The molecular mechanism of induction of unfolded protein response by Chlamydia

https://doi.org/10.1016/j.bbrc.2018.11.034Get rights and content

Highlights

  • Chlamydia induces UPR for ATP, nutrients and to protect host cell from apoptosis.

  • Chlamydial T3SS effectors activate non-muscle myosin heavy chain II (NMMHC-II).

  • Activated NMMHC-II binds UPR master regulator (BiP) or transducers to induce UPR.

  • Inhibition of UPR activation prevents Chlamydia replication and inclusion formation.

Abstract

The unfolded protein response (UPR) contributes to chlamydial pathogenesis, as a source of lipids and ATP during replication, and for establishing the initial anti-apoptotic state of host cell that ensures successful inclusion development. The molecular mechanism(s) of UPR induction by Chlamydia is unknown. Chlamydia use type III secretion system (T3SS) effector proteins (e.g, the Translocated Actin-Recruiting Phosphoprotein (Tarp) to stimulate host cell's cytoskeletal reorganization that facilitates invasion and inclusion development. We investigated the hypothesis that T3SS effector-mediated assembly of myosin-II complex produces activated non-muscle myosin heavy chain II (NMMHC-II), which then binds the UPR master regulator (BiP) and/or transducers to induce UPR. Our results revealed the interaction of the chlamydial effector proteins (CT228 and Tarp) with components of the myosin II complex and UPR regulator and transducer during infection. These interactions caused the activation and binding of NMMHC-II to BiP and IRE1α leading to UPR induction. In addition, specific inhibitors of myosin light chain kinase, Tarp oligomerization and myosin ATPase significantly reduced UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through T3SS effector-mediated activation of NMMHC-II components of the myosin complex to facilitate infectivity. The finding provides greater insights into chlamydial pathogenesis with the potential to identify therapeutic targets and formulations.

Introduction

Pelvic inflammatory disease, tubal factor infertility and ectopic pregnancy are serious complications of human genital infection by the Gram-negative intracellular bacterium Chlamydia trachomatis [[1], [2], [3]]. The rising infections with attendant healthcare cost is a major burden on the public healthcare system [4]. A better understanding of the molecular pathogenesis of Chlamydia diseases will aid the design of therapeutic measures. Chlamydia acquire nutrients such as lipids and ATP from host cells [1,2] and require living cells for their intracellular survival, replication and inclusion development [5]. Therefore, chlamydial infection produces conditions such as extra protein expression from the microbe that perturb the protein folding and modification functions of the endoplasmic reticulum (ER), causing ER stress that can lead to cell death if no cellular adaptation is induced [6,7]. The unfolded protein response (UPR) is a cellular response to ER stress due to calcium displacement, increased protein expression and demand for protein modification by an intracellular microbial parasite, or excessive accumulation of misfolded proteins [8,9]. The induction of UPR aims at restoring cellular homeostasis by enhancing host cell survival through autophagy promotion [10]; increases protein folding capacity of ER [11]; activates ER-associated protein degradation to relieve the stress on ER protein-folding machinery; or, if stress is not resolved, activates apoptotic pathways [12]. The three ER membrane proteins that are transducers of the UPR signaling are: inositol-requiring enzyme-1α (IRE1α), protein kinase RNA-activated (PKR)-like ER kinase (PERK), and the activating transcription factor-6α (ATF6α) [13]. These UPR transducers are kept inactive by binding to the ER chaperone GRP78/BiP (the master regulator of UPR) when the ER folding capacity is operating normally. Activators of UPR either cause the ER stress through depletion of Ca2+ from the ER [14], or the accumulation of unfolded/misfolded, overexpressed or modified proteins in the ER, any of which dissociates the UPR transducers from BiP to activate UPR signaling [15,16].

Indeed, recent reports have demonstrated that Chlamydia, like certain other obligate intracellular microbial agents such as Brucella melitensis, Listeria monocytogenes and Hepatitis C virus, activate the UPR pathways to enhance their intracellular survival and replication [15,[17], [18], [19]]. Mechanistically, Shiga toxigenic strains of Escherichia coli produce AB5 subtilase cytotoxin that binds to and inactivates BiP to activate the UPR transducers [20]; Hepatitis C virus activates UPR at least in part due to the accumulation of immature core protein (Core 199) in the ER lumen [15], while L. monocytogenes require its cholesterol-dependent cytolysin toxin (listeriolysin O) to induce UPR [18]. However, the mechanism of UPR activation by Chlamydia is not known. Chlamydia utilize various mechanisms including clathrin-mediated endocytosis for its uptake by host cells [21]. Upon host cell invasion, Chlamydia recruit and activate elements of the cytoskeleton such as actin, myosin complex and microtubules to enhance entry, facilitate the establishment of parasitophorous inclusion and its structural stability, replication and extrusion of EBs from host cell [[22], [23], [24], [25], [26]]. To achieve this, Chlamydia translocate certain T3SS effector proteins e.g., Tarp and CT166 into the host cell cytoplasm [27,28] to rapidly recruit and activate members of the host's GTPase proteins (such as Rac1 and Cdc42) at site of entry and around the vesicle [29,30]. Specifically, the Tarp protein possesses G and F actin binding domains [22], induces actin nucleation, polymerization and filament formation [31]. Also, Tarp regulates the recruitment and activation of host cell kinases (e.g., the ROCK and Src family kinases) around the inclusion and acts as a scaffold for Rac1 guanine nucleotide exchange [32]. The CT166 protein post-translationally modifies the GTPase itself [28] while the CT228 is involved in the recruitment of components of the myosin II complex and co-localizes with Src family of kinases around the inclusion membrane [23]. These kinases inactivate the myosin light chain phosphatase (MYPT1) through phosphorylation at Threo-852, causing its release from the myosin light chain 2 (MLC2) and also the activation of the myosin heavy chain II (NMMHC-II) [33]. Interestingly, activation of the NMMHC-II is required for the activation and modulation of the most conserved IRE1α arm of UPR in eukaryotes [[34], [35], [36]]. Besides, the chlamydial inclusion membrane protein CT813 also known as InaC [37] recruits ADP-ribosylation factor 1 (ARF1) and 4 (ARF4) to effect the acetylation and detyrosination of microtubules required for interaction with the Golgi complex and production of infectious EBs [24]. These findings suggest that chlamydial infection may activate the cytoskeletal network and associated molecules that are required for UPR activation and stabilization.

In this study, we investigated the hypothesis that the release of the T3SS effector proteins (e.g., Tarp and CT228) into host cells by Chlamydia leads to the recruitment and activation of the NMMHC-II which then binds to BiP and IRE1α, resulting in UPR activation. Our results identified NMMHC II as molecules that bind to BiP and IRE1α during Chlamydia infection. Specific inhibitors that block the activation and function of NMMHC II, also suppressed UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through the activation of NMMHC-II components of the myosin complex, providing greater insights into Chlamydia pathogenesis with potential therapeutic targets.

Section snippets

Chlamydia strains and cell cultures

Chlamydia muridarum Nigg (the agent of mouse pneumonitis, MoPn - animal specific strain), C. trachomatis (human specific strain) serovars L2/LGV-434 and D/UW-3 were grown in HeLa 229 cells (ATCC, Rockville, MD USA) and purified elementary bodies (EBs) were tittered as infectious forming units per millimeter (IFU/ml) using standard procedures previously described [19,38,39].

Immunoprecipitation assays

Immunoprecipitation kit (abcam Cambridge, MA, USA) was used in pull down experiments with UPR specific antibodies. M-PER®

Several species and strains of Chlamydia induce UPR

We extended previous studies that the mouse agent of pneumonitis (C. muridarum) activated all the three arms of UPR [19], by establishing that different strains and serovars of Chlamydia induce UPR. Thus, the assessment of UPR induction in epithelial cells using the human C. trachomatis (Ct.) serovars D and L. or the murine strain C. muridarum, revealed that all the chlamydial strains activated the IRE1α arm of the UPR (Fig. 1a). The activation of PERK arm of UPR was observed at 24hr post

Discussion

UPR plays a major role in chlamydial inclusion development and pathogenesis, since the its inhibition is deleterious to the survival and replication of Chlamydia [19]. UPR also plays a role in the replication of other bacterial pathogens such as Listeria [18] and Brucella spp [53]. However, the mechanism of UPR induction by Chlamydia remained unknown. The chlamydial T3SS system is essential for infection since Chlamydia uses T3SS effectors to recruit and modify host cell cytoskeletal components

Funding

This work was supported by the Centers for Disease Control and Prevention and PHS grants (AI41231, GM 08248, RR03034 and 1SC1GM098197) from the NIH.

Ethics statements

I declare that none of the authors has a financial interest related to this work. All of the authors consented to this submission and the results are not under consideration for publication elsewhere. All animal protocols were approved by the CDC Institutional Animal Care and Use Committee (IACUC) under Protocol # 2894IGIMOUC-A1. The CDC IACUC is guided by Title 9, Chapter I, Subchapter A--Animal Welfare (USDA Regulations).

Note: The conclusions in this report are those of the author(s) and do

Conflict of interest statements

I declare that none of the authors has a financial interest related to this work.

Acknowledgements

We thank Ted Hackstadt of the Laboratory of Intracellular Parasites, NIAID/NIH, Hamilton MT USA and Travis Jewett of the University of Central Florida, Orlando, FL USA for their resource and intellectual contributions to the study.

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