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

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. 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.

Download Full-text

The PGRS Domain of Mycobacterium tuberculosis PE_PGRS Protein Rv0297 Is Involved in Endoplasmic Reticulum Stress-Mediated Apoptosis through Toll-Like Receptor 4

mBio ◽

10.1128/mbio.01017-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 19

Author(s):

Sonam Grover ◽

Tarina Sharma ◽

Yadvir Singh ◽

Sakshi Kohli ◽

Manjunath P. ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mycobacterium Tuberculosis ◽

Er Stress ◽

Unfolded Protein Response ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Content Type ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

ABSTRACT The genome of Mycobacterium tuberculosis , the causal organism of tuberculosis (TB), encodes a unique protein family known as the PE/PPE/PGRS family, present exclusively in the genus Mycobacterium and nowhere else in the living kingdom, with largely unexplored functions. We describe the functional significance of the PGRS domain of Rv0297, a member of this family. In silico analyses revealed the presence of intrinsically disordered stretches and putative endoplasmic reticulum (ER) localization signals in the PGRS domain of Rv0297 (Rv0297PGRS). The PGRS domain aids in ER localization, which was shown by infecting macrophage cells with M. tuberculosis and by overexpressing the protein by transfection in macrophage cells followed by activation of the unfolded protein response, as evident from increased expression of GRP78/GRP94 and CHOP/ATF4, leading to disruption of intracellular Ca 2+ homeostasis and increased nitric oxide (NO) and reactive oxygen species (ROS) production. The consequent activation of the effector caspase-8 resulted in apoptosis of macrophages, which was Toll-like receptor 4 (TLR4) dependent. Administration of recombinant Rv0297PGRS (rRv0297PGRS) also exhibited similar effects. These results implicate a hitherto-unknown role of the PGRS domain of the PE_PGRS protein family in ER stress-mediated cell death through TLR4. Since this protein is already known to be present at later stages of infection in human granulomas it points to the possibility of it being employed by M. tuberculosis for its dissemination via an apoptotic mechanism. IMPORTANCE Apoptosis is generally thought to be a defense mechanism in protecting the host against Mycobacterium tuberculosis in early stages of infection. However, apoptosis during later stages in lung granulomas may favor the bacterium in disseminating the disease. ER stress has been found to induce apoptosis in TB granulomas, in zones where apoptotic macrophages accumulate in mice and humans. In this study, we report ER stress-mediated apoptosis of host cells by the Rv0297-encoded PE_PGRS5 protein of M. tuberculosis exceptionally present in the pathogenic Mycobacterium genus. The PGRS domain of Rv0297 aids the protein in localizing to the ER and induces the unfolded protein response followed by apoptosis of macrophages. The effect of the Rv0297PGRS domain was found to be TLR4 dependent. This study presents novel insights on the strategies employed by M. tuberculosis to disseminate the disease.

Download Full-text

Adaptation to Endoplasmic Reticulum Stress Requires Transphosphorylation within the Activation Loop of Protein Kinases Kin1 and Kin2, Orthologs of Human Microtubule Affinity-Regulating Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.00266-18 ◽

2018 ◽

Vol 38 (23) ◽

Author(s):

Chandrima Ghosh ◽

Leena Sathe ◽

Joel David Paprocki ◽

Valerica Raicu ◽

Madhusudan Dey

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Cellular Membrane ◽

Kinase Domain ◽

Activation Loop ◽

Content Type ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis

ABSTRACT Perturbations in endoplasmic reticulum (ER) homeostasis, a condition termed ER stress, activate the unfolded protein response (UPR), an intracellular network of signaling pathways. Recently, we have shown that protein kinase Kin1 and its paralog, Kin2, in the budding yeast Saccharomyces cerevisiae (orthologs of microtubule affinity-regulating kinase in humans) contribute to the UPR function. These Kin kinases contain a conserved kinase domain and an autoinhibitory kinase-associated 1 (KA1) domain separated by a long undefined domain. Here, we show that Kin1 or Kin2 protein requires minimally a kinase domain and an adjacent kinase extension region (KER) for UPR function. We also show that the functional mini-Kin2 protein is predominantly visualized inside the cells and precipitated with the cellular membrane fraction, suggesting its association with the cellular endomembrane system. Furthermore, we show that transphosphorylation of the Kin1 residue T302 and the analogous Kin2 residue T281 within the activation loop are important for full kinase activity. Collectively, our data suggest that, during ER stress, the Kin kinase domain is released from its autoinhibitory KA1 domain and is activated by transphosphorylation.

Download Full-text

Sensing of Bacterial Type IV Secretion via the Unfolded Protein Response

mBio ◽

10.1128/mbio.00418-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 60

Author(s):

Maarten F. de Jong ◽

Tregei Starr ◽

Maria G. Winter ◽

Andreas B. den Hartigh ◽

Robert Child ◽

...

Keyword(s):

Unfolded Protein Response ◽

Type Iv Secretion ◽

Host Cells ◽

Type Iv Secretion System ◽

Content Type ◽

Unfolded Protein ◽

Type Iv ◽

Er Chaperone ◽

The Unfolded Protein Response ◽

Protein Response

ABSTRACTHost cytokine responses toBrucella abortusinfection are elicited predominantly by the deployment of a type IV secretion system (T4SS). However, the mechanism by which the T4SS elicits inflammation remains unknown. Here we show that translocation of the T4SS substrate VceC into host cells induces proinflammatory responses. Ectopically expressed VceC interacted with the endoplasmic reticulum (ER) chaperone BiP/Grp78 and localized to the ER of HeLa cells. ER localization of VceC required a transmembrane domain in its N terminus. Notably, the expression of VceC resulted in reorganization of ER structures. In macrophages, VceC was required forB. abortus-induced inflammation by induction of the unfolded protein response by a process requiring inositol-requiring transmembrane kinase/endonuclease 1. Altogether, these findings suggest that translocation of the T4SS effector VceC induces ER stress, which results in the induction of proinflammatory host cell responses duringB. abortusinfection.IMPORTANCEBrucellaspecies are pathogens that require a type IV secretion system (T4SS) to survive in host cells and to maintain chronic infection. By as-yet-unknown pathways, the T4SS also elicits inflammatory responses in infected cells. Here we show that inflammation caused by the T4SS results in part from the sensing of a T4SS substrate, VceC, that localizes to the endoplasmic reticulum (ER), an intracellular site ofBrucellareplication. Possibly via binding of the ER chaperone BiP, VceC causes ER stress with concomitant expression of proinflammatory cytokines. Thus, induction of the unfolded protein response may represent a novel pathway by which host cells can detect pathogens deploying a T4SS.

Download Full-text

Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca2+-ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus

mBio ◽

10.1128/mbio.01060-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 1

Author(s):

Martin Weichert ◽

José Guirao-Abad ◽

Vishukumar Aimanianda ◽

Karthik Krishnan ◽

Christina Grisham ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Wall ◽

Er Stress ◽

Aspergillus Fumigatus ◽

Secretory Pathway ◽

Functional Coupling ◽

Content Type ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

ABSTRACT Many species of pathogenic fungi deploy the unfolded protein response (UPR) to expand the folding capacity of the endoplasmic reticulum (ER) in proportion to the demand for virulence-related proteins that traffic through the secretory pathway. Although Ca2+ plays a pivotal role in ER function, the mechanism by which transcriptional upregulation of the protein folding machinery is coordinated with Ca2+ homeostasis is incompletely understood. In this study, we investigated the link between the UPR and genes encoding P-type Ca2+-ATPases in the human-pathogenic mold Aspergillus fumigatus. We demonstrate that acute ER stress increases transcription of the srcA gene, encoding a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family, as well as that of pmrA, encoding a secretory pathway Ca2+-ATPase (SPCA) in the Golgi membrane. Loss of the UPR transcription factor HacA prevented the induction of srcA and pmrA transcription during ER stress, defining these ER/Golgi Ca2+ pumps as novel downstream targets of this pathway. While deletion of srcA alone caused no major deficiencies, a ΔsrcA/ΔpmrA mutant displayed a severe polarity defect, was hypersensitive to ER stress, and showed attenuated virulence. In addition, cell wall analyses revealed a striking reduction in mannose levels in the absence of both Ca2+ pumps. The ΔhacA mutant was hypersensitive to agents that block calcineurin-dependent signaling, consistent with a functional coupling between the UPR and Ca2+ homeostasis. Together, these findings demonstrate that the UPR integrates the need for increased levels of chaperone and folding enzymes with an influx of Ca2+ into the secretory pathway to support fungal growth, stress adaptation, and pathogenicity. IMPORTANCE The UPR is an intracellular signal transduction pathway that maintains homeostasis of the ER. The pathway is also tightly linked to the expression of virulence-related traits in diverse species of human-pathogenic and plant-pathogenic fungal species, including the predominant mold pathogen infecting humans, Aspergillus fumigatus. Despite advances in the understanding of UPR signaling, the linkages and networks that are governed by this pathway are not well defined. In this study, we revealed that the UPR is a major driving force for stimulating Ca2+ influx at the ER and Golgi membranes and that the coupling between the UPR and Ca2+ import is important for virulence, cell wall biosynthesis, and resistance to antifungal compounds that inhibit Ca2+ signaling.

Download Full-text

IreA controls endoplasmic reticulum stress-induced autophagy and survival through homeostasis recovery

Molecular and Cellular Biology ◽

10.1128/mcb.00054-18 ◽

2018 ◽

pp. MCB.00054-18 ◽

Cited By ~ 6

Author(s):

Eunice Domínguez-Martín ◽

Laura Ongay-Larios ◽

Laura Kawasaki ◽

Olivier Vincent ◽

Gerardo Coello ◽

...

Keyword(s):

Gene Expression ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Cell Survival ◽

Eukaryotic Cell ◽

Functional Link ◽

Unfolded Protein ◽

Transcriptional Changes ◽

The Unfolded Protein Response ◽

Protein Response

The Unfolded Protein Response (UPR) is an adaptive pathway that restores cellular homeostasis after endoplasmic reticulum (ER) stress. The ER-resident kinase/ribonuclease Ire1 is the only UPR sensor conserved during evolution. Autophagy, a lysosomal degradative pathway, also contributes to the recovery of cell homeostasis after ER-stress but the interplay between these two pathways is still poorly understood. We describe the Dictyostelium discoideum ER-stress response and characterize its single bonafide Ire1 orthologue, IreA. We found that tunicamycin (TN) triggers a gene-expression reprogramming that increases the protein folding capacity of the ER and alleviates ER protein load. Further, IreA is required for cell-survival after TN-induced ER-stress and is responsible for nearly 40% of the transcriptional changes induced by TN. The response of Dictyostelium cells to ER-stress involves the combined activation of an IreA-dependent gene expression program and the autophagy pathway. These two pathways are independently activated in response to ER-stress but, interestingly, autophagy requires IreA at a later stage for proper autophagosome formation. We propose that unresolved ER-stress in cells lacking IreA causes structural alterations of the ER, leading to a late-stage blockade of autophagy clearance. This unexpected functional link may critically affect eukaryotic cell survival under ER-stress.

Download Full-text

ER stress in neurodegenerative disease: from disease mechanisms to therapeutic interventions

Endoplasmic Reticulum Stress in Diseases ◽

10.1515/ersc-2017-0002 ◽

2017 ◽

Vol 4 (1) ◽

Cited By ~ 2

Author(s):

Felipe Cabral-Miranda ◽

Claudio Hetz

Keyword(s):

Endoplasmic Reticulum ◽

Neurodegenerative Diseases ◽

Er Stress ◽

Major Element ◽

Neuronal Loss ◽

Therapeutic Interventions ◽

Unfolded Protein ◽

Disease Mechanisms ◽

The Unfolded Protein Response ◽

Protein Response

AbstractThe conception that protein aggregates composed by misfolded proteins underlies the occurrence of several neurodegenerative diseases suggests that this phenomenon may have a common origin, ultimately driven by disruption of proteostasis control. The unfolded protein response (UPR) embodies a major element of the proteostasis network, which is engaged by endoplasmic reticulum (ER) stress. Chronic ER stress may operate as a possible mechanism of neurodegeneration, contributing to synaptic alterations, neuroinflammation and neuronal loss. In this review we discuss most recent findings relating ER stress and the development of distinct neurodegenerative diseases, and the possible strategies for disease intervention.

Download Full-text

Endoplasmic Reticulum Stress and Unfolded Protein Response in Breast Cancer: The Balance between Apoptosis and Autophagy and Its Role in Drug Resistance

International Journal of Molecular Sciences ◽

10.3390/ijms20040857 ◽

2019 ◽

Vol 20 (4) ◽

pp. 857 ◽

Cited By ~ 20

Author(s):

Lorenza Sisinni ◽

Michele Pietrafesa ◽

Silvia Lepore ◽

Francesca Maddalena ◽

Valentina Condelli ◽

...

Keyword(s):

Breast Cancer ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Unfolded Protein Response ◽

Inflammatory Diseases ◽

Unfolded Protein ◽

Chronic Activation ◽

The Unfolded Protein Response ◽

Protein Response ◽

The Relationship

The unfolded protein response (UPR) is a stress response activated by the accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER) and its uncontrolled activation is mechanistically responsible for several human pathologies, including metabolic, neurodegenerative, and inflammatory diseases, and cancer. Indeed, ER stress and the downstream UPR activation lead to changes in the levels and activities of key regulators of cell survival and autophagy and this is physiologically finalized to restore metabolic homeostasis with the integration of pro-death or/and pro-survival signals. By contrast, the chronic activation of UPR in cancer cells is widely considered a mechanism of tumor progression. In this review, we focus on the relationship between ER stress, apoptosis, and autophagy in human breast cancer and the interplay between the activation of UPR and resistance to anticancer therapies with the aim to disclose novel therapeutic scenarios. The hypothesis that autophagy and UPR may provide novel molecular targets in human malignancies is discussed.

Download Full-text

Endoplasmic reticulum stress and the unfolded protein response in pancreatic islet inflammation

Journal of Molecular Endocrinology ◽

10.1530/jme-15-0306 ◽

2016 ◽

Vol 57 (1) ◽

pp. R1-R17 ◽

Cited By ~ 31

Author(s):

Kira Meyerovich ◽

Fernanda Ortis ◽

Florent Allagnat ◽

Alessandra K Cardozo

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Unfolded Protein Response ◽

Diabetic Patients ◽

Β Cells ◽

Β Cell ◽

Unfolded Protein ◽

Islet Inflammation ◽

The Unfolded Protein Response ◽

Protein Response

Insulin-secreting pancreatic β-cells are extremely dependent on their endoplasmic reticulum (ER) to cope with the oscillatory requirement of secreted insulin to maintain normoglycemia. Insulin translation and folding rely greatly on the unfolded protein response (UPR), an array of three main signaling pathways designed to maintain ER homeostasis and limit ER stress. However, prolonged or excessive UPR activation triggers alternative molecular pathways that can lead to β-cell dysfunction and apoptosis. An increasing number of studies suggest a role of these pro-apoptotic UPR pathways in the downfall of β-cells observed in diabetic patients. Particularly, the past few years highlighted a cross talk between the UPR and inflammation in the context of both type 1 (T1D) and type 2 diabetes (T2D). In this article, we describe the recent advances in research regarding the interplay between ER stress, the UPR, and inflammation in the context of β-cell apoptosis leading to diabetes.

Download Full-text

Endoplasmic reticulum stress regulation of the Kar2p/BiP chaperone alleviates proteotoxicity via dual degradation pathways

Molecular Biology of the Cell ◽

10.1091/mbc.e11-04-0297 ◽

2012 ◽

Vol 23 (4) ◽

pp. 630-641 ◽

Cited By ~ 6

Author(s):

Chia-Ling Hsu ◽

Rupali Prasad ◽

Christie Blackman ◽

Davis T. W. Ng

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Target Genes ◽

Regulatory Element ◽

Unfolded Protein ◽

Single Target ◽

Transcriptional Regulatory ◽

The Unfolded Protein Response ◽

Protein Response ◽

Stress Activation

The unfolded protein response (UPR) monitors and maintains protein homeostasis in the endoplasmic reticulum (ER). In budding yeast, the UPR is a transcriptional regulatory pathway that is quiescent under normal conditions. Under conditions of acute ER stress, activation of UPR targets is essential for cell viability. How individual target genes contribute to stress tolerance is unclear. Uncovering these roles is hampered because most targets also play important functions in the absence of stress. To differentiate stress-specific roles from everyday functions, a single target gene was uncoupled from UPR control by eliminating its UPR-specific regulatory element. Through this approach, the UPR remains intact, aside from its inability to induce the designated target. Applying the strategy to the major ER chaperone Kar2p/BiP revealed the physiological function of increasing its cellular concentration. Despite hundreds of target genes under UPR control, we show that activation of KAR2 is indispensable to alleviate some forms of ER stress. Specifically, activation is essential to dispose misfolded proteins that are otherwise toxic. Surprisingly, induced BiP/Kar2p molecules are dedicated to alleviating stress. The inability to induce KAR2 under stress had no effect on its known housekeeping functions.

Download Full-text

Cu, Zn Superoxide Dismutase and NADP(H) Homeostasis Are Required for Tolerance of Endoplasmic Reticulum Stress inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e08-07-0697 ◽

2009 ◽

Vol 20 (5) ◽

pp. 1493-1508 ◽

Cited By ~ 44

Author(s):

Shi-Xiong Tan ◽

Mariati Teo ◽

Yuen T. Lam ◽

Ian W. Dawes ◽

Gabriel G. Perrone

Keyword(s):

Endoplasmic Reticulum ◽

Superoxide Dismutase ◽

Er Stress ◽

Stress Sensitivity ◽

Pentose Phosphate ◽

Unfolded Protein ◽

Genome Wide ◽

The Unfolded Protein Response ◽

Protein Response

Genome-wide screening for sensitivity to chronic endoplasmic reticulum (ER) stress induced by dithiothreitol and tunicamycin (TM) identified mutants deleted for Cu, Zn superoxide dismutase (SOD) function (SOD1, CCS1) or affected in NADPH generation via the pentose phosphate pathway (TKL1, RPE1). TM-induced ER stress led to an increase in cellular superoxide accumulation and an increase in SOD1 expression and Sod1p activity. Prior adaptation of the hac1 mutant deficient in the unfolded protein response (UPR) to the superoxide-generating agent paraquat reduced cell death under ER stress. Overexpression of the ER oxidoreductase Ero1p known to generate hydrogen peroxide in vitro, did not lead to increased superoxide levels in cells subjected to ER stress. The mutants lacking SOD1, TKL1, or RPE1 exhibited decreased UPR induction under ER stress. Sensitivity of the sod1 mutant to ER stress and decreased UPR induction was partially rescued by overexpression of TKL1 encoding transketolase. These data indicate an important role for SOD and cellular NADP(H) in cell survival during ER stress, and it is proposed that accumulation of superoxide affects NADP(H) homeostasis, leading to reduced UPR induction during ER stress.

Download Full-text