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. 2019 Jul 23;10(4):e01538-19.
doi: 10.1128/mBio.01538-19.

Brucella abortus Infection of Placental Trophoblasts Triggers Endoplasmic Reticulum Stress-Mediated Cell Death and Fetal Loss via Type IV Secretion System-Dependent Activation of CHOP

Affiliations

Brucella abortus Infection of Placental Trophoblasts Triggers Endoplasmic Reticulum Stress-Mediated Cell Death and Fetal Loss via Type IV Secretion System-Dependent Activation of CHOP

Mariana X Byndloss et al. mBio. .

Abstract

Subversion of endoplasmic reticulum (ER) function is a feature shared by multiple intracellular bacteria and viruses, and in many cases this disruption of cellular function activates pathways of the unfolded protein response (UPR). In the case of infection with Brucella abortus, the etiologic agent of brucellosis, the unfolded protein response in the infected placenta contributes to placentitis and abortion, leading to pathogen transmission. Here we show that B. abortus infection of pregnant mice led to death of infected placental trophoblasts in a manner that depended on the VirB type IV secretion system (T4SS) and its effector VceC. The trophoblast death program required the ER stress-induced transcription factor CHOP. While NOD1/NOD2 expression in macrophages contributed to ER stress-induced inflammation, these receptors did not play a role in trophoblast death. Both placentitis and abortion were independent of apoptosis-associated Speck-like protein containing a caspase activation and recruitment domain (ASC). These studies show that B. abortus uses its T4SS to induce cell-type-specific responses to ER stress in trophoblasts that trigger placental inflammation and abortion. Our results suggest further that in B. abortus the T4SS and its effectors are under selection as bacterial transmission factors.IMPORTANCEBrucella abortus infects the placenta of pregnant cows, where it replicates to high levels and triggers abortion of the calf. The aborted material is highly infectious and transmits infection to both cows and humans, but very little is known about how B. abortus causes abortion. By studying this infection in pregnant mice, we discovered that B. abortus kills trophoblasts, which are important cells for maintaining pregnancy. This killing required an injected bacterial protein (VceC) that triggered an endoplasmic reticulum (ER) stress response in the trophoblast. By inhibiting ER stress or infecting mice that lack CHOP, a protein induced by ER stress, we could prevent death of trophoblasts, reduce inflammation, and increase the viability of the pups. Our results suggest that B. abortus injects VceC into placental trophoblasts to promote its transmission by abortion.

Keywords: Brucella; effector functions; endoplasmic reticulum; placenta; trophoblast; type IV secretion.

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Figures

FIG 1
FIG 1
B. abortus infection induces cell death in the placenta. (A) Representative images of spleen and placenta from pregnant mice infected with wild-type B. abortus for 13 days. The white arrow and left bottom inset show microgranulomas, the black arrow and right bottom inset show trophoblast death, and "N" shows areas of neutrophilic infiltrate (×ばつ). (B) Cell death assessed by blinded histopathology scoring in spleen and placenta from mice in panel A. Values represent individual mice (black circles) and means (black line). *, P < 0.05 using Mann-Whitney statistical analysis. (C) Representative images of TUNEL-stained placenta tissue from panel B. (D) Pathology scoring of TUNEL staining in placentas of B. abortus-infected mice treated with a vehicle or the ER stress inhibitor TUDCA.
FIG 2
FIG 2
Death of trophoblast giant cells is dependent on the T4SS and its effector VceC. (A) Representative images of hematoxylin and eosin-stained (left) and TUNEL-stained (right) placental tissue from mock-infected or B. abortus-infected pregnant mice. The black arrow and inset show trophoblasts (×ばつ). (B) Trophoblast death measured by TUNEL assay in placenta from pregnant mice infected with wild-type (WT) B. abortus or isogenic virB2 and vceC mutants for 13 days (n = 6). Values represent individual mice (black circles) and means (black lines). *, P < 0.05 using Mann-Whitney statistical analysis. (C) Fetal viability in pregnant mice infected with wild-type B. abortus or isogenic virB2 and vceC mutants for 13 days (n = 6). Values represent individual mice (black circles) and means (black lines). *, P < 0.05 using one-way ANOVA. (D) Placental colonization with wild-type B. abortus or its isogenic virB2 and vceC mutants in pregnant mice infected for 13 days. Values represent means ± SEM. *, P < 0.05 using one-way ANOVA. ns, nonsignificant.
FIG 3
FIG 3
Role of NOD1 and NOD2 in death of trophoblast giant cells. (A) Role of NOD1/NOD2 in trophoblast death. Nod1+/+ Nod2+/+ or Nod1−/− Nod2−/− dams were bred to sires of the same genotype and inoculated with B. abortus. Trophoblast death in the placentas at 13d postinfection was assessed by TUNEL staining. (B) Contribution of NOD1/NOD2 in maternally derived immune cells to B. abortus-induced abortion. Nod1+/+ Nod2+/+ or Nod1−/− Nod2−/− dams (n = 4–5) were bred to Nod1+/+ Nod2+/+ sires to generate fetuses carrying immune cells with the maternal Nod1 Nod2 genotype. Each dot represents the percent viability of pups in one litter and the bar indicates the geometric mean of the group.
FIG 4
FIG 4
The T4SS effector VceC is not required for intracellular replication in BeWo trophoblast-like cells. (A) Fluorescence images of BeWo cells infected with B. abortus 2308 and isogenic ΔvirB9 and ΔvceC mutants (red) showing colocalization with LAMP-1 (green) at 4 h and 24 h postinfection. Scale bar in lower right panel represents 10 μm, and scale bar in inset represents 2 μm. Images are representative of three independent replicates. (B) Quantification of LAMP-1-positive BCVs in BeWo cells infected with B. abortus 2308 or ΔvirB9 and ΔvceC mutants at 4, 8, 12, and 24 h after B. abortus infection. Data are compiled from three independent experiments per strain and time point. (C) CFU of wild-type B. abortus, the ΔvirB mutant, and the ΔvceC mutant in BeWo cells infected for 0 h and 24 h (n = 4). Values represent means ± SEM. *, P < 0.05 using one-way ANOVA.
FIG 5
FIG 5
VceC induces expression of CHOP in the placenta. (A and B) RT-PCR analysis of transcripts for CHOP (Ddit3) in placentas (A) and spleens (B) of pregnant mice infected with wild-type B. abortus or the vceC mutant for 13 days (n = 7). Values represent means ± SEM. *, P < 0.05 using unpaired t test. (C and D) Detection of CHOP by Western blotting in placentas of uninfected mice or mice infected with wild-type B. abortus or the vceC mutant for 13 days (n = 4). (C) Densitometric quantification of CHOP signal relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Significance of differences was determined by a Kruskal-Wallis test with Dunn’s post hoc test. *, P < 0.05. (D) Western blots used for quantification in panel C. (E to G) Induction of ER stress responses in HEK293 cells after ectopic expression of VceC1-418 or VceC38-418 or after thapsigargin treatment. (E) Expression of DDIT3 measured by qRT-PCR. Bars represent geometric means ± SEM. (F) Abundance of spliced XBP1 transcript measured by qRT-PCR. Bars represent geometric means ± SEM. (G) Agarose gel showing spliced (S) and unspliced (U) XBP1 transcripts.
FIG 6
FIG 6
CHOP induction in the placenta contributes to fetal loss, trophoblast death, and extracellular release of B. abortus. (A) Viability of pups born to C57BL/6J or congenic CHOP knockout (CHOP-KO) dams infected for 13 days with B. abortus 2308 or isogenic vceC mutant (n = 4 for the mock-infected and n = 6 for the infected group). (B) Histologic scoring of TUNEL staining of trophoblasts from CHOP-KO mice. (C) B. abortus colonization of the placentas in mice from panel A. (D) Proportion of intracellular (gentamicin-resistant) versus extracellular (gentamicin-sensitive) B. abortus organisms after incubation of tissue ex vivo for 30 min in 50 mg/ml of gentamicin (n = 6). *, P < 0.05. Significance of differences was analyzed using a Mann-Whitney test.

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