The roles of mucD and alginate in the virulence of Pseudomonas aeruginosa in plants, nematodes and mice

We are exploiting the broad host range of the human opportunistic pathogen Pseudomonas aeruginosa strain PA14 to elucidate the molecular basis of bacterial virulence in plants, nematodes, insects and mice. In this report, we characterize the role that two PA14 gene products, MucD and AlgD, play in virulence. MucD is orthologous to the Escherichia coli periplasmic protease and chaperone DegP. DegP homologues are known virulence factors that play a protective role in stress responses in various species. AlgD is an enzyme involved in the biosynthesis of the exopolysaccharide alginate, which is hyperinduced in mucD mutants. A PA14 mucD mutant was significantly impaired in its ability to cause disease in Arabidopsis thaliana and mice and to kill the nematode Caenorhabditis elegans. Moreover, MucD was found to be required for the production of an extracellular toxin involved in C. elegans killing. In contrast, a PA14 algD mutant was not impaired in virulence in plants, nematodes or mice. A mucDalgD double mutant had the same phenotype as the mucD single mutant in the plant and nematode pathogenesis models. However, the mucDalgD double mutant was synergistically reduced in virulence in mice, suggesting that alginate can partially compensate for the loss of MucD function in mouse pathogenesis.     

Use of cell wall stress to characterize σ22 (AlgT/U) activation by regulated proteolysis and its regulon in Pseudomonas aeruginosa

MucA sequesters extracytoplasmic function (ECF) σ²² ( algT/U encoded) from target promoters including P algD for alginate biosynthesis. We have shown that cell wall stress (e.g. d -cycloserine) is a potent inducer of the algD operon. Here we showed that MucB, encoded by the algT-mucABCD operon, interacts with MucA in the sigma–sequestration complex. We hypothesized that AlgW protease (a DegS homologue) is activated by cell wall stress to cleave MucA and release σ²². When strain PAO1 was exposed to d -cycloserine, MucA was degraded within just 10 min, and σ²² was activated. However, in an algW mutant, MucA was stable with no increased σ²² activity. Studies on a yaeL mutant, defective in an RseP/YaeL homologue, suggest that YaeL protease cleaves MucA only after cleavage by AlgW. A defect in mucD , encoding a periplasmic HtrA/DegP homologue, caused MucA instability, suggesting MucD degrades cell wall stress signals. Overall, these data indicate that cell wall stress signals release σ²² by regulated intramembrane proteolysis (RIP). Microarray analyses identified genes of the early and late cell wall stress stimulon, which included genes for alginate production. The subset of genes in the σ²² regulon was then determined, which included gene products predicted to contribute to recovery from cell wall stress.     

MicroRNA-217 promotes ethanol-induced fat accumulation in hepatocytes by down-regulating SIRT1

Ethanol-mediated inhibition of hepatic sirtuin 1 (SIRT1) plays a crucial role in the pathogenesis of alcoholic fatty liver disease. Here, we investigated the underlying mechanisms of this inhibition by identifying a new hepatic target of ethanol action, microRNA-217 (miR-217). The role of miR-217 in the regulation of the effects of ethanol was investigated in cultured mouse AML-12 hepatocytes and in the livers of chronically ethanol-fed mice. In AML-12 hepatocytes and in mouse livers, chronic ethanol exposure drastically and specifically induced miR-217 levels and caused excess fat accumulation. Further studies revealed that overexpression of miR-217 in AML-12 cells promoted ethanol-mediated impairments of SIRT1 and SIRT1-regulated genes encoding lipogenic or fatty acid oxidation enzymes. More importantly, miR-217 impairs functions of lipin-1, a vital lipid regulator, in hepatocytes. Taken together, our novel findings suggest that miR-217 is a specific target of ethanol action in the liver and may present as a potential therapeutic target for treating human alcoholic fatty liver disease.     

Multiple positive and negative elements involved in the regulation of expression of GSY1 in Saccharomyces cerevisiae

GSY1 is one of the two genes encoding glycogen synthase in Saccharomyces cerevisiae. Both the GSY1 message and the protein levels increased as cells approached stationary phase. A combination of deletion analysis and site-directed mutagenesis revealed a complex promoter containing multiple positive and negative regulatory elements. Expression of GSY1 was dependent upon the presence of a TATA box and two stress response elements (STREs). Expression was repressed by Mig1, which mediates responses to glucose, and Rox1, which mediates responses to oxygen. Characterization of the GSY1 promoter also revealed a novel negative element. This element, N1, can repress expression driven by either an STRE or a heterologous element, the UAS of CYC1. Repression by N1 is dependent on the number of these elements that are present, but is independent of their orientation. N1 repressed expression when placed either upstream or downstream of the UAS, although the latter position is more effective. Gel shift analysis detected a factor that appears to bind to the N1 element. The complexity of the GSY1 promoter, which includes two STREs and three distinct negative elements, was surprising. This complexity may allow GSY1 to respond to a wide range of environmental stresses.     

Biochemical analysis of alginate biosynthesis protein AlgX from Pseudomonas aeruginosa: purification of an AlgX-MucD (AlgY) protein complex

AlgX was found to be an essential protein for alginate biosynthesis, but its function is unknown. In this study, an isogenic, marker-free algX-knock out mutant was generated. In-frame fusions of algX with phoA and lacZ were analysed, respectively. No LacZ-activity was detected, but the PhoA fusion showed alkaline phosphatase activity. These data indicated that the C-terminus of AlgX is located in the periplasm, but is not required for protein function. Accordingly, AlgX with C-terminal fusion of strep tag II restored alginate production in the algX-negative mutant and was purified under native conditions from periplasmic and crude cell extracts, respectively. AlgX was identified by MALDI/TOF-MS analysis of tryptic peptides. TritonX-100 mediated solubilisation of cytoplasmic membrane and subsequent strep tag II affinity chromatography led to purification of an AlgX-MucD (AlgY) protein complex as identified by MALDI/TOF-MS analysis. This data suggested a protein-protein interaction between AlgX and MucD (AlgY) with a 1:1 stoichiometry. Thus AlgX might exert its function via interaction with MucD (AlgY). Immunoelectron microscopic localisation of AlgX-strep tag II suggested a localisation close to the cytoplasmic membrane.     

Chicken liver and muscle carnitine palmitoyltransferase 1: nutritional regulation of messengers

In mammals, carnitine palmitoyltransferase 1 (CPT1) is a rate limiting enzyme of fatty acid oxidation. Two isoforms are present. We characterized a full-length cDNA sequence encoding chicken liver L-CPT1 isoform and a partial cDNA sequence encoding chicken muscle M-CPT1 isoform. CPT1 messengers showed the expected tissue specificity. M-CPT1 messenger and CPT1 activity were higher in oxidative than in glycolytic muscle. Expression of both isoforms was assessed in various tissues of genetically fat or lean chickens. Fasting considerably increased L-CPT1 mRNA expression and beta-hydroxyacyl CoA dehydrogenase (HAD) activity in the liver of fat or lean chickens. Unexpectedly, fasting did not increase M-CPT1 mRNA levels nor HAD activity in muscles of either chicken genotype. It however increased succinyl-CoA:3-ketoacid CoA transferase (SCOT) mRNA expression (an enzyme related to ketone body utilization) in oxidative muscle. SCOT messenger was slightly more abundant in oxidative muscle of lean chickens but not in glycolytic muscle. In conclusion, the regulation of fatty acid oxidation is probably not impaired in fat chicken. The absence of fasting stimulation of M-CPT1 mRNA expression, which is at variance with the situation observed in mammals, suggests that during fasting, chicken muscles preferentially use ketone bodies as fuel, at least in the short term.     

Novel gene encoding a Ca2+-binding protein and under hexokinase-dependent sugar regulation

A cDNA encoding a predicted 15-kDa protein was earlier isolated from sugar-induced genes in rice embryos (Oryza sativa L.) by cDNA microarray analysis. Here we report that this cDNA encodes a novel Ca2+-binding protein, named OsSUR1 (for Oryza sativa sugar-up-regulated-1). The recombinant OsSUR1 protein expressed in Escherichia coli had 45Ca2+-binding activity. Northern analysis showed that the OsSUR1 gene was expressed mainly in the internodes of mature plants and in embryos at an early stage of germination. Expression of the OsSUR1 gene was induced by sugars that could serve as substrates of hexokinase, but expression was not repressed by Ca2+ signaling inhibitors, calmodulin antagonists and inhibitors of protein kinase or protein phosphatase. These results suggested that Os-SUR1 gene expression was stimulated by a hexokinase-dependent pathway not mediated by Ca2+.     

Differential expression of the pro-inflammatory cytokines IL-1beta-1, TNFalpha-1 and IL-8 in vaccinated pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon juveniles

Laboratory-reared pink and chum salmon juveniles (approximately 2g) received an intraperitoneal injection with a commercial, unadjuvanted Aeromonas salmonicida bacterin or sterile saline. Relative to elongation factor-1A, expression levels of genes encoding the proinflammatory cytokines interleukin-1beta-1 (IL-1beta), tumour necrosis factor-alpha-1 (TNFalpha) and interleukin-8 (IL-8) in pools of kidney and liver were examined 6- and 24-h after injection. Expression of IL-1beta was significantly elevated in pink and chum salmon by 6-h, and declined in pink salmon but not in chum salmon by 24-h. Similarly, expression of TNFalpha was significantly elevated in both species at 6h and only in chum salmon after 24-h. Expression of IL-8 was significantly elevated in both species at 6- and 24-h after injection. Expression of the three proinflammatory cytokine genes differed between salmon species both in the timing and magnitude of their expression. The significance of these differences with respect to immune function in these fish requires further research.     

Molecular characterization of a pathogenesis-related protein 8 gene encoding a class III chitinase in rice

A cDNA encoding a class III chitinase (Oschib1) was isolated from a cDNA library constructed from rice leaves infected with the blast fungus Magnaporthe grisea. The cDNA contains an open reading frame of 861 nucleotides encoding 286 amino acid residues with a pI of 5.06. The deduced amino acid sequence of Oschibl has a high level of similarity with class IIIb chitinases of Gladiolus gandavensis (46%) and Tulipa bakeri (49%). A high level of Oschibl mRNA was detected after inoculation with M. grisea or Xanthomonas oryzae pv. oryzae. Expression of Oschib1 was induced more rapidly when an avirulent strain of M. grisea was inoculated (incompatible interaction) than when a virulent strain was used (compatible interaction). Expression of Oschibl was also induced by treatment of signaling molecules such as salicylic acid, ethylene, and methyl jasmonic acid, and by treatment with H2O2 or CuSO4. The induction patterns of Oschibl expression suggest that Oschib1 may be involved in defense response against pathogen infections and may be classified as a member of pathogenesis-related protein 8 in rice.     

Expression pattern of the coparyl diphosphate synthase gene in developing rice anthers

Rice anthers contain high concentrations of gibberellins A(4) and A(7). To understand their physiological roles, we examined the site of their biosynthesis by analyzing the expression pattern of a gene (OsCPS) encoding coparyl diphosphate synthase in developing rice flowers. Expression was apparent in the anthers 1-2 days before flowering, and CPS mRNA accumulated in the maturing pollen.     

烟草NtCBL1基因的克隆、表达载体构建及表达分析

CBL是一类Ca²⁺感受蛋白,在植物适应或抵制逆境胁迫的过程中发挥重要的作用。从烟草品种K326中克隆到了一个CBL1的同源基因,该序列包含了一个642 bp的开放阅读框,编码一个由213个氨基酸残基组成的蛋白,预测分子量为24.5 kDa,等电点为5.03。同源性分析结果显示,该基因与林烟草CBL1、甜辣椒CBL1等具有较高的同源性,故命名为NtCBL1。生物信息学分析表明,NtCBL1具有CBL家族保守的EF-hand钙结合结构域。组织表达分析发现该基因在成熟期的根、茎、叶、花中均有表达,在根中的表达量最高。逆境胁迫实验表明,该基因表达受低钾、高盐、干旱、ABA和低温诱导调控,参与烟草生物与非生物逆境胁迫的响应。并成功构建了NtCBL1-pBI121过表达载体。研究结果为解析NtCBL1在响应逆境胁迫的功能奠定一定理论基础。     

Molecular determinants of nuclear protein phosphatase-1 regulation by NIPP-1.

NIPP-1 is a subunit of the major nuclear protein phosphatase-1 (PP-1) in mammalian cells and potently inhibits PP-1 activity in vitro. Using yeast two-hybrid and co-sedimentation assays, we mapped a PP-1-binding site and the inhibition function to the central one-third domain of NIPP-1. Full-length NIPP-1 (351 residues) and the central domain, NIPP-1(143-217), were equally potent PP-1 inhibitors (IC50 = 0.3 nM). Synthetic peptides spanning the central domain of NIPP-1 further narrowed the PP-1 inhibitory function to residues 191-200. A second, noninhibitory PP-1-binding site was identified by far-Western assays with digoxygenin-conjugated catalytic subunit (PP-1C) and included a consensus RVXF motif (residues 200-203) found in many other PP-1-binding proteins. The substitutions, V201A and/or F203A, in the RVXF motif, or phosphorylation of Ser199 or Ser204, which are established phosphorylation sites for protein kinase A and protein kinase CK2, respectively, prevented PP-1C-binding by NIPP-1(191-210) in the far-Western assay. NIPP-1(191-210) competed for PP-1 inhibition by full-length NIPP-1(1-351), inhibitor-1 and inhibitor-2, and dissociated PP-1C from inhibitor-1- and NIPP-1(143-217)-Sepharose but not from full-length NIPP-1(1-351)-Sepharose. Together, these data identified some of the key elements in the central domain of NIPP-1 that regulate PP-1 activity and suggested that the flanking sequences stabilize the association of NIPP-1 with PP-1C.     

Large-scale propagation of recombinant adherent cells that secrete a stable form of human glandular kallikrein, hK2

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease that is expressed predominantly in the prostate epithelium and has 78% aa identity with prostate-specific antigen (PSA). hK2 has been recognized as a potential prostate cancer marker and has been demonstrated to be highly expressed in prostate cancer compared to benign prostatic tissue. Purification and characterization of hK2 have been impeded due to its lower expression in bodily fluids and tissues compared to PSA and its ability to autodegrade. Therefore, to study biochemical and biological characteristics of hK2, a stable and enzymatically inactive mutant form of hK2, hK2(A217V), was expressed in a hamster cell line, AV12-664 (AV12-hK2(A217V)). AV12-hK2(A217V) cells secreted prohK2(A217V) (phK2(A217V)) in the spent medium at approximately 2.5 microgram/ml. Since AV12-hK2(A217V) are adherent cells, it was necessary to develop an efficient system to propagate large numbers of cells to obtain significant quantities of phK2(A217V). In this paper, we compared ceramic core bioreactor and microcarrier beads as alternatives to static culture to propagate adherent cells. Considering production levels, ease of operation, cost effectiveness, and labor, microcarrier beads were found to be a better alternative. Our findings led to the development of a general protocol for large-scale propagation of adherent cells on microcarrier beads eliminating the need for propagating AV12-hK2(A217V) in culture flasks or bioreactors. Microcarrier beads coated with AV12-hK2(A217V) cells could be propagated in 1- or 3-liter spinner flasks and were passed from one spinner to the next in a manner analogous to static culture or could be frozen and later used as inoculum for subsequent spinners. Using this protocol, >40 liters of spent medium was harvested within 30 days, which in turn was used to purify phK2(A217V). phK2(A217V) purified from spent medium of cells grown either on microcarrier beads or in culture flasks were biochemically similar as indicated by HIC-HPLC profile followed by sequencing of relevant peaks.