Oksana Ocheretina

Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator

ABSTRACT Infection with adenovirus vectors (AdV) results in rapid activation of innate immunity, which serves the dual purpose of stimulating inflammatory antiviral host defenses and the adaptive immune system. Viral recognition by macrophages, dendritic cells, and other cell types requires an ability to sense the presence of a foreign molecular pattern by “pattern recognition receptors.” The nature of the adenoviral sensor, the target ligand of the sensor, and the downstream antiviral signaling response triggered by virus infection have not been defined for this nonenveloped double-stranded DNA (dsDNA) virus. We have identified four critical links involved in AdV recognition by murine antigen-presenting cells (APC) and primary lung fibroblasts: (i) viral recognition occurs chiefly via a Toll-like receptor (TLR)-independent nucleic acid-sensing mechanism recognizing the viral dsDNA genome, (ii) the intact viral particle and capsid proteins are required for efficient intracellular delivery of the viral genome, (iii) delivery of the viral genome triggers interferon regulatory factor 3 (IRF3) phosphorylation, and (iv) IRF3 activation is the required dominant antiviral signaling pathway used by APC, whereas the “primary” involvement of NF-κB, mitogen-activated protein kinase, or Akt pathways is less prominent. In this study we provide the first direct evidence that infection by a dsDNA virus stimulates an IRF3-mediated interferon and proinflammatory response through a TLR-independent DNA-sensing mechanism.

A novel, non-redox-regulated NAD-dependent malate dehydrogenase from chloroplasts of Arabidopsis thaliana L.

We report a novel plastidic NAD-dependent malate dehydrogenase (EC 1.1.1.37), which is not redox-regulated in contrast to its NADP-specific counterpart (EC1.1.1.82). Analysis of isoenzyme patterns revealed a single NAD-MDH associated with highly purified chloroplasts isolated fromArabidopsis and spinach. A cDNA clone encoding the novel enzyme was found in the Arabidopsis EST data base by sorting all putative clones for NAD-dependent malate dehydrogenase. A derived amino acid sequence is very similar to mitochondrial and peroxisomal NAD-MDHs within the region coding for the mature protein but possesses a 80-amino acid long N-terminal domain with typical characteristics of a chloroplast transit peptide. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to a mature enzyme subunit of 34 kDa. Expressed in Escherichia coli, the recombinant enzyme exhibited the same distinctive isoelectric point of 5.35 as the original enzyme from Arabidopsis chloroplasts. Northern analysis revealed that the protein is expressed in both autotrophic and heterotrophic tissues. The findings reported here indicate that the “malate valve” operates not only in the illuminated chloroplasts but also in dark chloroplasts and in heterotrophic plastids and is therefore a general mechanism to maintain the optimal ratio between ATP and reducing equivalents in plastids.

Correlation between genotypic and phenotypic testing for resistance to rifampin in Mycobacterium tuberculosis clinical isolates in Haiti: investigation of cases with discrepant susceptibility results.

The World Health Organization has recommended use of molecular-based tests MTBDRplus and GeneXpert MTB/RIF to diagnose multidrug-resistant tuberculosis in developing and high-burden countries. Both tests are based on detection of mutations in the Rifampin (RIF) Resistance-Determining Region of DNA-dependent RNA Polymerase gene (rpoB). Such mutations are found in 95–98% of Mycobacterium tuberculosis strains determined to be RIF-resistant by the “gold standard” culture-based drug susceptibility testing (DST). We report the phenotypic and genotypic characterization of 153 consecutive clinical Mycobacterium tuberculosis strains diagnosed as RIF-resistant by molecular tests in our laboratory in Port-au-Prince, Haiti. 133 isolates (86.9%) were resistant to both RIF and Isoniazid and 4 isolates (2.6%) were RIF mono-resistant in MGIT SIRE liquid culture-based DST. However the remaining 16 isolates (10.5%) tested RIF-sensitive by the assay. Five strains with discordant genotypic and phenotypic susceptibility results had RIF minimal inhibitory concentration (MIC) close to the cut-off value of 1 µg/ml used in phenotypic susceptibility assays and were confirmed as resistant by DST on solid media. Nine strains had sub-critical RIF MICs ranging from 0.063 to 0.5 µg/ml. Finally two strains were pan-susceptible and harbored a silent rpoB mutation. Our data indicate that not only detection of the presence but also identification of the nature of rpoB mutation is needed to accurately diagnose resistance to RIF in Mycobacterium tuberculosis. Observed clinical significance of low-level resistance to RIF supports the re-evaluation of the present critical concentration of the drug used in culture-based DST assays.

Redox equilibria between the regulatory thiols of light/dark-modulated chloroplast enzymes and dithiothreitol: fine-tuning by metabolites

Three light/dark-modulated chloroplast enzymes, namely NADP-dependent malate dehydrogenase (EC 1.1.1.82), D-fructose 1,6-bisphosphatase (EC 3.1.3.11), and phosphoribulokinase (EC 2.7.1.19) were purified to apparent homogeneity from spinach leaves. Equilibrium constants for the covalent modification of the regulatory disulfide bonds of these enzymes in dithiothreitol (DTT)-redox buffer were determined according to a previously published method in the literature (Clancey and Gilbert (1987) J. Biol. Chem. 262, 13545-13549). The thiol/disulfide-redox potential (Kox) was defined as the ratio of reduced to oxidized dithiothreitol at which 50% of the maximal enzyme activity was observed after equilibrium had been established. All Kox values were very high, comparable to those of extracellular disulfide containing proteins: 0.23 +/- 0.02 for NADP-malate dehydrogenase, 0.59 +/- 0.17 for phosphoribulokinase, and 0.70 +/- 0.16 for D-fructose 1,6-bisphosphatase. The equilibrium constants for the reactions between these enzymes and the redox buffers were also determined in the presence of various concentrations of specific metabolites known to influence the rates of reduction and oxidation. Increasing concentrations of D-fructose 1,6-bisphosphate in the presence of Ca2+ shift the equilibrium constant between D-fructose 1,6-bisphosphatase and the DTT-redox buffer to much lower values. A decreasing NADPH/(NADP + NADPH) ratio increases the Kox of NADP-malate dehydrogenase in the redox buffer to very high values. For PRK, low concentrations of ATP result in a slight decrease of the Kox that is not further affected by higher ATP concentrations. The differences of the equilibrium constants of NADP-malate dehydrogenase and D-fructose 1,6-bisphosphatase as dependent upon the NADPH/(NADP + NADPH) ratio and the concentration of D-fructose 1,6-bisphosphate, respectively, reflect a mechanism of feed-back and feed-forward regulation by the product NADP and the substrate D-fructose 1,6-bisphosphate, respectively. Thus the actual activation state of these two key enzymes of chloroplast metabolism are determined in an independent manner. The relatively small effect of the ATP concentration upon the redox potential of phosphoribulokinase indicates that fine-regulation at this step might be achieved on another level (e.g., catalysis or aggregation state).

Adenovirus Induction of IRF3 Occurs through a Binary Trigger Targeting Jun N-Terminal Kinase and TBK1 Kinase Cascades and Type I Interferon Autocrine Signaling

ABSTRACT Pathogen recognition is a critical function of immune sentinel cells. Naïve macrophages or dendritic cells (DCs) undergo pathogen-directed activation and maturation, and as mature antigen-presenting cells (APCs), they contribute essential functions to both innate and adaptive immunity. Using recombinant adenovirus (rAdV) as a model for murine APC activation by DNA viruses, we demonstrate a critical role for stress kinase activation in cell intrinsic and extrinsic antiviral signaling cascades. We propose two viral triggers, viral capsid and viral DNA, are required for APC activation. Endosomal escape and presentation of cytosolic rAdV DNA induces phosphorylation of TANK-binding kinase 1 (TBK1) at serine 172 but does not induce IκB kinase ε activity as determined by in vitro kinase assays. However, induction of TBK1 alone is not sufficient for interferon regulatory factor 3 (IRF3) phosphorylation. We show that capsid-dependent activation of Jun N-terminal kinase (JNK) stress kinase is a necessary step, licensing TBK1 phosphorylation of IRF3 at Ser 396. A second later phase of JNK activity is required to coordinate phosphorylation of JNK-dependent transcription factors (c-Jun/ATF2) with activated IRF3 in the induction of primary IRF3-responsive transcripts. Finally, we demonstrate that maximal JNK/TBK1/IRF3 stimulation by rAdV depends on an intact type I interferon (IFN) signaling cascade. By requiring multiple viral triggers and type I IFN autocrine regulation, APCs have an inherent fail-safe mechanism against inappropriate activation and maturation.

The Effect of HIV and HPV Coinfection on Cervical COX-2 Expression and Systemic Prostaglandin E2 Levels

Human immunodeficiency virus (HIV-1) infection causes chronic inflammation. COX-2–derived prostaglandin E2 (PGE2) has been linked to both inflammation and carcinogenesis. We hypothesized that HIV-1 could induce COX-2 in cervical tissue and increase systemic PGE2 levels and that these alterations could play a role in AIDS-related cervical cancer. Levels of cervical COX-2 mRNA and urinary PGE-M, a biomarker of systemic PGE2 levels, were determined in 17 HIV-negative women with a negative cervical human papilloma virus (HPV) test, 18 HIV-infected women with a negative HPV test, and 13 HIV-infected women with cervical HPV and high-grade squamous intraepithelial lesions on cytology. Cervical COX-2 levels were significantly associated with HIV and HPV status (P = 0.006 and 0.002, respectively). Median levels of urinary PGE-M were increased in HIV-infected compared with uninfected women (11.2 vs. 6.8 ng/mg creatinine, P = 0.02). Among HIV-infected women, urinary PGE-M levels were positively correlated with plasma HIV-1 RNA levels (P = 0.003). Finally, levels of cervical COX-2 correlated with urinary PGE-M levels (P = 0.005). This study shows that HIV-1 infection is associated with increased cervical COX-2 and elevated systemic PGE2 levels. Drugs that inhibit the synthesis of PGE2 may prove useful in reducing the risk of cervical cancer or systemic inflammation in HIV-infected women. Cancer Prev Res; 5(1); 34–40. ©2011 AACR.

Cloning and sequence analysis of cDNAs encoding plant cytosolic malate dehydrogenase

Here we report the first complete sequence of plant cytosolic malate dehydrogenase (EC 1.1.1.37). The phylogenetic relationships between malate dehydrogenases from different cell compartments are discussed. The constructed phylogenetic tree shows that cytosolic NAD-MDH and chloroplast NADP-MDH have evolved through gene duplication of the pre-existing nuclear gene.

Effects of N-terminal truncations upon chloroplast NADP-malate dehydrogenases from pea and spinach

Using the purification procedure of Fickenscher and Scheibe (Biochim. Biophys. Acta 749 (1983), 249-254) and a modification of the method, we produced a series of NADP-MDH forms from spinach and pea-leaf extracts that were characterized by a stepwise shortening of the N-terminal sequences. Limited proteolysis of the enzymes resulted in the generation of even shorter forms. Immunoprecipitation of the NADP-MDH from crude extracts revealed that the sequences of the intact enzymes from pea, spinach and maize started at a position (Ser) identical with that established for the Sorghum enzyme (Crétin, C., et al. (1990) Eur. J. Biochem. 192, 299-303). Spinach NADP-MDH isolated by conventional methods was shown to represent the intact form. Thus, the kinetic, regulatory and structural properties of the various truncated forms could be compared with those of an intact form. Removal of 5 or 11 amino acids, as occurred during isolation of the pea NADP-MDH, was without any significant effect. The enzymes were all dimeric and still exhibited the characteristic redox-regulatory properties. However, removal of 31 and 37 amino acids using aminopeptidase K resulted in the formation of active monomers characterized by only slightly lowered affinities towards the substrates, a shift of their pH optimum from 8 to 7, the loss of oxaloacetate inhibition and an increased maximal velocity. Although these forms lacked most or all of the N-terminal extra-peptide, including the 2 cysteines involved in redox-modification, they were still sensitive to the redox-potential. However, the low concentration of thiol required for immediate and complete restoration of any lost activity (40 mM beta-mercaptoethanol) suggested that this reaction might not be relevant for redox-regulation in vivo.

Epidemiology and genetic diversity of multidrug-resistant tuberculosis in East Africa.

Multidrug-resistant tuberculosis (MDR-TB) is an emerging problem in many parts of the world, and levels of MDR-TB among new TB patients are increasing in sub-Saharan Africa. We reviewed the prevalence and molecular epidemiology of MDR-TB in East Africa, including Burundi, Kenya, Rwanda, Tanzania, and Uganda. In 16 epidemiologic surveys, the prevalence of MDR among new cases ranges from 0.4% in Tanzania to 4.4% in Uganda, and among recurrent cases ranges from 3.9% in Tanzania to 17.7% in Uganda. There is a gap of 5948 cases between the estimated number of MDR-TB cases in East Africa and the number actually diagnosed. The only confirmed risk factors for MDR-TB are prior treatment for TB and refugee status. HIV has not been reported as a risk factor, and there are no reports of statistical association between spoligotype and drug resistance pattern. Increased capacity for diagnosis and treatment of MDR-TB is needed, with an emphasis on recurrent TB cases and refugees.

Use of Luminex MagPlex Magnetic Microspheres for High-Throughput Spoligotyping of Mycobacterium tuberculosis Isolates in Port-au-Prince, Haiti

ABSTRACT Genotyping of Mycobacterium tuberculosis strains became indispensable for understanding tuberculosis transmission dynamics and designing measures to combat the disease. Unfortunately, typing involves sophisticated laboratory analysis, is expensive, and requires a high level of technical expertise, which limited its use in the resource-poor countries where the majority of tuberculosis cases occur. Spoligotyping is a PCR-based M. tuberculosis complex genotyping method with advantages of technical simplicity, numerical output, and high reproducibility. It is based on the presence or absence of 43 distinct “spacers” separating insertion elements in the direct repeat region of the M. tuberculosis genome. The spoligotyping assay involves reverse hybridization of PCR products to the capture spacers attached to nitrocellulose membranes or to microspheres. Here we report modification of the classic 43-spacer method using the new generation of Luminex multiplexing technology with magnetic microspheres. The method was successfully established and validated on strains with known spoligotypes in our laboratory in Haiti. The distribution of spoligotypes determined in a collection of 758 recent M. tuberculosis isolates was in accordance with previous data for Haitian isolates in the SITWITWEB international database, which were obtained with the traditional membrane-based method. In the present form, spoligotyping may be suitable as a high-throughput, first-line tool for genotyping of Mycobacterium tuberculosis in countries with limited resources.