Shiga Toxins Trigger the Secretion of Lysyl-tRNA Synthetase to Enhance Proinflammatory Responses

Abstract

Shiga toxins (Stxs) produced by Shiga toxin-producing Escherichia coli (STEC) strains are major virulence factors that cause fatal systemic complications, such as hemolytic uremic syndrome and disruption of the central nervous system. Although numerous studies report proinflammatory responses to Stx type 1 (Stx1) or Stx type 2 (Stx2) both in vivo and in vitro, none have examined dynamic immune regulation involving cytokines and/or unknown inflammatory mediators during intoxication. Here, we showed that enzymatically active Stxs trigger the dissociation of lysyl-tRNA synthetase (KRS) from the multi-aminoacyl-tRNA synthetase complex in human macrophage-like differentiated THP-1 cells and its subsequent secretion. The secreted KRS acted to increase the production of proinflammatory cytokines and chemokines. Thus, KRS may be one of the key factors that mediate transduction of inflammatory signals in the STEC-infected host.

Introduction

Shiga toxins (Stxs) are major virulence factors produced by Shigella dysenteriae serotype 1 and human pathogenic Shiga toxin-producing Escherichia coli (STEC). In particular, STEC infection is a significant threat to public health as it can cause life-threatening illnesses such as hemolytic uremic syndrome (HUS), which is characterized by acute renal failure, thrombocytopenia, and damage to endothelial cells lining the glomeruli and kidney arterioles [20,29,30]. STEC produces Stxs, which are divided into two classes (Stx1 and Stx2) based on their antigenic similarity to the prototypical Stx expressed by S. dysenteriae serotype 1 [28]. All Stxs have an AB5 structure comprising an enzymatic A subunit that is non-covalently associated with homopentameric B subunits [2,27]. The pentameric B subunits of the holotoxin are responsible for its binding to the neutral glycolipid receptor globotriaosylceramide (Gb3) on the surface of host cells, thereby mediating cytoplasmic delivery of the A subunit, which possesses N-glycosidase activity [15]. Following retrograde transport to the trans-Golgi network and endoplasmic reticulum, Stxs activate host stress responses to trigger apoptosis [1], inflammatory responses [11], or autophagy [10] in different cell types. Notably, Stx-induced proinflammatory cytokines and chemokines contribute to damage in the colon and to the development of life-threatening conditions such as acute renal failure (e.g., HUS) and neurological abnormalities [31]. Differentiated macrophage-like THP-1 (D-THP-1) cells respond to Stxs by activating mitogen-activated protein kinase (MAPK) cascades and by producing proinflammatory tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, macrophage inflammatory protein (MIP)-1α, macrophage chemoattractant monocyte chemoattractant protein 1, IL-8, and growth-related oncogene β [4,14].

Aminoacyl-tRNA synthetases (ARSs) are key enzymes involved in protein synthesis. In mammals (including humans), ARSs are organized into a macromolecular protein complex (called the multiaminoacyl-tRNA synthetase complex; MSC), which comprises nine different ARSs and three ARS-interacting multifunctional proteins (AIMP 1–3) [16]. The MSC facilitates highly organized protein synthesis [9] and serves as a reservoir of regulatory molecules that have functions beyond protein synthesis [21]. For example, the bifunctional molecule glutamyl-prolyl-tRNA synthetase (EPRS) is released from the MSC upon stimulation by IFN-γ and forms a component of a translation complex that directs gene-specific silencing of translation [23]. Another component of the complex, lysyl-tRNA synthetase (KRS), is secreted, whereupon it triggers a proinflammatory response [19]. In addition, the accessory protein, AIMP1, is also secreted and upregulates expression of proinflammatory genes [8,18].

Here, we show for the first time that Stxs trigger the dissociation and secretion of KRS from the MSC in toxin-sensitive human macrophage-like D-THP-1 cells to enhance proinflammatory responses.

 

Materials and Methods

Antibodies and Reagents

Anti-Myc-HRP, anti-KRS, anti-β-actin, anti-EPRS, and anti-AIMP2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and from Neomics (Seoul, Korea), respectively. Anti-c-Myc-conjugated agarose beads (Sigma Aldrich, St. Louis, MO, USA) were used to immunoprecipitate c-Myc-tagged proteins. The Strep-tag-specific HRP-conjugated monoclonal antibody used to detect the Strep-tag was purchased from IBA (Goettingen, Germany). Cells were transiently transfected using the X-tremeGENE HP DNA transfection reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. Expression vectors (pcDNA3-AIMP2-Myc (to express Myc-tagged AIMP2), pEXPR IBA105-AIMP2-Strep (to express Strep-tagged AIMP2), and pEXPR IBA103-KRS-Strep (to express Strep-tagged KRS)) were kindly provided by Dr. Sunghoon Kim (Seoul National University, Seoul, Korea). Stx1, Stx2, and holotoxins harboring a mutation in the A subunit (Stx1A- and Stx2A-) or comprising the B subunit only (Stx1B-sub and Stx2B-sub) were provided by Dr. Vernon L. Tesh (Texas A&M University, TX, USA).

Cell Culture

The human myelogenous leukemia cell line THP-1 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and differentiated into macrophage-like (D-THP-1) cells as previously described [11]. HeLa cells were maintained in MEM (Gibco, Grand Island, NY, USA) supplemented with 10% FBS and 1% antibiotic-antimycotic solution. Human T84 colon carcinoma cells were obtained from European Collection of Cell Cultures (Salisbury, UK). Cells were cultured in DMEM/F-12 (1:1) supplemented with 10% FBS.

Preparation of Cell Lysates and Western Blotting

Cells treated with Stx1 (400 ng/ml) or Stx2 (10 ng/ml) were lysed in radio-immunoprecipitation assay buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 2 mM EDTA) supplemented with protease inhibitor and phosphatase inhibitor cocktails. The collected culture supernatants were pre-cleared by centrifugation at 16,000 ×g for 20 min at 4℃ and concentrated using the Amicon Ultra centrifugal filter system prior to western blotting. Western blot analysis was performed as previously described [11].

Isolation of RNA and Quantitative RT-PCR

Total RNA was isolated from Stx-treated D-THP-1 cells using the PureLink RNA Mini Kit column-based purification system (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Purified RNA samples were treated with TURBO DNase (Ambion) for 30 min to remove contaminating genomic DNA. The purity and concentration of the RNA were measured spectrophotometrically (Nanodrop Technologies, Wilmington, DE, USA). Quantitative RT-PCR assays were performed as previously described [11] using the following primers: KRS, 5’-GCCTCAAAGACAAGGAAACAAG-3’ (forward) and 5’-TGTCCAGCTCGTTGTGATAAG-3’ (reverse); β-Actin, 5’-CCTGGCACCCAGCACAAT-3’ (forward) and 5’-GCCGATCCACACGGAGTACT-3’ (reverse).

Isolation of Nuclear and Cytoplasmic Extracts

KRS-transfected D-THP-1 cells were treated with Stxs for 0 to 240 min followed by processing using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, Waltham, MA, USA) to yield nuclear and cytoplasmic fractions, which were then treated with protease and phosphatase inhibitors.

Immunoprecipitation

After transfecting HeLa cells with the pcDNA3-AIMP2-Myc vector to induce overexpression of the AIMP2 gene, cells were treated with Stx1 (400 ng/ml) or Stx2 (10 ng/ml) for 6 h before harvesting with RIPA lysis buffer. Equal amounts of cell lysate were incubated with anti-c-Myc agarose beads for 3 h at 4℃, washed with cold DPBS, and centrifuged at 500 ×g for 3 min (three times). The beads were then boiled for 10 min at 95℃ in SDS loading buffer and samples were loaded onto SDS-PAGE gels.

Pull-Down Assay

Strep-AIMP2 complexes were pulled down by Strep-tag affinity purification as previously described [25]. Briefly, HeLa cells were cultured to 80% confluence in 10 × 150 mm plates and then transfected with pEXPR IBA105-AIMP2-Strep expression plasmids. After 48 h, cells were washed twice with DPBS and stimulated with Stx1 (400 ng/ml) for an additional 12 h. Cells were then washed twice with DPBS, harvested, and lysed with lysis buffer A (250 mM Tris-HCl (pH 7.4), 37.5% glycerol, 750 mM NaCl, and 5 mM EDTA) supplemented with protease inhibitor and phosphatase inhibitor cocktails. The soluble fraction was separated by centrifugation at 17,000 × g for 5 min at 4℃, diluted 1:5 with binding buffer (20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and complete EDTA-free protease inhibitor cocktail), and incubated overnight at 4℃ with 200 μl of Strep-Tactin Superflow high-capacity resin (IBA GmbH). The resin was washed extensively with buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.1% Igepal CA-630, 10% glycerol, and 1 mM PMSF) and the proteins were eluted with 2 ml of buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.1% Igepal CA-630, 10% glycerol, EDTA-free protease inhibitor cocktail, and 2 mM d-desthiobiotin) for 2 h at 4℃ on a rotator. Eluates were analyzed by SDS-PAGE followed by western blotting with anti-AIMP2 or anti-KRS antibodies.

Purification of Recombinant KRS

FreeStyle 293-F cells (Life Technologies, Grand Island, NY, USA) were cultured in 500 ml (2 × 106 cells/ml) of FreeStyle 293 Expression Medium (Gibco) at 37℃ in 5% CO2 in a humidified incubator with suspension at 120 rpm, and then transfected with the pEXPR IBA103-KRS-Strep plasmids. Two days later, the cells were harvested by centrifugation at 130 ×g and lysed in the lysis buffer A. Cell lysates were then loaded onto a Strep-Tactin column (IBA, Goettingen, Germany), washed with buffer (100 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA), and eluted with another buffer (100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 2.5 mM desthiobiotin) at room temperature. Purified KRS was analyzed on 4–12% gradient SDS-PAGE gels (Life Technologies).

ELISA

Following stimulation with Stx1, Stx2, Stx1A, Stx2A, Stx1B-sub, or Stx2B-sub for 0–24 h, the amount of KRS protein secreted by D-THP-1 cells was measured using the lysyl-tRNA synthetase ELISA kit (Neomics, Seoul, Korea). Supernatants collected from D-THP-1 cells (2.5 × 106 cells) treated with different concentrations of purified Strep-KRS (0, 10 nM, 100 nM, and 1 μM) for 24 h in serum-free RPMI medium were analyzed for the presence of inflammatory cytokines (IL-8, IL-1β, TNF-α, and MIP-1α) using specific ELISA kits (KOMA Biotech, Seoul, Korea). To examine synergic effects on the production of inflammatory cytokines, D-THP-1 cells (2.5 × 106 cells) were treated with purified Strep-KRS (100 nM) plus Stx1 (400 ng/ml) or Stx2 (10 ng/ml) for 0 to 24 h.

Statistical Analysis

Data are expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments. Student’s t-test was used to measure differences between samples. P < 0.05 (#) or < 0.001 (*) were considered statistically significant.

 

Results

Stxs Trigger the Secretion of KRS in Human Macrophage-Like THP-1 Cells

To explore the possibility that Stxs trigger the secretion of endogenous KRS from macrophages, we treated undifferentiated monocytic (UD-THP-1) or D-THP-1 cells with Stx1 or Stx2 for 0–24 h. Secreted KRS was detected in culture media of only D-THP-1 cells (Figs. 1 A and 1B). No β-actin was detected in the media, indicating that the KRS secretion was not caused by Stx-mediated cell lysis (Fig. 1A ). Of note, UD-THP-1 cells secrete proinflammatory cytokines in response to Stxs upon differentiation into the macrophage-like state [4,5,11]. No secretion of the MSC components EPRS and AIMP2 was observed (Fig. 1A). In addition, functionally defective toxin mutants (Stx1A− and Stx2A−) and B subunits alone (Stx1B-sub and Stx2B-sub) did not trigger KRS secretion (Figs. 1A and 1B). Neither Stx1 nor Stx2 significantly increased expression of KRS mRNA, suggesting that the presence of KRS in the media was not simply due to increased expression of KRS (Fig. 1C) . Upon exposure to Stx2, KRS remained in the cytosol and was not translocated to the nucleus (Fig. 1D). Thus, these results indicate that enzymatically functional Stxs stimulate the secretion of KRS from human macrophage-like differentiated THP-1 cells.

Fig. 1.Stxs induce KRS secretion in macrophage-like THP-1 cells. (A) Undifferentiated THP-1 (UD-THP-1) or differentiated THP-1 (D-THP-1) cells were either not treated (Cont) or treated with Stx1, Stx2, enzymatically defective mutants (Stx1A− and Stx2A−), or B subunits (Stx1B-sub and Stx2B-sub) alone. KRS was detected in the culture media 24 h after exposure to Stx1 or Stx2. The upper and lower panels show the results of immunoblot analysis of EPRS, KRS, AIMP2, and β-actin expression in the culture media and cell lysates, respectively. Results are representative of three independent experiments. (B) ELISA to detect KRS secreted from D-THP-1 cells, either not stimulated (Cont) or stimulated with active or inactive toxins for 0–24 h. Standards provided with the ELISA kit were used to calculate the amount of soluble KRS. Data are expressed as the mean ± SEM from three independent experiments. Statistical significance was calculated using Student’s t-test (*p < 0.001). (C) Levels of KRS mRNA in D-THP-1 cells following exposure to Stx1 or Stx2. Quantitative RT-PCR was performed with primers specific for human KRS. Neither Stx1 nor Stx2 induced a significant increase in KRS mRNA expression. Data are expressed as the mean ± SEM of three separate experiments. (D) Stx2 does not induce the translocation of KRS from the cytosol to the nucleus in D-THP-1 cells. Cytosolic (Cyto) and nuclear (NE) fractions were analyzed by western blotting. β-Actin and lamin A/C were used as markers for the Cyto and NE fractions, respectively.

Stxs Induce the Dissociation of KRS from AIMP2 Within the MSC in Cells Expressing the Toxin Receptor Gb3

The interaction of Stxs with the membrane receptor Gb3 is a prerequisite for activation of multiple stress responses and proinflammatory cytokine production [11]. Fujii et al. [3] showed that Stx induces apoptosis in Gb3-positive HeLa cells. To further investigate Stx-induced KRS secretion in toxin-sensitive cells, we treated HeLa cells with Stx2 for 20 h. KRS was released into the supernatant (Fig. 2A). However, Stx-mediated KRS secretion was undetectable in the crypt-like colon carcinoma-derived T84 cell line, which does not express Gb3 (Fig. 2B).

Fig. 2.Stxs trigger dissociation of KRS from AIMP2 within the MSC in cells expressing Gb3. (A) Stx2-induced KRS secretion by toxin receptor Gb3-positive HeLa cells. HeLa cells were either not treated (Cont) or treated with Stx2 for 20 h. Equivalent media or lysates samples were subjected to SDS-PAGE followed by western blotting with anti-KRS or β-actin antibodies. (B) Toxin receptor Gb3-dependent KRS secretion. Toxin receptor Gb3-deficient T84 cells were either not treated (Cont) or stimulated with Stx1 for 12 and 24 h or with Stx2 for 20 h. (C) HeLa cells were transfected with Myc-AIMP2 or empty vector (EV) followed by exposure to Stx1 or Stx2 for 6 h. AIMP2 was then immunoprecipitated with an anti-Myc antibody. Co-immunoprecipitation of KRS was examined with an anti-KRS antibody. Expression of KRS and Myc-AIMP2 in whole cell lysates was examined by western blotting with anti-KRS and Myc antibodies, respectively. (D) Pull-down of Strep-AIMP2 by KRS from Stx1-stimulated HeLa cells. HeLa cells were transfected with either empty vector (EV) or Strep-AIMP2 expression plasmids, and then treated with Stx1 for 12 h. Strep-AIMP2 eluates from the Strep-Tactin resin were analyzed by SDS-PAGE (Coomassie Blue staining, CB stain) and western blotting (WB) with anti-KRS, AIMP2, or Strep antibodies.

A structural biological study reported that KRS is held within the MSC by binding to the N-terminal region of AIMP2 [17]. The study also showed that phosphorylation of KRS at Ser207 in allergen-stimulated mast cells disrupts the interaction with AIMP2, thereby releasing KRS from the MSC [17]. Based on this result and the finding that KRS was secreted from Stxs-treated cells (Figs. 1A, 1B, and 2A), we hypothesized that Stxs may trigger the release of KRS from AIMP2; KRS would then be secreted. Therefore, we treated HeLa cells expressing Myc-AIMP2 with Stx1 or Stx2 followed by immunoprecipitation with Myc-conjugated agarose beads. Precipitates were then blotted with antibodies against AIMP2 and KRS. Although binding between KRS and AIMP2 was clearly detected in Myc-AIMP2-transfected cells not exposed to toxin (Cont), stimulation with Stx1 or Stx2 led to a marked reduction in the interaction between the two molecules (Fig. 2C). Next, Strep-AIMP2 proteins extracted from Stx1-treated HeLa cells were subjected to Strep-Tactin pull-down assays followed by immunoblotting with anti-KRS or anti-AIMP2. The results showed that Stx1 induced the dissociation of KRS from AIMP2 (Fig. 2D). Collectively, these data suggest that Stxs induce the dissociation of KRS from AIMP2 within the MSC, which subsequently leads to the secretion of KRS from toxin-sensitive cells.

Stx-Mediated KRS Secretion Increases the Production of Proinflammatory Chemokines and Cytokines

Previous studies show that KRS is secreted by various cancer cell lines in response to oncogenic stimuli, thereby triggering proinflammatory responses and increased TNF-α production [19]. To examine proinflammatory responses induced by KRS, D-THP-1 cells were treated with the purified secreted form of KRS (Fig. 3A). Cell-free supernatants were then analyzed by ELISA. There were marked and dose-dependent increases in the levels of proinflammatory MIP-1α (Fig. 3B) and IL-8 (Fig. 3C). KRS also triggered release of the proinflammatory cytokines IL-1β (Fig. 3D) and TNF-α (Fig. 3E). To further demonstrate the effects of KRS after exposure to Stx, D-THP-1 cells were treated with both KRS and Stx1. The results showed that co-treatment significantly increased the production of MIP-1α (Fig. 3F) and IL-8 (Fig. 3G). In addition, co-treatment with KRS and Stx1 induced the production of IL-1β (Fig. 3H ) and TNF-α (Fig. 3I) to a greater extent than treatment with Stx1 alone. Taken together, these results suggest that KRS acts synergistically with Stx1 to increase the production of proinflammatory chemokines and cytokines in D-THP-1 cells, suggesting that KRS acts as a proinflammatory mediator in the presence of Stxs.

Fig. 3.Stx-mediated KRS secretion triggers increased production of proinflammatory chemokines and cytokines. (A) Strep-KRS purified from FreeStyle 293-F cells. HEK293-F cells were transfected with either empty vector (EV) or Strep-KRS expression plasmids and KRS protein purified on Strep-tag beads. Purified proteins were then analyzed by SDS-PAGE. (B–E) D-THP-1 cells were treated with different concentrations of purified Strep-KRS for 24 h. The supernatants were then examined for the presence of MIP-1α (B), IL-8 (C), IL-1β (D), or TNF-α (E) by ELISA. (F–I) Cells were incubated with Stx1 in the presence or absence of purified KRS (100 nM). Secreted MIP-1α (F), IL-8 (G), IL-1β (H), or TNF-α (I) were measured by ELISA. Standards provided with the ELISA kits were used to calculate the amounts of soluble cytokine/chemokine protein. Data are expressed as the mean ± SEM of three independent experiments. Statistical significance was calculated using Student’s t-test (p < 0.01 (#), control (Cont) versus KRS treatment; p < 0.001 (*), Stx1 or Stx2 treatment versus Stx1 and KRS or Stx2 and KRS treatment).

 

Discussion

Infection with STEC leads to the development of HUS and acute renal failure, both of which increase patient morbidity and mortality. Cytokines and chemokines secreted from macrophages in response to Stxs may damage primary target organs in vivo. For example, studies of murine models of Stx-mediated renal damage report that challenge with the toxins elicits chemotactic responses in macrophages in the kidney, which may contribute to pathophysiology [6,7,13]. These results highlight the need to clarify the mechanisms underlying Stxs-induced production of cytokines or cytokine-like molecules involved in inflammation and in the activation of innate immune responses.

Beyond their primary role in protein synthesis, human ARSs (unlike prokaryotic ARSs) have additional domains that play critical roles in regulating important biological processes, including intracellular signaling, metabolic processes, tissue differentiation, angiogenesis, and inflammation [19,22,24,32]. Secreted human KRS triggers proinflammatory responses, implying that it is a cytokine-like molecule [19]. Here, we showed that Stxs induced the secretion of KRS, which then induced a proinflammatory immune response. Our primary observations suggest that in the toxin-sensitive cells, KRS is dissociated from AIMP2 within the MSC in response to Stx1 or Stx2; presumably, dissociated KRS is then secreted from the cell, whereupon it exacerbates proinflammatory responses.

Park et al. [19] showed that secreted human KRS triggers TNF-α production by activating monocytes/macrophages, suggesting that human KRS acts as a cytokine. Recent studies show that human KRS regulates mast cell responses by activating the MAPK cascade via regulation of target gene expression [12,17,33]. Here, we showed that HeLa (Fig. 2A) and D-THP-1 (Figs. 1A and 1B) cells secreted KRS in response to the Stxs, but not in response to N-glycosidase-deficient Stx1A−, Stx2A−, or the Stx1B or Stx2B subunits (Figs. 1 A and 1B). Because the N-glycosidase activity of Stxs is required for KRS secretion, Stxs may induce dissociation of KRS from AIMP2 in the cytosol (Figs. 2 B and 2C) in response to toxin-mediated ribotoxic stress [26]. As mentioned above, phosphorylation of KRS in allergen-stimulated mast cells causes it to dissociate from AIMP2 [17]. Thus, toxin-mediated ribotoxic stress may transduce signals that induce a modification of KRS to dissociate from AIMP2.

Further studies should examine the regulation of Stxs-activated proinflammatory cytokines and their mediators in cases of serious infectious disease caused by bacteria that produce the toxins. The results of the present study may provide valuable insight into the immunological mechanism underlying toxin-induced inflammation in various target organs. Studies to see whether secreted human KRS is related to the coordination of various signaling pathways with pathophysiological implications will be necessary to evaluate the precise physiological significance of KRS.