Genome Sequence of Pectin-Degrading Alishewanella agri, Isolated from Landfill Soil

Alishewanella agri BL06(T) (= KCTC 22400(T) = JCM 15597(T)) was isolated from landfill soil in Pohang, South Korea. A. agri showed the ability to degrade pectin, a structural heteropolysaccharide present in the cell wall of plants. Here we report the genome sequence of Alishewanella agri BL06(T), the second sequenced strain in the genus Alishewanella.     

嗜盐碱球菌湛江新种的系统分类鉴定

根据经典微生物划线纯培养方法从湛江一个盐田泥样中分离获得1个嗜盐碱古菌菌株SCSIOCD13T,通过形态、细胞化学分类及分子发育等对其进行系统分类鉴定研究。经鉴定该菌细胞球形,好氧,革兰染色不定,在最适pH 8.5和37℃培养条件下,能够耐受2.5～5.5 mol/L盐浓度生长。16S rRNA基因序列比对分析显示,该菌株与嗜盐碱古菌Natronococcus属成员N.jeotgali B1T(99.2%),N.occultus NCMB 2192T(96.8%)和N.amylolyticus Ah-36T(95.8%)最相近,且系统发育树和细胞化学特征与Natronococcus属相一致。然而,系统发育树上显示该菌株与最相似菌种形成单独分支,且生理学特性显示出与最相似的3个物种具有明显的差异,包括盐浓度、pH耐受,镁离子需求,H2S产生,淀粉、明胶水解,抗生素敏感性等。另外,杂交结果显示,菌株SCSIO CD13T与最相似菌种Natronococcus jeotgali B1T的杂交值为23%;综合多相分类结果,菌株SCSIO CD13T应为Natronococcus属的一个新成员,将其命名为湛江嗜盐碱球菌新种(Natronococcus zhanjian-gensis),典型菌株SCSIO CD13T。     

Genome sequence of Lentibacillus jeotgali Grbi(T), isolated from traditional Korean salt-fermented seafood

Lentibacillus jeotgali Grbi(T), isolated from a traditional Korean salt-fermented seafood, is a strictly aerobic, Gram-positive, nonmotile, endospore-forming, moderately halophilic bacterium belonging to the family Bacillaceae in the phylum Firmicutes. Here, the draft genome sequence of L. jeotgali Grbi(T) (3,775,822 bp with a G+C content of 42.5%) is reported. This is the first reported genome sequence from a Lentibacillus species.     

Genome Sequence of Pectin-Degrading Alishewanella aestuarii Strain B11T, Isolated from Tidal Flat Sediment

We present the genome sequence of Alishewanella aestuarii B11(T) (=KCTC 22051(T)=DSM 19476(T)). This species, isolated from tidal flat sediment, was reported to be a novel species. A. aestuarii is known to degrade pectin, an important component of plant cell wall. The presence of the genes related to pectin metabolism in this strain indicates its capability to utilize pectin.     

Isolation and characterization of Bacillus thioparus sp. nov., chemolithoautotrophic, thiosulfate-oxidizing bacterium

A novel bacterium, strain BMP-1(T), was isolated from a continuous wastewater treatment culture system operating with a bacterial consortium. Cells of the isolate were Gram-variable, aerobic, moderately halotolerant, motile and endospore-forming rods. Strain BMP-1(T) grew chemolithoautotrophically by oxidation of thiosulfate to sulfate with a growth yield of 1.07 g protein mol(-1) of thiosulfate consumed. DNA G+C content was 43.8 mol%. Its cell wall had peptidoglycan based on m-diaminopimelic acid, and the major component of fatty acid was C(15 : 0). The 16S rRNA gene analysis showed that strain belongs to the genus Bacillus, sharing a 99.5% of sequence similarity with Bacillus jeotgali CCM 7133(T). DNA-DNA hybridization between the isolate of this study and this strain was 44%. Thus, the inclusion of strain BMP-1(T) in the genus Bacillus is suggested as a novel species and the name Bacillus thioparus sp. nov. (Type strain BMP-1(T)=BM-B-436(T)=CECT 7196(T)) is proposed. The sequence of the 16S rRNA gene has been deposited in GenBank with accession number DQ371431.     

Cadmium and zinc removal from aqueous solutions by Bacillus jeotgali: pH, salinity and temperature effects

Effects of pH, salinity and temperature on biosorption of Cd and Zn by bacteria Bacillus jeotgali strain U3 were evaluated in batch experiments. Traditional and Subsequent Addition Methods (SAM) were used to carry out the bioassays. Sorption of metals was higher when pH or temperature was increased, or when salinity was reduced. The Langmuir isotherm better fit the biosorption data for Cd, while the Freundlich model fitted better for Zn biosorption. A comparison with similar biosorbents suggested that Bacillus jeotgali strain U3 could be considered a good biosorbent for Cd and Zn recovery.     

Arsukibacterium ikkense gen. nov., sp. nov, a novel alkaliphilic, enzyme-producing gamma-Proteobacterium isolated from a cold and alkaline environment in Greenland

A novel aerobic, Gram-negative, non-pigmented bacterium, GCM72(T), was isolated from the alkaline, low-saline ikaite columns in the Ikka Fjord, SW Greenland. Strain GCM72(T) is a motile, non-pigmented, amylase- and protease-producing, oxidase-positive, and catalase-negative bacterium, showing optimal growth at pH 9.2-10.0, at 15 degrees C, and at 3% (w/v) NaCl. Major fatty acids were C(12:0) 3-OH (12.2+/-0.1%), C(16:00) (18.0+/-0.1%), C(18:1)omega7c (10.7+/-0.5%), and summed feature 3 comprising C(16:1)omega7c and/or iso-C(15:0) 2-OH (36.3+/-0.7%). Phylogenetic analysis based on 16S rRNA gene sequences showed that isolate GCM72(T) was most closely related to Rheinheimera baltica and Alishewanella fetalis of the gamma-Proteobacteria with a 93% sequence similarity to both. The G+C content of DNA isolated from GCM72(T) was 49.9mol% and DNA-DNA hybridization between GCM72T and R. baltica was 9.5%. Fatty acid analysis and G+C content supports a relationship primarily to R. baltica, but several different features, such as a negative catalase-response and optimal growth at low temperature and high pH, together with the large phylogenetic distance and low DNA similarity to its closest relatives, lead us to propose a new genus, Arsukibacterium, gen. nov., with the new species Arsukibacterium ikkense sp. nov. (type strain is GCM72(T)).     

Limited pattern of TCR delta chain gene rearrangement on the RNA level in multiple sclerosis

Susceptibility to multiple sclerosis (MS) is most likely affected by a number of genes, including HLA and T-cell receptor (TCR) genes. T cells expressing gamma/delta receptors seem to contribute to autoagression in MS, as evidenced by their localization in the MS plaques in the brain. The aim of this study was to analyse the TCRdelta chain gene rearrangement at the RNA (cDNA) level and compare to the DNA pattern rearrangement. TCRdelta gene rearrangement was analysed in MS patients and healthy individuals with the use of primers specific for Vdelta1-6 and Jdelta1 genes (at the DNA level) and specific for Vdelta1-6 and Cdelta1 genes (at the cDNA level). The size of PCR products was analysed on agarose gel and by ALF-Express (Pharmacia). Additionally, the lymphocyte surface immunophenotype was studied with specific monoclonal antibodies. At the DNA level a restricted pattern of Vdelta3-Jdelta1 and Vdelta5-Jdelta1 was found only in MS patients. Contrary to DNA, mono-, oligoclonal RNA (cDNA) rearrangements were limited to Vdelta1-Cdelta1, Vdelta2-Cdelta1 and Vdelta3-Cdelta1 only in MS patients as well. Surface immunophenotype analysis revealed in MS a much higher frequency of activated gamma/delta T lymphocytes, i.e. expressing HLA-DR and CD25. An elevated level of CD56 positive cells in MS was recorded. Mono-oligoclonal pattern of TCRdelta gene rearrangement at the RNA level, along with increase in activated gamma/delta T cells, strongly argue for a significant role of gamma/delta T lymphocytes in the pathogenesis of MS.     

IL2RA Genetic Heterogeneity in Multiple Sclerosis and Type 1 Diabetes Susceptibility and Soluble Interleukin-2 Receptor Production

Multiple sclerosis (MS) and type 1 diabetes (T1D) are organ-specific autoimmune disorders with significant heritability, part of which is conferred by shared alleles. For decades, the Human Leukocyte Antigen (HLA) complex was the only known susceptibility locus for both T1D and MS, but loci outside the HLA complex harboring risk alleles have been discovered and fully replicated. A genome-wide association scan for MS risk genes and candidate gene association studies have previously described the IL2RA gene region as a shared autoimmune locus. In order to investigate whether autoimmunity risk at IL2RA was due to distinct or shared alleles, we performed a genetic association study of three IL2RA variants in a DNA collection of up to 9,407 healthy controls, 2,420 MS, and 6,425 T1D subjects as well as 1,303 MS parent/child trios. Here, we report “allelic heterogeneity” at the IL2RA region between MS and T1D. We observe an allele associated with susceptibility to one disease and risk to the other, an allele that confers susceptibility to both diseases, and an allele that may only confer susceptibility to T1D. In addition, we tested the levels of soluble interleukin-2 receptor (sIL-2RA) in the serum from up to 69 healthy control subjects, 285 MS, and 1,317 T1D subjects. We demonstrate that multiple variants independently correlate with sIL-2RA levels.     

The C609T inborn polymorphism in NAD(P)H:quinone oxidoreductase 1 is associated with susceptibility to multiple sclerosis and affects the risk of development of the primary progressive form of the disease

Oxidative stress plays a pivotal role in the pathogenesis of multiple sclerosis (MS). Inactivating polymorphisms of genes encoding detoxification enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), could influence susceptibility to MS. To test this hypothesis we performed a case-control study in which we compared the distribution of NQO1 genotypes between 231 MS patients and 380 controls, using both PCR-RFLP and real-time PCR assays. Correlations with MS clinical subtype classification and gender were also evaluated. A significantly higher frequency of the homozygous (T/T) and heterozygous (C/T) NQO1 C⁶⁰⁹T variant genotypes was observed among MS patients compared to controls (P = 0.01), with MS patients showing a 1.5-fold increased risk of carrying at least one variant T allele (P = 0.009). Interestingly, patients belonging to the primary progressive subgroup exhibited a significantly higher incidence of the heterozygous C/T variant genotype, compared to the other forms of MS (P = 0.019). There was no correlation of the NQO1 polymorphism with gender. These results provide the first evidence for a pathogenetic role for the NQO1 C⁶⁰⁹T polymorphism in MS susceptibility and suggest a possible role for the NQO1 genetic background in the development of primary progressive MS.     

Inactivation of hepatitis A HM-175/18f, reovirus T1 Lang and MS2 during alkaline stabilization of human biosolids

AIM: To compare the inactivation rates of male-specific bacteriophage-2 (MS2), hepatitis A HM-175/18f (HM-175) and reovirus T1 Lang (T1 L) during alkaline stabilization of wastewater residues. METHODS AND RESULTS: A bench scale alkaline stabilization model was used to evaluate the inactivation of MS2 seeded into raw sludge simultaneously with HM-175 or T1 L. Stabilization was performed in triplicate at 28 and 4 degrees C for both viral combinations. During stabilization at 28 and 4 degrees C, MS2 and T1 L concentrations were similar at each time point (t = 0.1, 2, 12 and 24 h). MS2 and HM-175 concentrations were also similar at each time point during stabilization at 28 degrees C. At 4 degrees C, MS2 and HM-175 concentrations were not similar at the first two time points (t = 0.1 and 2 h), but were similar at later time points (t = 12 and 24 h). CONCLUSIONS: The inactivation rates of T1 L at 4 degrees C and both T1 L and HM-175 at 28 degrees C were similar to the inactivation rate of MS2 at all time points. At 4 degrees C, MS2 was inactivated at a faster rate during the first two time points (t = 0.1 and 2 h) than HM-175, but was inactivated similarly at later time points (t = 12 and 24 h). SIGNIFICANCE AND IMPACT OF THE STUDY: Phages, such as MS2, would be ideal indicators for the presence of enteric viruses in wastewater residues because of their ubiquity, nonpathogenic nature, low cost and time associated with their detection. The findings of this study suggest that MS2 could serve as an indicator for monitoring the persistence of enteric viruses, such as HM-175 and T1 L, during alkaline stabilization performed at moderate temperatures (28 degrees C), but may not serve as an indicator for HM-175 at reduced temperature (4 degrees C). The utility of MS2 as an indicator of viral persistence during biosolids treatment should be further evaluated, as the increased efficiency and frequency of pathogen monitoring associated with their use may reduce the potential public health risk associated with biosolids, facilitating a greater acceptance for their land application.     

Increased CD8+ T Cell Response to Epstein-Barr Virus Lytic Antigens in the Active Phase of Multiple Sclerosis

It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.     

T and B cell responses to myelin-oligodendrocyte glycoprotein in multiple sclerosis

The pathogenesis of multiple sclerosis (MS) is believed to involve an autoimmune component directed against the myelin sheath. One potential target Ag for such autoimmune attack is the myelin-oligodendrocyte glycoprotein (MOG) because an anti-MOG mAb has profound influence on the course of experimental autoimmune encephalomyelitis, which to some extent represents an experimental model of MS. Using single cell assays, we have evaluated T and B cell reactivities to MOG in MS patients and controls. T cell reactivity was estimated by counting the number of cells that secreted IFN-gamma in response to MOG, whereas B cell reactivity was estimated by enumerating cells secreting antibodies that bound to MOG. MOG reactive T cells were detected in the peripheral blood of the majority of the 16 MS patients examined (mean 1/7299 mononuclear cells), but infrequently and at lower numbers in samples from neurologic controls. MOG-reactive T cells were more frequent among MS patients' cerebrospinal fluid (CSF) mononuclear cells (mean 1/450 cells). The T cell response to MOG was evidently MHC class II restricted, because Fab fragments of a rabbit polyclonal anti HLA-DR antibodies abrogated the Ag-induced increase in number of cells that secreted IFN-gamma, as analyzed on CSF and PBMC from three patients with MS. Anti-MOG IgG antibody-secreting cells were detected in blood in 8 of 16 MS patients (mean 1/25,641 cells), but they were also strongly accumulated in CSF, being detected in 8 of 10 MS patients examined (mean 1/265 cells), while rarely found in controls. The findings imply that MOG may represent a pathogenetically important target Ag in MS.     

Increased frequencies of myelin oligodendrocyte glycoprotein/MHC class II-binding CD4 cells in patients with multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease characterized by infiltration of pathogenic immune cells in the CNS resulting in destruction of the myelin sheath and surrounding axons. We and others have previously measured the frequency of human myelin-reactive T cells in peripheral blood. Using T cell cloning techniques, a modest increase in the frequency of myelin-reactive T cells in patients as compared with control subjects was observed. In this study, we investigated whether myelin oligodendrocyte glycoprotein (MOG)-specific T cells could be detected and their frequency was measured using DRB1*0401/MOG(97-109(107E-S)) tetramers in MS subjects and healthy controls expressing HLA class II DRB1*0401. We defined the optimal culture conditions for expansion of MOG-reactive T cells upon MOG peptide stimulation of PMBCs. MOG(97-109)-reactive CD4(+) T cells, isolated with DRB1*0401/MOG(97-109) tetramers, and after a short-term culture of PMBCs with MOG(97-109) peptides, were detected more frequently from patients with MS as compared with healthy controls. T cell clones from single cell cloning of DRB1*0401/MOG(97-109(107E-S)) tetramer(+) cells confirmed that these T cell clones were responsive to both the native and the substituted MOG peptide. These data indicate that autoantigen-specific T cells can be detected and enumerated from the blood of subjects using class II tetramers, and the frequency of MOG(97-109)-reactive T cells is greater in patients with MS as compared with healthy controls.     

Expression of TRAIL receptors in human autoreactive and foreign antigen-specific T cells

Deletion of T cells due to apoptosis induction is a regulatory mechanism in the human immune system that may be impaired in autoimmune diseases such as multiple sclerosis (MS). Involvement of the apoptosis-mediating CD95/CD95 ligand system in MS has been demonstrated. Here, we report that (auto)antigen-specific human T cells are not killed in vitro by soluble TNF-related apoptosis-inducing ligand (TRAIL) although expressing death-inducing receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2. Apoptosis was assessed by caspase activation and DNA fragmentation, receptor expression was detected by RT - PCR and flow cytometry. The (auto)antigen-specific T cells were also resistant to specific TRAIL-R1/TRAIL-R2-directed induction of apoptosis, indicating that coexpression of the truncated TRAIL-R3 and TRAIL-R4 in these T cells is not responsible for the observed resistance. Upon stimulation, levels of death-inducing TRAIL receptors decreased whereas TRAIL was up-regulated on the cell surface. In contrast to CD95, the role of TRAIL receptors in MS might not involve regulation of T cell vulnerability.     

Lack of TIM-3 immunoregulation in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory disease of the CNS white matter associated with T cell infiltrates and alterations of immune functions that can be measured in the peripheral immune system. TIM-3 has been identified as a central regulator of IFN-gamma-secreting type 1 Th (Th1) cells and immune tolerance. In this study, using a newly generated mAb against human TIM-3, we examined TIM-3 function on ex vivo CD4(+) T cells isolated from the circulation of healthy subjects and patients with MS. Blocking TIM-3 during T cell stimulation significantly enhanced IFN-gamma secretion in control subjects but had no effect in untreated patients with MS, demonstrating a defect in TIM-3 immunoregulation. Treatment with glatiramer acetate or IFN-beta reversed this functional defect. Reduced levels and altered kinetics of T cell TIM-3 expression, which was restored in treated patients, is one mechanism that can explain the loss of TIM-3 regulation of T cell function in untreated patients with MS. These data provide functional, mechanistic data for dysregulated TIM-3 immunoregulation in a human autoimmune disease and suggest that approved therapies for the treatment of MS may function in part by restoring TIM-3 immunoregulation of T cell function.     

CD28 costimulation is crucial for the development of spontaneous autoimmune encephalomyelitis

Multiple sclerosis (MS) is a severe central nervous system disease. Experimental autoimmune encephalomyelitis (EAE) mimics MS in mice. We report that spontaneous development of EAE in RAG-1-deficient mice transgenic for a myelin basic protein (MBP)-specific TCR (TgMBP+/RAG-1-/-) requires expression of the T cell costimulatory molecule CD28. Surprisingly, T cells from CD28-/-TgMBP+/RAG-1-/- mice proliferate and produce IL-2 in response to MBP1-17 peptide in vitro, excluding clonal anergy as the mechanism of CD28-regulated pathogenesis. Proliferation of autoaggressive T cells was dependent on the concentration of the MBP peptide, as was the development of MBP-induced EAE in CD28-deficient PL/J mice. These results provide the first genetic evidence that CD28 costimulation is crucial for MBP-specific T cell activation in vivo and the initiation of spontaneous EAE.     

Analysis of the association of allelic variants of apolypoprotein E and interleukin 1 beta genes with multiple sclerosis in ethnic Tatars

Multiple sclerosis (MS) is a multifactorial disease of the central nervous system. The apolipoprotein E (APOE) and interleukin 1 beta (IL1B) genes are considered to be candidate genes of MS. The aim of the study was to examine the hypothesis of the importance of APOE and IL1B gene polymorphisms in MS development in ethnic Tatars. DNA samples isolated by phenol-chloroform extraction from peripheral blood of 383 ethnic Tatars (120 MS patients and 263 healthy donors) were studied. 112C/R and 158R/C APOE gene polymorphisms as well as -511T/C IL1B gene polymorphism were analyzed by polymerase chain reaction (PCR) followed by PCR product digestion by endonuclease. Odds ratio (OR) values were used for evaluation of the relative risk of alleles and(or) genotype combinations. It has been shown that APOE*2/*3 genotype is associated with low risk of the disease development (OR = 0.20) in women. A combined effect of APOE and IL1B allelic variants has been discovered indicating the increased risk of the disease development in the carriers of APOE*4 and IL1B*T/*T alleles (OR = 4.76).     

LncRNAs associated with multiple sclerosis expressed in the Th1 cell lineage

Multiple sclerosis (MS) is a type of inflammatory and demyelinating disorder of the central nervous system in which immune-mediated inflammatory processes are elicited by secreted cytokines from T helper (Th)-1 and Th17 cells. While some protein-coding genes expressed in T cell types have established involvement in MS disease progression, little is understood about the roles of long noncoding RNAs (lncRNAs) within the disease landscape. LncRNAs, noncoding RNAs longer than 200 nucleotides, likely control gene expression and function of Th1 cells, and offer the potential to act as therapeutic and biomarker candidates for MS. We identified lncRNAs in Th1 cells linked to MS. Expression levels of candidate lncRNAs and genes were evaluated in 50 MS patients and 25 healthy controls using quantitative real-time polymerase chain reaction, and their correlations were assessed. LncRNAs encoded by AC007278.2 and IFNG-AS1-001 showed significantly higher expression in relapsing Phase MS patients whereas IFNG-AS1-003 was elevated in patients in the remitting phase compared with relapsing patients. Collectively, these misregulated lncRNAs may provide valuable tools to understand the relationships between lncRNAs and MS, and possibly other related disorders.