Evidence of Extensive Circulation of Yersinia enterocolitica in Rodents and Shrews in Natural Habitats from Retrospective and Perspective Studies in South Caucasus.

Yersinia enterocolitica culture-positive rodents and shrews were reported in different territories across Georgia during 14 of 17 years of investigations conducted for the period of 1981-1997. In total, Y. enterocolitica was isolated from 2052 rodents (15 species) and 33 shrews. Most isolates were obtained from Microtus arvalis, Rattus norvegicus, Mus musculus, and Apodemus spp. During the prospective study (2017-2019), isolates of Yersinia-like bacteria were cultured from 53 rodents collected in four parts of Georgia. All the Yersinia-like isolates were confirmed as Y. enterocolitica based on the API 20E and the BD Phenix50 tests. Whole-genome (WG) sequencing of five rodents and one shrew strain of Y. enterocolitica revealed that they possessed a set of virulence genes characteristic of the potentially pathogenic strains of biogroup 1A. All isolates lacked distinguished virulence determinants for YstA, Ail, TccC, VirF, and virulence plasmid pYV but carried the genes for YstB, YmoA, HemPR-HmuVSTU, YaxAB, PhlA, PldA, ArsCBR, and a flagellar apparatus. One strain contained a gene highly homologous to heat-labile enterotoxin, a chain of E. coli, a function not previously described for Y. enterocolitica. The WG single-nucleotide polymorphism-based typing placed the isolates in four distinct phylogenetic clusters.

Identification of a Novel Yersinia enterocolitica Strain from Bats in Association with a Bat Die-Off That Occurred in Georgia (Caucasus).

Yersinia entercolitica is a bacterial species within the genus Yersinia, mostly known as a human enteric pathogen, but also recognized as a zoonotic agent widespread in domestic pigs. Findings of this bacterium in wild animals are very limited. The current report presents results of the identification of cultures of Y. entercolitica from dead bats after a massive bat die-off in a cave in western Georgia. The growth of bacterial colonies morphologically suspected as Yersinia was observed from three intestine tissues of 11 bats belonging to the Miniopterus schreibersii species. These three isolates were identified as Y. enterocolitica based on the API29 assay. No growth of Brucella or Francisella bacteria was observed from tissues of dead bats. Full genomes (a size between 4.6-4.7 Mbp) of the Yersinia strains isolated from bats were analyzed. The phylogenetic sequence analyses of the genomes demonstrated that all strains were nearly identical and formed a distinct cluster with the closest similarity to the environmental isolate O:36/1A. The bat isolates represent low-pathogenicity Biotype 1A strains lacking the genes for the Ail, Yst-a, Ysa, and virulence plasmid pYV, while containing the genes for Inv, YstB, and MyfA. Further characterization of the novel strains cultured from bats can provide a clue for the determination of the pathogenic properties of those strains.

Seroepidemiology, Spatial Distribution, and Risk Factors of Francisella tularensis in Jordan.

There is a paucity of data on Francisella tularensis in the Middle East and North Africa. This is the first countrywide study to determine the seroprevalence, spatial distribution, and risk factors for F. tularensis in Jordan. A total of 828 Jordanians were serologically tested for F. tularensis by ELISA. These individuals filled out a self-administered questionnaire to collect demographic and risk factor information. Bivariate and multivariate logistic regressions were performed to determine which variables are associated with seropositivity. The overall seroprevalence of F. tularensis was 7.7% (95% CI: 6.10-9.75). The bivariate analyses showed that age, region of residence, small ruminant ownership, and practicing horticulture were significantly associated with seropositivity, and these variables were controlled for in the multivariate analysis. The multivariate analysis showed an increased odds of seropositivity among individuals living in northern desert, middle, and northern highland areas, compared with individuals living in the drier southern area, as 7.27 (95% CI: 2.49-21.19), 3.79 (95% CI: 1.53-9.39), and 3.52 (95% CI: 1.45-388.55), respectively. Individuals owning a small ruminant had 1.86 (95% CI: 1.02-3.40) greater odds for seropositivity than individuals who do not own a small ruminant. Individuals practicing horticulture had 2.10 (95% CI: 1.20-3.66) greater odds for seropositivity than individuals who do not practice horticulture. This is the first study to address the seroprevalence of F. tularensis in Jordan and the Middle East. Further research is needed to identify clinical cases of tularemia in Jordan and to determine the circulating F. tularensis subspecies.

Seroprevalence and Risk Factors for Coxiella burnetii in Jordan.

This is the first cross-sectional study of the seroprevalence and risk factors for Coxiella burnetii in Jordan. A total of 781 individuals from 11 governorates of Jordan were tested by SERION ELISA classic C. burnetii IgG Phase 2. A validated and pretested questionnaire was used to collect risk factors and demographic data. The overall seroprevalence for C. burnetii was 24.2% (95% CI; 21.3-27.3%). Unadjusted odds ratios showed that governorate of residence, consumption of raw milk, and ownership of sheep, goats, and dogs were significantly (P ≤ 0.05) associated with C. burnetii seropositivity. The multivariate logistic regression showed that individuals who own small ruminants had three times greater odds of seropositivity than those who do not own a small ruminant, after controlling for age, gender, raw milk consumption, and ownership of dogs. In addition, individuals who live in Al-Karak, Az-Zarqa, and Al-Tafilah had significantly greater odds of seropositivity compared with individuals who live in the capital city, Amman (OR = 3.6, 4.8, and 2.7, respectively). This study suggests that preventive measures should be practiced in ruminant farms in Jordan to avoid C. burnetii infection. Coxiella burnetii should also be considered in the differential diagnosis of febrile-like illnesses in Jordan, especially among farmers and veterinarians.

Synthesis and evaluation of the anti-inflammatory properties of selenium-derivatives of celecoxib.

Celecoxib is a selective cyclooxygenase (COX)-2 inhibitor used to treat inflammation, while selenium is known to down-regulate the transcription of COX-2 and other pro-inflammatory genes. To expand the anti-inflammatory property, wherein celecoxib could inhibit pro-inflammatory gene expression at extremely low doses, we incorporated selenium (Se) into two Se-derivatives of celecoxib, namely; selenocoxib-2 and selenocoxib-3. In vitro kinetic assays of the inhibition of purified human COX-2 activity by these compounds indicated that celecoxib and selenocoxib-3 had identical K(I) values of 2.3 and 2.4µM; while selenocoxib-2 had a lower K(I) of 0.72µM. Furthermore, selenocoxib-2 inhibited lipopolysaccharide-induced activation of NF-κB leading to the down-regulation of expression of COX-2, iNOS, and TNFα more effectively than selenocoxib-3 and celecoxib in RAW264.7 macrophages and murine bone marrow-derived macrophages. Studies with rat liver microsomes followed by UPLC-MS-MS analysis indicated the formation of selenenylsulfide conjugates of selenocoxib-2 with N-acetylcysteine. Selenocoxib-2 was found to release minor amounts of Se that was effectively inhibited by the CYP inhibitor, sulphaphenazole. While these studies suggest that selenocoxib-2, but not celecoxib and selenocoxib-3, targets upstream events in the NF-κB signaling axis, the ability to effectively suppress NF-κB activation independent of cellular selenoprotein synthesis opens possibilities for a new generation of COX-2 inhibitors with significant and broader anti-inflammatory potential.

Gambogic acid covalently modifies IkappaB kinase-beta subunit to mediate suppression of lipopolysaccharide-induced activation of NF-kappaB in macrophages.

GA (gambogic acid) is a polyprenylated xanthone abundant in the resin of Garcinia morella and Garcinia hanburyi with a long history of use as a complementary and alternative medicine. The antitumour activity of GA has been well demonstrated and is thought to arise partly from the associated anti-inflammatory activity. Recent studies have indicated that the antitumour activity of GA is mediated by its ligation of TfR1 (transferrin receptor-1). Since the cellular expression of TfR1 is down-regulated by LPS (lipopolysaccharide), we hypothesized that an alternative pathway exists in immune cells, such as macrophages, where GA could mitigate the expression of pro-inflammatory genes. Here we demonstrate that GA inhibits the LPS-dependent expression of NF-kappaB (nuclear factor kappaB) target pro-inflammatory genes in macrophages. Western immunoblot, NF-kappaB-luciferase reporter and gel-shift analyses revealed that GA strongly blocked the activation of NF-kappaB induced by LPS, whereas 9,10-dihydro-GA, which lacks the reactive alpha,beta-unsaturated carbonyl group, was ineffective. Moreover, GA was able to decrease nuclear p65 levels in RAW264.7 macrophages, where the expression of TfR1 was down-regulated by RNA interference. in vitro kinase assays coupled with interaction studies using biotinylated GA as well as proteomic analysis demonstrated that IKKbeta [IkappaB (inhibitory kappaB) kinase-beta], a key kinase of the NF-kappaB signalling axis, was covalently modified by GA at Cys-179, causing significant inhibition of its kinase activity. Taken together, these results demonstrate the potent anti-inflammatory activity of GA.

Selenium attenuates pro-inflammatory gene expression in macrophages.

Selenium (Se) is an important element required for the optimal functioning of the immune system. Particularly in macrophages, which play a pivotal role in immune regulation, Se acts as a major antioxidant in the form of selenoproteins to mitigate the cytotoxic effects of reactive oxygen species. Here we describe the role of Se as an anti-inflammatory agent and its effect on the macrophage signal transduction pathways elicited by bacterial endotoxin, LPS. Our studies demonstrate that supplementation of Se to macrophages (Se-deficient) leads to a significant decrease in the LPS-induced expression of two important pro-inflammatory genes, cyclooxygenase-2 (COX-2) and tumor necrosis factor-alpha (TNF-alpha) via the inhibition of MAP kinase pathways. Furthermore, Se-deficiency in mice exacerbated the LPS-mediated infiltration of macrophages into the lungs suggesting that Se status is a crucial host factor that regulates inflammation. In summary, our results indicate that Se plays an important role as an anti-inflammatory agent by tightly regulating the expression of pro-inflammatory genes in immune cells.

The anti-inflammatory effects of selenium are mediated through 15-deoxy-Delta12,14-prostaglandin J2 in macrophages.

Selenium is an essential micronutrient that suppresses the redox-sensitive transcription factor NF-kappaB-dependent pro-inflammatory gene expression. To understand the molecular mechanisms underlying the anti-inflammatory property of selenium, we examined the activity of a key kinase of the NF-kappaB cascade, IkappaB-kinase beta (IKKbeta) subunit, as a function of cellular selenium status in murine primary bone marrow-derived macrophages and RAW264.7 macrophage-like cell line. In vitro kinase assays revealed that selenium supplementation decreased the activity of IKKbeta in lipopolysaccharide (LPS)-treated macrophages. Stimulation by LPS of selenium-supplemented macrophages resulted in a time-dependent increase in 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) formation, an endogenous inhibitor of IKKbeta activity. Further analysis revealed that inhibition of IKKbeta activity in selenium-supplemented cells correlated with the Michael addition product of 15d-PGJ2 with Cys-179 of IKKbeta, while the formation of such an adduct was significantly decreased in the selenium-deficient macrophages. In addition, anti-inflammatory activities of selenium were also mediated by the 15d-PGJ2-dependent activation of the peroxisome proliferator-activated nuclear receptor-gamma in macrophages. Experiments using specific cyclooxygenase (COX) inhibitors and genetic knockdown approaches indicated that COX-1, and not the COX-2 pathway, was responsible for the increased synthesis of 15d-PGJ2 in selenium-supplemented macrophages. Taken together, our results suggest that selenium supplementation increases the production of 15d-PGJ2 as an adaptive response to protect cells against oxidative stress-induced pro-inflammatory gene expression. More specifically, modification of protein thiols by 15d-PGJ2 represents a previously undescribed code for redox regulation of gene expression by selenium.

A coupled dinuclear iron cluster that is perturbed by substrate binding in myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) uses iron as its cofactor and dioxygen as its cosubstrate to effect the unique, ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate to d-glucuronate. The nature of the iron cofactor and its interaction with the substrate, myo-inositol (MI), have been probed by electron paramagnetic resonance (EPR) and Mössbauer spectroscopies. The data demonstrate the formation of an antiferromagnetically coupled, high-spin diiron(III/III) cluster upon treatment of solutions of Fe(II) and MIOX with excess O(2) or H(2)O(2) and the formation of an antiferromagnetically coupled, valence-localized, high-spin diiron(II/III) cluster upon treatment with either limiting O(2) or excess O(2) in the presence of a mild reductant (e.g., ascorbate). Marked changes to the spectra of both redox forms upon addition of MI and analogy to changes induced by binding of phosphate to the diiron(II/III) cluster of the protein phosphatase, uteroferrin, suggest that MI coordinates directly to the diiron cluster, most likely in a bridging mode. The addition of MIOX to the growing family of non-heme diiron oxygenases expands the catalytic range of the family beyond the two-electron oxidation (hydroxylation and dehydrogenation) reactions catalyzed by its more extensively studied members such as methane monooxygenase and stearoyl acyl carrier protein Delta(9)-desaturase.

Oxygen activation by a mixed-valent, diiron(II/III) cluster in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) catalyzes the ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate (myo-inositol, MI) to d-glucuronate (DG). The preceding paper [Xing, G., Hoffart, L. M., Diao, Y., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C., and Bollinger, J. M., Jr. (2006) Biochemistry 45, 5393-5401] demonstrates by Mössbauer and electron paramagnetic resonance (EPR) spectroscopies that MIOX can contain a non-heme dinuclear iron cluster, which, in its mixed-valent (II/III) and fully oxidized (III/III) states, is perturbed by binding of MI in a manner consistent with direct coordination. In the study presented here, the redox form of the enzyme that activates O(2) has been identified. l-Cysteine, which was previously reported to accelerate turnover, reduces the fully oxidized enzyme to the mixed-valent form, and O(2), the cosubstrate, oxidizes the fully reduced form to the mixed-valent form with a stoichiometry of one per O(2). Both observations implicate the mixed-valent, diiron(II/III) form of the enzyme as the active state. Stopped-flow absorption and freeze-quench EPR data from the reaction of the substrate complex of mixed-valent MIOX [MIOX(II/III).MI] with limiting O(2) in the presence of excess, saturating MI reveal the following cycle: (1) MIOX(II/III).MI reacts rapidly with O(2) to generate an intermediate (H) with a rhombic, g < 2 EPR spectrum; (2) a form of the enzyme with the same absorption features as MIOX(II/III) develops as H decays, suggesting that turnover has occurred; and (3) the starting MIOX(II/III).MI complex is then quantitatively regenerated. This cycle is fast enough to account for the catalytic rate. The DG/O(2) stoichiometry in the reaction, 0.8 +/- 0.1, is similar to the theoretical value of 1, whereas significantly less product is formed in the corresponding reaction of the fully reduced enzyme with limiting O(2). The DG/O(2) yield in the latter reaction decreases as the enzyme concentration is increased, consistent with the hypothesis that initial conversion of the reduced enzyme to the MIOX(II/III).MI complex and subsequent turnover by the mixed-valent form is responsible for the product in this case. The use of the mixed-valent, diiron(II/III) cluster by MIOX represents a significant departure from the mechanisms of other known diiron oxygenases, which all involve activation of O(2) from the II/II manifold.

Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) activates O2 at a mixed-valent nonheme diiron(II/III) cluster to effect oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate [myo-inositol (MI)] by four electrons to d-glucuronate. Abstraction of hydrogen from C1 by a formally (superoxo)diiron(III/III) intermediate was previously proposed. Use of deuterium-labeled substrate, 1,2,3,4,5,6-[2H]6-MI (D6-MI), has now permitted initial characterization of the C-H-cleaving intermediate. The MIOX.1,2,3,4,5,6-[2H]6-MI complex reacts rapidly and reversibly with O2 to form an intermediate, G, with a g = (2.05, 1.98, 1.90) EPR signal. The rhombic g-tensor and observed hyperfine coupling to 57Fe are rationalized in terms of a (superoxo)diiron(III/III) structure with coordination of the superoxide to a single iron. G decays to H, the intermediate previously detected in the reaction with unlabeled substrate. This step is associated with a kinetic isotope effect of > or =5, showing that the superoxide-level complex does indeed cleave a C-H(D) bond of MI.

Expression of myo-inositol oxygenase in tissues susceptible to diabetic complications.

Alterations of intracellular levels of myo-inositol (MI) have the potential to impact such cellular processes as signaling pathways and osmotic balance. Depletion of MI has been implicated in the etiology of diabetic complications; however, the mechanistic details remain sketchy. myo-Inositol oxygenase (MIOX-EC 1.13.99.1) catalyzes the first committed step of the only pathway of MI catabolism. In the present study, extra-renal tissues and cell types, including those affected by diabetic complications, were examined for MIOX expression. Western blotting results indicated that kidney is the only major organ where MIOX protein is expressed at detectable levels. Immunohistochemical examination of the kidney revealed that the proximal tubular epithelial cells are the only site of MIOX expression in the kidney. Reverse-transcription-polymerase chain reaction (RT-PCR) and Western immunoblot analyses, however, revealed that the cell lines ARPE-19 and HLE-B3, representing human retinal pigmented epithelium and lens epithelium, respectively, also express MIOX. In addition, quantitative real-time RT-PCR analysis of all major tissues in the mouse showed that the sciatic nerve contained MIOX transcript, which was found to be significantly higher than that observed in other non-renal organs. These results indicate that MIOX is found at lower levels in extra-renal tissues where diabetic complications, including nephropathy, neuropathy, retinopathy, and cataract, are frequently observed.

Up-regulation of human myo-inositol oxygenase by hyperosmotic stress in renal proximal tubular epithelial cells.

myo-Inositol oxygenase (MIOX) catalyzes the oxidative cleavage of myo-inositol (MI) to give d-glucuronic acid, a committed step in MI catabolism. d-Glucuronic acid is further metabolized to xylitol via the glucuronate-xylulose pathway. Although accumulation of polyols such as xylitol and sorbitol is associated with MI depletion in diabetic complications, no causal relationship has been established. Therefore we are examining the role of MIOX in diabetic nephropathy. Here we present evidence that the basis for the depletion of MI in diabetes is likely to be mediated by the increased expression of MIOX, which is induced by sorbitol, mannitol, and xylitol in a porcine renal proximal tubular epithelial cell line, LLC-PK1. To understand the molecular mechanism of regulation of MIOX expression by polyols, we have cloned the human MIOX gene locus of 10 kb containing 5.6 kb of the 5' upstream sequence. Analysis of the 5' upstream sequence led to the identification of an osmotic response element (ORE) in the promoter region, which is present approximately 2 kb upstream of the translation start site. Based on luciferase reporter and electrophoretic mobility shift assays, polyols increased the ORE-dependent expression of MIOX. In addition, we demonstrate that the activity of the promoter is dependent on the binding of the transcription factor, tonicity element-binding protein, or osmotic response element-binding protein, to the ORE site. These results suggest that the expression of MIOX is up-regulated by a positive feedback mechanism where xylitol, one of the products of MI catabolism via the glucuronate-xylulose pathway, induces an overexpression of MIOX.

Molecular cloning, expression, and characterization of myo-inositol oxygenase from mouse, rat, and human kidney.

myo-Inositol oxygenase (MIOX) is a non-heme iron enzyme, which catalyzes the conversion of myo-inositol to d-glucuronic acid, the first committed step in myo-inositol catabolism. Full-length cDNAs of 858bp each coding for 33kDa protein were cloned from kidney cDNA libraries of mouse, rat, and human. The individual clones were expressed in Escherichia coli and recombinant MIOX proteins were purified to electrophoretic homogeneity. A hydrophobic interaction chromatography step yielded multiple conformers, with mouse and human MIOX showing three peaks and rat enzyme revealing two peaks. Individual MIOX peaks exhibited distinct V(max) and K(m) values. Interestingly, upon storage, the 33kDa protein was degraded to a approximately 30kDa truncated protein in each species, and formed small amounts of dimers of identical subunits. While MIOX is a highly conserved enzyme in all mammalian species, the labile nature and tendency to degrade in solution may be the source of significant differences in size previously reported in the literature. Regardless of the source, our results strongly dispel previous conflicting literature reports on the size of the protein and confirm that MIOX is a 33kDa protein.