ATF6β is a host cellular target of the Toxoplasma gondii virulence factor ROP18

Toxoplasma virulence factor ROP18 targets endoplasmic reticulum–bound transcription factor ATF6β in the host cell, leading to the detrimental loss of ATF6β through proteasome-dependent degradation

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

Role of Mouse and Human Autophagy Proteins in IFN-gamma-Induced Cell-Autonomous Responses against Toxoplasma gondii

IFN-?? mediates cellular innate immunity against an intracellular parasite, Toxoplasma gondii, by inducing immunity-related GTPases such as p47 IFN-??-regulated GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), which also participate in antibacterial responses via autophagy. An essential autophagy protein, Atg5, was previously shown to play a critical role in anti- T. gondii cell-autonomous immunity. However, the involvement of other autophagy proteins remains unknown. In this study, we show that essential autophagy proteins differentially participate in anti-T. gondii cellular immunity by recruiting IFN-??-inducible GTPases. IFN-??-induced suppression of T. gondii proliferation and recruitment of an IRG Irgb6 and GBPs are profoundly impaired in Atg7- or Atg16L1-deficient cells. In contrast, cells lacking other essential autophagy proteins, Atg9a and Atg14, are capable of mediating the anti-T. gondii response and recruiting Irgb6 and GBPs to the parasites. Although IFN-?? also stimulates anti-T. gondii cellular immunity in humans, whether this response requires GBPs and human autophagy proteins remains to be seen. To analyze the role of human ATG16L1 and GBPs in IFN-??-mediated anti-T. gondii responses, human cells lacking ATG16L1 or GBPs were generated by the Cas9/CRISPR genome-editing technique. Although both ATG16L1 and GBPs are dispensable for IFN-??-induced inhibition of T. gondii proliferation in the human cells, human ATG16L1 is also required for the recruitment of GBPs. Taken together, human ATG16L1 and mouse autophagy components Atg7 and Atg16L1, but not Atg9a and Atg14, participate in the IFN-??-induced recruitment of the immunity-related GTPases to the intracellular pathogen.close3

A Cluster of Interferon-γ-Inducible p65 GTPases Plays a Critical Role in Host Defense against Toxoplasma gondii

Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates ∼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity

CREBH Determines the Severity of Sulpyrine-Induced Fatal Shock

<div><p>Although the pyrazolone derivative sulpyrine is widely used as an antipyretic analgesic drug, side effects, including fatal shock, have been reported. However, the molecular mechanism underlying such a severe side effect is largely unclear. Here, we report that the transcription factor CREBH that is highly expressed in the liver plays an important role in fatal shock induced by sulpyrine in mice. CREBH-deficient mice were resistant to experimental fatal sulpyrine shock. We found that sulpyrine-induced expression of cytochrome P450 2B (CYP2B) family genes, which are involved in sulpyrine metabolism, in the liver was severely impaired in CREBH-deficient mice. Moreover, introduction of CYP2B in CREBH-deficient liver restored susceptibility to sulpyrine. Furthermore, ectopic expression of CREBH up-regulated CYP2B10 promoter activity, and <em>in vivo</em> knockdown of CREBH in wild-type mice conferred a significant resistance to fatal sulpyrine shock. These data demonstrate that CREBH is a positive regulator of CYP2B in response to sulpyrine administration, which possibly results in fatal shock.</p> </div

RNAi-mediated CREBH-knockdown mice are resistant to the sulpyrine shock.

<p>(A) Livers of wild-type mice transfected with RNAi vectors for CREBH (n = 4) or negative control vectors (n = 4) were taken at 24 hr after transfection. Gene expression of CREBH was analysed by a quantitative RT-PCR assay. ***, P<0.001. (B, C) Wild-type mice transfected with RNAi vectors for CREBH (n = 14) or negative control vectors (n = 14) were intraperitoneally injected with 2.7 mg/g of sulpyrine at 24 hr after transfection. Survival rate was monitored for 48 hr. *, P<0.05. Sera were taken at 2 hr 40 min after sulpyrine injection. Serum concentrations of 4-AA and 4-FAA were measured by a HPLC assay. ***, P<0.001. Data are representative of two (A, C) independent experiments or pooled from three (B) independent experiments.</p

Cleavage of CREBH and its translocation into nucleus were induced by sulpyrine.

<p>(A) 293T cells were transiently transfected with 2 µg HA-tagged hCREBH-F expression vectors, and they were cultured with sulpyrine (2 mM) or tunicamycin (5 µg/ml) for 6 hr. Whole-cell lysates were prepared and the size of CREBH was analysed by western blot analysis using anti-CREBH monoclonal (upper panel) and anti-actin antibodies (lower panel). **, CREBH-full ; *, CREBH-ΔC. (B) 293T cells were transiently transfected with 2 µg HA-tagged hCREBH-F expression vectors, and they were cultured with sulpyrine (2 mM) or tunicamycin (5 µg/ml) for 6 hr. The cell lysates of nuclear fractions were prepared. The nuclear translocation of CREBH was analyzed by western blot analysis using anti-CREBH monoclonal (upper panel) and anti-Sp1 monoclonal antibodies (lower panel). (C) 293T cells were transiently transfected with HA-tagged hCREBH-F expression vectors and HA-tagged hCREBH-ΔC expression vectors, and they were cultured with mock, sulpyrine (2 mM) and tunicamycin (5 µg/ml) for 4 hr. Cells were stained with anti-HA mouse antibody, then stained with Alexa Fluor 594-conjugated anti-mouse IgG (red) together with DAPI (blue). Stained cells were analysed using a confocal microscope. Bars, 10 µm. Data are representative of three (A) and two (B,C) independent experiments.</p

Sulpyrine treatment causes ER stress.

<p>(A) Huh7 cells were transiently transfected with luciferase reporter plasmids containing the CYP2B10 promoter together with control, hCREBH-F, or hCREBH-ΔC expression vector. Relative luciferase activities were shown as fold increases over the background levels shown by lysates prepared from control vector-transfected cells. Error bars are means ± S.D. of triplicates. (B) Huh7 cells were transiently transfected with luciferase reporter plasmids containing the indicated length CYP2B10 promoter together with control or hCREBH-F expression vector. Relative luciferase activities were shown as fold increases over the background levels shown by lysates prepared from control vector-transfected cells. Error bars are means ± S.D. of triplicates. (C) Wild-type mice were intraperitoneally injected with 2.7 mg/g of sulpyrine (n = 3) or 200 µg/kg tunicamycin (n = 3) or vehicle alone (control) (n = 3). At 2 hr 40 min after injection, hepatocytes were taken from these mice by a perfusion apparatus. Gene expression of CHOP, spliced XBP1, and CYP2B10 was measured by a quantitative RT-PCR assay. **, P<0.01; ***, P<0.001. Data are representative of three (A, B) and two (C) independent experiments.</p

The blood concentration of 4-AA is involved in sulpyrine-induced shock.

<p>(A) Calibration curves for relationship between the administration volumes of 4-AA, 4-FAA and the concentrations of in the sera. (B) Wild-type mice (n = 5) were intraperitoneally injected with indicated volumes of 4-AA and 4-FAA. Survival rate was monitored for 180 min. (C) Wild-type mice (n = 5) were intraperitoneally injected with indicated volumes (0, 0.5, 0.9, 1.0, 1.2, 1.3, 1.4, and 1.5 mg/g) of 4-AA and 4-FAA. Survival rate was monitored for 180 min. Data are representative of two (A–C) independent experiments.</p