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.

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.IMPORTANCE Brucella 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

Hypertension and hand-foot skin reactions related to VEGFR2 genotype and improved clinical outcome following bevacizumab and sorafenib

BACKGROUND: Hypertension (HT) and hand-foot skin reactions (HFSR) may be related to the activity of bevacizumab and sorafenib. We hypothesized that these toxicities would correspond to favorable outcome in these drugs, that HT and HFSR would coincide, and that VEGFR2 genotypic variation would be related to toxicity and clinical outcomes. METHODS: Toxicities (≥ grade 2 HT or HFSR), progression-free survival (PFS), and overall survival (OS) following treatment initiation were evaluated. Toxicity incidence and VEGFR2 H472Q and V297I status were compared to clinical outcomes. RESULTS: Individuals experiencing HT had longer PFS following bevacizumab therapy than those without this toxicity in trials utilizing bevacizumab in patients with prostate cancer (31.5 vs 14.9 months, n = 60, P = 0.0009), and bevacizumab and sorafenib in patients with solid tumors (11.9 vs. 3.7 months, n = 27, P = 0.052). HT was also linked to a > 5-fold OS benefit after sorafenib and bevacizumab cotherapy (5.7 versus 29.0 months, P = 0.0068). HFSR was a marker for prolonged PFS during sorafenib therapy (6.1 versus 3.7 months respectively, n = 113, P = 0.0003). HT was a risk factor for HFSR in patients treated with bevacizumab and/or sorafenib (OR(95%CI) = 3.2(1.5-6.8), P = 0.0024). Carriers of variant alleles at VEGFR2 H472Q experienced greater risk of developing HT (OR(95%CI) = 2.3(1.2 - 4.6), n = 170, P = 0.0154) and HFSR (OR(95%CI) = 2.7(1.3 - 5.6), n = 170, P = 0.0136). CONCLUSIONS: This study suggests that HT and HFSR may be markers for favorable clinical outcome, HT development may be a marker for HFSR, and VEGFR2 alleles may be related to the development of toxicities during therapy with bevacizumab and/or sorafenib

The transcription factor CHOP, an effector of the integrated stress response, is required for host sensitivity to the fungal intracellular pathogen <i>Histoplasma capsulatum</i>

<div><p>The ability of intracellular pathogens to manipulate host-cell viability is critical to successful infection. Some pathogens promote host-cell survival to protect their replicative niche, whereas others trigger host-cell death to facilitate release and dissemination of the pathogen after intracellular replication has occurred. We previously showed that the intracellular fungal pathogen <i>Histoplasma capsulatum</i> (<i>Hc</i>) uses the secreted protein Cbp1 to actively induce apoptosis in macrophages; interestingly, <i>cbp1</i> mutant strains are unable to kill macrophages and display severely reduced virulence in the mouse model of <i>Hc</i> infection. To elucidate the mechanism of Cbp1-induced host-cell death, we performed a comprehensive alanine scanning mutagenesis and identified all amino acid residues that are required for Cbp1 to trigger macrophage lysis. Here we demonstrate that <i>Hc</i> strains expressing lytic <i>CBP1</i> alleles activate the integrated stress response (ISR) in infected macrophages, as indicated by an increase in eIF2α phosphorylation as well as induction of the transcription factor <i>CHOP</i> and the pseudokinase <i>Tribbles 3</i> (<i>TRIB3</i>). In contrast, strains bearing a non-lytic allele of <i>CBP1</i> fail to activate the ISR, whereas a partially lytic <i>CBP1</i> allele triggers intermediate levels of activation. We further show that macrophages deficient for <i>CHOP</i> or <i>TRIB3</i> are partially resistant to lysis during <i>Hc</i> infection, indicating that the ISR is critical for susceptibility to <i>Hc</i>-mediated cell death. Moreover, we show that CHOP-dependent macrophage lysis is critical for efficient spread of <i>Hc</i> infection to other macrophages. Notably, <i>CHOP</i> knockout mice display reduced macrophage apoptosis and diminished fungal burden and are markedly resistant to <i>Hc</i> infection. Together, these data indicate that Cbp1 is required for <i>Hc</i> to induce the ISR and mediate a CHOP-dependent virulence pathway in the host.</p></div

<i>Hc</i> does not cause ER stress in infected BMDMs.

<p>BMDMs were infected with the indicated <i>Hc</i> strains at an MOI of 5, mock infected (uninf), or treated with 2.5 μg/mL tunicamycin (Tm). <b>(A)</b> Unspliced (<i>Xbp1u</i>) and spliced (<i>Xbp1s</i>) isoforms of <i>Xbp1</i> were detected by non-quantitative RT-PCR. <b>(B-D)</b> Relative abundances of <b>(B)</b> <i>ERdj4</i>, <b>(C)</b> <i>SEL1L</i>, and <b>(D)</b> <i>BiP</i> were measured by RT-qPCR. <b>(E)</b> Phosphorylated PERK (Thr 980) was detected by Western blot at 12 hpi. <b>(F)</b> BMDMs were treated with the PERK-specific inhibitor GSK2606414 (3 μM) or vehicle (DMSO) for 12 hpi, and caspase-3/7 activity was assessed and normalized to vehicle-treated uninfected cells. Each value is an average of triplicate wells ± standard deviation. **p<0.01, two-tailed t test.</p

Model of Cbp1-mediated host-cell death during <i>Hc</i> infection.

<p>During infection, <i>Hc</i> yeast produce a large amount of the secreted protein Cbp1 (red triangle). Cbp1 induces phosphorylation of the mammalian protein eIF2α through an unknown mechanism. The increase in phospho-eIF2 leads to the preferential translation of the transcription factor ATF4, which leads to the expression of <i>CHOP</i> and <i>TRIB3</i>. The pseudokinase Tribbles 3 inhibits Akt phosphorylation, thereby promoting Bax/Bak oligomerization, ultimately resulting in caspase-3/7 activation, which is enhanced by caspase-8 activation. The activation of the executioner caspases ultimately results in macrophage death, allowing for the release of live fungal cells.</p

CHOP is required for optimal spread of <i>Hc</i> during macrophage infection.

<p><b>(A)</b> Uninfected wildtype BMDMs were seeded in 6-well plates. Above them, transwells with wildtype or <i>CHOP</i>^<i>-/-</i> BMDMs, either uninfected or infected with wildtype <i>Hc</i>, were placed with the macrophages on the underside of the transwell. After the onset of lysis of the infected macrophages, the transwells were removed. The fungal burdens immediately after transwell removal <b>(B)</b> and lysis kinetics <b>(C)</b> of the bottom macrophages were assessed by CFU enumeration and LDH release assay, respectively. For CFUs, ***p<0.005 by two-tailed t-test; for macrophage lysis, *p<0.05 by two-tailed Mann-Whitney.</p

Lytic <i>CBP1</i> alleles activate the integrated stress response in infected macrophages.

<p>BMDMs were treated with 2.5 μg/mL tunicamycin (Tm), infected with indicated <i>Hc</i> strains at an MOI of 5, or mock infected (uninf). Phosphorylated eIF2α (Ser51) <b>(A)</b> and ATF4 <b>(B)</b> were assessed by Western blot at 12 hpi. Representative blots are shown. The average signal intensity of phospho-eIF2α or ATF4 relative to the corresponding α-tubulin loading control from three replicates is shown in the bar graphs.*p<0.05, **p<0.01, compared to wildtype, two-tailed t-test. <b>(C)</b> Relative abundances of <i>CHOP</i> and <i>TRIB3</i> transcripts were assessed by RT-qPCR at the indicated time points and normalized to uninfected BMDMs at 3 hpi.</p

Macrophage Cell Death and Transcriptional Response are Actively Triggered by the Fungal Virulence Factor Cbp1 During H. capsulatum Infection

Microbial pathogens induce or inhibit death of host cells during infection, with significant consequences for virulence and disease progression. Death of an infected host cell can either facilitate release and dissemination of intracellular pathogens or promote pathogen clearance.Histoplasma capsulatum is an intracellular fungal pathogen that replicates robustly within macrophages and triggers macrophage lysis by unknown means. To identify H. capsulatumeffectors of macrophage lysis, we performed a genetic screen and discovered three mutants that grew to wild-type levels within macrophages but failed to elicit host-cell death. Each mutant was defective in production of the previously identified secreted protein Cbp1 (calcium-binding protein 1), whose role in intracellular growth had not been fully investigated. We found that Cbp1 was dispensable for high levels of intracellular growth but required to elicit a unique transcriptional signature in macrophages, including genes whose induction was previously associated with endoplasmic reticulum stress and host-cell death. Additionally, Cbp1 was required for activation of cell-death caspases-3/7, and macrophage death during H. capsulatuminfection was dependent on the pro-apoptotic proteins Bax and Bak. Taken together, these findings strongly suggest that the ability of Cbp1 to actively program host-cell death is an essential step in H. capsulatum pathogenesis