Deletion of a gene encoding an amino acid transporter in the midgut membrane causes resistance to a Bombyx parvo-like virus.
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ABSTRACT:
SUBMITTER: Ito K
PROVIDER: S-EPMC2391135 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bombyx mori densovirus type 1 (BmDV) is a pathogen that causes flacherie disease in the silkworm. The absolute nonsusceptibility to BmDV among certain silkworm strains is determined independently by two genes, nsd-1 and Nid-1. However, neither of these genes has been molecularly identified to date. Here, we isolated the nsd-1 gene by positional cloning and characterized the properties of its product, NSD-1. Sequence and biochemical analyses revealed that this gene encodes a Bombyx-specific mucin-like glycoprotein with a single transmembrane domain. The NSD-1 protein was specifically expressed in the larval midgut epithelium, the known infection site of BmDV. Sequence analysis of the nsd-1 gene from 13 resistant and 12 susceptible strains suggested that a specific arginine residue in the extracellular tail of the NSD-1 protein was common among susceptible strains. Germline transformation of the susceptible-type nsd-1 (with a single nucleotide substitution) conferred partial susceptibility to resistant larvae, indicating that the + nsd-1 gene is required for the susceptibility of B. mori larvae to BmDV and the susceptibility is solely a result of the substitution of a single amino acid with arginine. Taken together, our results provide striking evidence that a novel membrane-bound mucin-like protein functions as a cell-surface receptor for a densovirus.
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Project description:Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93, which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis.
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Project description:Baculoviruses are virulent pathogens that infect a wide range of insects. They initiate infections via specific interactions between the structural proteins on the envelopes of occlusion-derived virions (ODVs) and the midgut cell surface receptors in hosts. However, host factors that are hijacked by baculoviruses for efficient infection remain largely unknown. In this study, we identified a membrane-associated protein sucrose hydrolase (BmSUH) as an ODV binding factor during Bombyx mori nucleopolyhedrovirus (BmNPV) primary infection. BmSUH was specifically expressed in the midgut microvilli where the ODV-midgut fusion happened. Knockout of BmSUH by CRISPR/Cas9 resulted in a significantly higher survival rate after BmNPV orally infection. Liquid chromatography-tandem mass spectrometry analysis and co-immunoprecipitation analysis demonstrated that PIF protein complex required for ODV binding could interact with BmSUH. Furthermore, fluorescence dequenching assay showed that the amount of ODV binding and fusion to the midgut decreased in BmSUH mutants compared to wild-type silkworm, suggesting the role of BmSUH as an ODV binding factor that mediates the ODV entry process. Based on a multilevel survey, the data showed that BmSUH acted as a host factor that facilitates BmNPV oral infection. More generally, this study indicated that disrupting essential protein-protein interactions required for baculovirus efficient entry may be broadly applicable to against viral infection.
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Project description:SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl(-)/HCO3(-) exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.
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Project description:We recently characterized a transposon-induced NaCl-sensitive mutant of Staphylococcus aureus (U. Vijaranakul, M. J. Nadakavukaren, D. O. Bayles, B. J. Wilkinson, and R. K. Jayaswal, Appl. Environ. Microbiol. 63:1889-1897, 1997). To further characterize this mutant, we determined the nucleotide sequence at the insertion site of the transposon on the S. aureus chromosome. Nucleotide sequencing revealed a 1,326-bp open reading frame (ORF442) encoding a hydrophobic 442-amino-acid polypeptide with a calculated molecular mass of 49,058 Da. The hydrophilicity profile of the gene product revealed the existence of 12 hydrophobic domains predicted to form membrane-associated alpha-helices. Comparison of the amino acid sequence of ORF442 with amino acid sequences in the GenBank database showed extensive homology with the branched-chain-amino-acid transport genes of gram-positive and gram-negative bacteria. This is the first brnQ gene in staphylococci to be described.
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Project description:Epidemiological and experimental data implicate branched-chain amino acids (BCAAs) in the development of insulin resistance, but the mechanisms that underlie this link remain unclear. Insulin resistance in skeletal muscle stems from the excess accumulation of lipid species, a process that requires blood-borne lipids to initially traverse the blood vessel wall. How this trans-endothelial transport occurs and how it is regulated are not well understood. Here we leveraged PPARGC1a (also known as PGC-1?; encoded by Ppargc1a), a transcriptional coactivator that regulates broad programs of fatty acid consumption, to identify 3-hydroxyisobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, as a new paracrine regulator of trans-endothelial fatty acid transport. We found that 3-HIB is secreted from muscle cells, activates endothelial fatty acid transport, stimulates muscle fatty acid uptake in vivo and promotes lipid accumulation in muscle, leading to insulin resistance in mice. Conversely, inhibiting the synthesis of 3-HIB in muscle cells blocks the ability of PGC-1? to promote endothelial fatty acid uptake. 3-HIB levels are elevated in muscle from db/db mice with diabetes and from human subjects with diabetes, as compared to those without diabetes. These data unveil a mechanism in which the metabolite 3-HIB, by regulating the trans-endothelial flux of fatty acids, links the regulation of fatty acid flux to BCAA catabolism, providing a mechanistic explanation for how increased BCAA catabolic flux can cause diabetes.
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Project description:Postexercise protein or amino acid ingestion restores muscle protein synthesis in older adults and represents an important therapeutic strategy for aging muscle. However, the precise nutritional factors involved are unknown.The purpose of this study was to determine the role of increased postexercise Leu ingestion on skeletal muscle myofibrillar protein synthesis (MyoPS), mammalian/mechanistic target of rapamycin complex 1 signaling, and amino acid transporter (AAT) mRNA expression in older men over a 24-h post-resistance exercise (RE) time course.During a stable isotope infusion trial (l-[ring-(13)C6]Phe; l-[1-(13)C]Leu), older men performed RE and, at 1 h after exercise, ingested 10 g of essential amino acids (EAAs) containing either a Leu content similar to quality protein (control, 1.85 g of Leu, n = 7) or enriched Leu (LEU; 3.5 g of Leu, n = 8). Muscle biopsies (vastus lateralis) were obtained at rest and 2, 5, and 24 h after exercise.p70 S6 kinase 1 phosphorylation was increased in each group at 2 h (P < 0.05), whereas 4E binding protein 1 phosphorylation increased only in the LEU group (P < 0.05). MyoPS was similarly increased (?90%) above basal in each group at 5 h (P < 0.05) and remained elevated (?90%) at 24 h only in the LEU group (P < 0.05). The mRNA expression of select AATs was increased at 2 and 5 h in each group (P < 0.05), but AAT expression was increased at 24 h only in the LEU group (P < 0.05).Leu-enriched EAA ingestion after RE may prolong the anabolic response and sensitivity of skeletal muscle to amino acids in older adults. These data emphasize the potential importance of adequate postexercise Leu ingestion to enhance the response of aging muscle to preventive or therapeutic exercise-based rehabilitation programs. This trial was registered at clinicaltrials.gov as NCT00891696.
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Project description:Proteins undergo structural fluctuations between nearly isoenergetic substates. Such fluctuations are often intimately linked with the functional properties of proteins. However, in some cases, such as in transmembrane ion transporters, the control of the ion transport requires that the protein is designed to restrict the motions in specific regions. In this study, we have investigated the dynamics of a membrane-bound respiratory oxidase, which acts both as an enzyme catalyzing reduction of O(2) to H(2)O and as a transmembrane proton pump. The segment of the protein where proton translocation is controlled ("gating" region) overlaps with a channel through which O(2) is delivered to the catalytic site. We show that the replacement of an amino acid residue with a small side chain (Gly) by one with a larger side chain (Val), in a narrow part of this channel, completely blocks the O(2) access to the catalytic site and results in formation of a compartment around the site that is impermeable to small gas molecules. Thus, the protein motions cannot counter the blockage introduced by the mutation. These results indicate that the protein motions are restricted in the proton-gating region and that rapid O(2) delivery to the catalytic site requires a gas channel, which is confined within a rigid protein body.
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