extends life but is not available to all patients, and inadequately corrects the most devastating features of the disease including mental retardation and skeletal deformities. Recent developments suggest that stem cells can be used to deliver needed enzymes to the central nervous system. To test this concept, we transplanted bone marrow-derived normal adult human MultiStem® cells into the cerebral lateral ventricles of immunodeficient MPS-I neonatal mice. Transplanted cells and human-specific DNA were detected in the hippocampal formation, striatum, and other areas of the central nervous system. Brain tissue assays revealed significant long-term decrease in GAG levels in the hippocampus and striatum. Sensorimotor testing 6 months after transplantation demonstrated significantly improved rotarod performance of transplanted mice in comparison to nontransplanted and sham-transplanted control animals. These results suggest that a single injection of MultiStem cells into the cerebral ventricles of neonatal MPS-I mice induces sustained reduction in GAG accumulation within the brain, and modest long-term improvement in sensorimotor function.

Endogenous heparan sulfate and heparin modulate bone morphogenetic protein-4 signaling and activity.

Bone morphogenetic proteins (BMPs) and their endogenous antagonists are important for brain and bone development and tumor initiation and progression. Heparan sulfate (HS) proteoglycans (HSPG) modulate the activities of BMPs and their antagonists. How glycosaminoglycans (GAGs) influence BMP activity in various malignancies and in inherited abnormalities of GAG metabolism, and the structural features of GAGs essential for modulation of BMP signaling, remain incompletely defined. We examined whether chemically modified soluble heparins, the endogenous HS in malignant cells and the HS accumulated in Hurler syndrome cells influence BMP-4 signaling and activity. We show that both exogenous (soluble) and endogenous GAGs modulate BMP-4 signaling and activity, and that this effect is dependent on specific sulfate residues of GAGs. Our studies suggest that endogenous sulfated GAGs promote the proliferation and impair differentiation of malignant human cells, providing the rationale for investigating whether pharmacological agents that inhibit GAG synthesis or function might reverse this effect. Our demonstration of impairment of BMP-4 signaling by GAGs in multipotent stem cells in human Hurler syndrome identifies a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses.

Functional abnormalities of heparan sulfate in mucopolysaccharidosis-I are associated with defective biologic activity of FGF-2 on human multipotent progenitor cells.

In mucopolysaccharidosis-I (MPS-I), alpha-L-iduronidase deficiency leads to progressive heparan sulfate (HS) and dermatan sulfate (DS) glycosaminoglycan (GAG) accumulation. The functional consequences of these accumulated molecules are unknown. HS critically influences tissue morphogenesis by binding to and modulating the activity of several cytokines (eg, fibroblast growth factors [FGFs]) involved in developmental patterning. We recently isolated a multipotent progenitor cell from postnatal human bone marrow, which differentiates into cells of all 3 embryonic lineages. The availability of multipotent progenitor cells from healthy volunteers and patients with MPS-I (Hurler syndrome) provides a unique opportunity to directly examine the functional effects of abnormal HS on cytokine-mediated stem-cell proliferation and survival. We demonstrate here that abnormally sulfated HS in Hurler multipotent progenitor cells perturb critical FGF-2-FGFR1-HS interactions, resulting in defective FGF-2-induced proliferation and survival of Hurler multipotent progenitor cells. Both the mitogenic and survival-promoting activities of FGF-2 were restored by substitution of Hurler HS by normal HS. This perturbation of critical HS-cytokine receptor interactions may represent a mechanism by which accumulated HS contributes to the developmental pathophysiology of Hurler syndrome. Similar mechanisms may operate in the pathogenesis of other diseases where structurally abnormal GAGs accumulate.

Identification of an MIP-1alpha -binding heparan sulfate oligosaccharide that supports long-term in vitro maintenance of human LTC-ICs.

We previously showed that heparan sulfate (HS) is required for in vitro cytokine + chemokine-mediated maintenance of primitive human hematopoietic progenitors. However, HS preparations are mixtures of polysaccharide chains of varying size, structure, and protein-binding abilities. Therefore, we examined whether the long-term culture-initiating cells (LTC-IC) supportive capability of HS is attributable to an oligosaccharide of defined length and protein-binding ability. Oligosaccharides of a wide range of sizes were prepared, and their capability to support human marrow LTC-IC maintenance in the presence of low-dose cytokines and a single chemokine, macrophage inflammatory protein-1alpha (MIP-1alpha), was examined. LTC-IC supportive capability of HS oligosaccharides correlated directly with size and MIP-1alpha binding ability. A specific MIP-1alpha-binding HS oligosaccharide preparation of M(r) 10 kDa that optimally supported LTC-IC maintenance was identified. This oligosaccharide had the structure required for MIP-1alpha binding, which we have recently described. The present study defines the minimum size and structural features of LTC-IC supportive HS.