Biochemical Studies of Mitochondrial Malate: Quinone Oxidoreductase from Toxoplasma gondii

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis and infects almost one-third of the global human population. A lack of effective drugs and vaccines and the emergence of drug resistant parasites highlight the need for the development of new drugs. The mitochondrial electron transport chain (ETC) is an essential pathway for energy metabolism and the survival of T. gondii. In apicomplexan parasites, malate:quinone oxidoreductase (MQO) is a monotopic membrane protein belonging to the ETC and a key member of the tricarboxylic acid cycle, and has recently been suggested to play a role in the fumarate cycle, which is required for the cytosolic purine salvage pathway. In T. gondii, a putative MQO (TgMQO) is expressed in tachyzoite and bradyzoite stages and is considered to be a potential drug target since its orthologue is not conserved in mammalian hosts. As a first step towards the evaluation of TgMQO as a drug target candidate, in this study, we developed a new expression system for TgMQO in FN102(DE3)TAO, a strain deficient in respiratory cytochromes and dependent on an alternative oxidase. This system allowed, for the first time, the expression and purification of a mitochondrial MQO family enzyme, which was used for steady-state kinetics and substrate specificity analyses. Ferulenol, the only known MQO inhibitor, also inhibited TgMQO at IC50 of 0.822 μM, and displayed different inhibition kinetics compared to Plasmodium falciparum MQO. Furthermore, our analysis indicated the presence of a third binding site for ferulenol that is distinct from the ubiquinone and malate sites

Various Types of Dirofilaria immitis Polyproteins Selectively Induce a Th2-Type Immune Response

Dirofilaria immitis polyproteins (DiAgs) are found as 15-kDa monomeric and 30-kDa dimeric forms in exceretory-secretory products of the adult worm. We evaluated the ability of various types of recombinant DiAg (rDiAg; V1 and V2 as monomers and V1V2, V2V1, V1V1, and V2V2 as dimers) to influence Th1/Th2 immune responses. V1-, V1Vx- and V2-, V2Vx-driven nonspecific immunoglobulin E (IgE) production peaked at 21 and 14 days after administration, respectively. Dimer-induced IgE response was an interesting biphasic pattern with the second peaks on days 35 (V2Vx) or 42 (V1Vx). Absolute amounts of nonspecific IgE production induced with monomers were larger than those observed with dimers at the first peak. The magnitude of cell expansion and interleukin-10 (IL-10) production in mesenteric lymph node (MLN) B-cell induced with rDiAgs was linked to the levels of the first IgE peak in vivo and IgE produced by rDiAg plus IL-4-stimulated B cells in vitro. All rDiAgs failed to augment IgG2c production. V2 and V2Vx elicited IL-4 production by MLN cells more rapidly than V1 and V1Vx. The inhibitory effect of rDiAg on gamma interferon (IFN-γ) production was stronger in monomers than in dimers. Neutralization of IL-10 restored IFN-γ production, whereas the expression of IL-4 and IgE was partly prevented by depletion of IL-10. These results indicate that monomer rather than dimer is an efficient form of DiAg and suggest that the difference of IgE-inducing capacity among these DiAgs is closely associated with the pattern of both B-cell activation and IL-4 production

Secondary Unconjugated Bile Acids Induce Hepatic Stellate Cell Activation

Hepatic stellate cells (HSCs) are key players in liver fibrosis, cellular senescence, and hepatic carcinogenesis. Bile acids (BAs) are involved in the activation of HSCs, but the detailed mechanism of this process remains unclear. We conducted a comprehensive DNA microarray study of the human HSC line LX-2 treated with deoxycholic acid (DCA), a secondary unconjugated BA. Additionally, LX-2 cells were exposed to nine BAs and studied using immunofluorescence staining, enzyme-linked immunosorbent assay, and flow cytometry to examine the mechanisms of HSC activation. We focused on the tumor necrosis factor (TNF) pathway and revealed upregulation of genes related to nuclear factor kappa B (NF-κB) signaling and senescence-associated secretory phenotype factors. α-Smooth muscle actin (α-SMA) was highly expressed in cells treated with secondary unconjugated BAs, including DCA, and a morphological change associated with radial extension of subendothelial protrusion was observed. Interleukin-6 level in culture supernatant was significantly higher in cells treated with secondary unconjugated BAs. Flow cytometry showed that the proportion of cells highly expressing α-SMA was significantly increased in HSCs cultured with secondary unconjugated BAs. We demonstrated that secondary unconjugated BAs induced the activation of human HSCs

Susceptibility of Aedes flavopictus miyarai and Aedes galloisi mosquito species in Japan to dengue type 2 virus

Objective: To evaluate the potential of local mosquitoes to act as vectors for dengue transmission in Japan. Methods: Serotype 2 ThNH28/93 was used to test the dengue susceptibility profiles of Aedes flavopictus miyarai (Ae. f. miyarai), Aedes galloisi (Ae. galloisi) and Aedes albopictus (Ae. albopictus), which were collected in Japan. We used Aedes aegypti from Thailand as a positive control. The mosquitoes were infected with the virus intrathoracically or orally. At 10 or 14 days post infection, the mosquitoes were dissected and total RNA was extracted from their abdomens, thoraxes, heads and legs. Mosquito susceptibility to dengue virus was evaluated using RT-PCR with dengue virus-specific primers. Differences in the infection and mortality rates of the different mosquito species were tested using Fisher's exact probability test. Results: The infection rates for dengue virus administered intrathoracically to Ae. f. miyarai, Ae. galloisi and Aedes aegypti mosquitoes were identical by RT-PCR on Day 10 post infection. All of the body parts we tested were RT-PCR-positive for dengue virus. For the orally administered virus, the infection rates in the different body parts of the Ae. f. miyarai mosquitoes were slightly higher than those of Ae. albopictus mosquitoes, but were similar to the control mosquitoes (P > 0.05). The mortality rates for Ae. f. miyarai and Ae. albopictus mosquitoes were similar (P = 0.19). Our data indicated that dengue virus was able to replicate and disseminate to secondary infection sites in all of the four mosquito species (Japanese and Thai). Conclusions: Ae. albopictus is a well-known candidate for dengue transmission in Japan. However, our data suggest that Ae. f. miyarai from Ishigaki Island (near Okinawa Island) and Ae. galloisi from Hokkaido (Northern Japan) should also be regarded as potential vectors for dengue transmission in these regions. Further studies on these mosquitoes should be conducted

The Orphan Nuclear Receptor TLX Is an Enhancer of STAT1-Mediated Transcription and Immunity to <i>Toxoplasma gondii</i>

<div><p>The protozoan parasite, <i>Toxoplasma</i>, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited this well-defined host–pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite <i>Toxoplasma</i> results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such “modifiers.”</p></div

TLX depletion in astrocyte cells impairs a subset of IFN-γ-inducible targets.

<p>(A) Quantitative reverse transcription PCR (RT-qPCR) measuring TLX expression in U251 cells following treatment with either control siRNA or siRNA targeting TLX. (B) Heatmap and hierarchical clustering analysis of 1,418 genes differentially regulated (≥1.5-fold, FDR ≤ 5%) plus or minus IFN-γ stimulation of cells treated with either control siRNA or siRNA targeting TLX. Row Z-scores of the mean expression data for duplicate arrays are shown. (C) Bubble plot showing enriched GO terms for each of the three clusters indicated in panel A. Bubble size indicates number of genes associated with each term. Bubble color indicates whether genes associated with each term were up-regulated (red) or down-regulated (blue), while color intensity indicates fold enrichment. (D–E) Heatmaps showing (D) TLX-dependent genes associated with CNS development and function (from cluster 3) and (E) TLX-dependent, IFN-γ-regulated immune genes (from cluster 1). Asterisks in panel D indicate genes previously shown to be TLX dependent in the CNS [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002200#pbio.1002200.ref047" target="_blank">47</a>].</p

Meis1 regulates cell cycle of the HSC compartment.

<p>(A) Representative flow cytometric profiles showing Annexin V and 7-AAD staining of LSK cells from <i>Mx1-Cre⁺ Meis1</i>^fl/fl and control mice one week after poly(I:C) treatment. Bar graphs on the right represent the percentages of apoptotic LSK cells (anexin V⁺ 7-AAD⁻) cells from poly(I:C)–treated <i>Mx1-Cre</i>⁺<i>Meis1</i>^fl/fl (solid bars) and control <i>Meis1</i>^fl/fl (open bars) mice (mean and SD; n = 3). (B) Representative flow cytometric profiles showing BrdU incorporation and 7-AAD staining of LSK cells from <i>Mx1-Cre Meis1</i>^fl/fl and control mice one week after poly(I:C) treatment. Bar graphs shown on the right represent the percentages of cells in G0/G1-, S- and G2/M-phase of the cell cycle in the LSK cell population from poly(I:C)–treated <i>Mx1-Cre</i>⁺<i>Meis1</i>^fl/fl (solid bars) and control <i>Meis1</i>^fl/fl (open bars) mice (mean and SD; n = 3). **p<0.01.</p

Meis1 controls expression of genes involved in HSC cell cycle and maintenance.

<p>(A) Alterations of gene expression in LSK cells induced by <i>Meis1</i> loss were analyzed by quantitative RT-PCR. Histograms show the indicated transcripts in sorted LSK cells from <i>Mx1-Cre⁺ Meis1</i>^fl/fl mice (open bars) and control <i>Meis1</i>^fl/fl mice (solid bars) one week post poly(I:C) treatment. Data were normalized to <i>Gapdh</i> expression and the level of each transcript in LSK cells from control mice was arbitrarily set to 1. Data are the means and standard deviations of three independent experiments. *p<0.05 and **p<0.01. (B) Models illustrating potential mechanisms of Meis1 function in the maintenance of HSC.</p

TLX is induced during <i>Toxoplasma</i> CNS infection and is required for parasite control.

<p>(A–D) Immunohistochemical staining of TLX in formalin-fixed paraffin-embedded sections from <i>Toxoplasma</i>-infected mouse brain. Sections were stained with anti-TLX (A and C) or secondary antibody as a control (B and D). (A) TLX positive cell (arrow) adjacent to <i>Toxoplasma</i> cyst. (C) Large cells with neuronal morphology are TLX positive. (E) Representative plots showing flow cytometric detection of intracellular interleukin-12 (IL-12) p40 in CD11c positive splenocytes from Mx1-cre TLX^f/f and TLX^f/f controls pretreated with Poly(I:C) prior to infection with values shown for the individual animal. (F) Graph of data from panel E for four-to-six mice per group. (G–H) Immunohistochemical detection of parasites in formalin-fixed paraffin embedded section. Free parasites are indicated by arrows. (I) qPCR-based measurement of parasite DNA in brain. Experiments were repeated two-to-three times with similar results. Data for panel F and I can be found in file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002200#pbio.1002200.s001" target="_blank">S1 Data</a>.</p

TLX expression enhances occupancy of pSTAT1 on the CXCL9 and CXCL10 promoters.

<p>ChIP with rabbit monoclonal antibody to phosphorylated STAT1 (Y701) or control rabbit IgG. U2OS cells expressing either control vector or TLX were either left untreated or stimulated with IFN-γ for 2 h. The experiment was repeated twice with similar results. Data can be found in file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002200#pbio.1002200.s001" target="_blank">S1 Data</a>.</p