Attachment of LcrV from Yersinia pestis at Dual Binding Sites to

Apr 19, 2007 - Attachment of LcrV from Yersinia pestis at Dual Binding Sites to Human TLR-2 and Human IFN-γ Receptor. Vyacheslav ... Two TLR2/IFN-γ ...
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Attachment of LcrV from Yersinia pestis at Dual Binding Sites to Human TLR-2 and Human IFN-γ Receptor Vyacheslav M. Abramov,‡ Valentin S. Khlebnikov,‡ Anatoly M. Vasiliev,‡ Igor V. Kosarev,‡ Raisa N. Vasilenko,‡ Nataly L. Kulikova,‡ Anna V. Khodyakova,‡ Valentin I. Evstigneev,‡ Vladimir N. Uversky,‡ Vladimir L. Motin,# Georgy B. Smirnov,§ and Robert R. Brubaker*,¶ Institute of Immunological Engineering, 142380 Lyubuchany, Russia, Departments of Pathology/Microbiology & Immunology, University of Texas Medical Branch, Galveston, Texas 77555, The Gamaleya Institute of Epidemiology and Microbiology, 123098 Moscow, Russia, and Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824 Received January 22, 2007

The virulence antigen (V-antigen, LcrV) of Yersinia pestis, the causative agent of bubonic plague, is an established protective antigen known to regulate, target, and mediate type III translocation of cytotoxic yersiniae outer proteins termed Yops; LcrV also prompts TLR2-dependent upregulation of antiinflammatory IL-10. In this study, we determined the parameters of specific interaction of LcrV with TLR2 expressed on human transfected HEK293 cells (TLR2+/CD14-), VTEC2.HS cells (TLR2+/CD14-), primary monocytes (TLR2+/CD14+), and THP-1 cells (TLR2+/CD14+). The IRRL314-317 motif of the extracellular domain of human and mouse TLR2 accounted for high-affinity binding of LcrV. The CD14 co-receptor did not influence this interaction. LcrV did not bind to human U937 (TLR2-/CD14-) and alveolar macrophages (TLR2-/CD14+) in the absence of receptor-bound human IFN-γ or a synthetic C-terminal fragment (hIFN-γ132-143). The latter, but not mouse IFN-γ (or synthetic control peptides), shared a GRRA138-141 site necessary for high-affinity specific binding. LcrV of Y. pestis shares the N-terminal LEEL32-35 binding site of Yersinia enterocolitica and also has an exposed internal DEEI203-206 binding site. Comparison of binding constants and consideration of steric restrictions indicate that binding is not cooperative and only the internal site binds LcrV to target cells. Both the LEEL32-35 and DEEI203-206 binding sites are removed by five amino acids from DKN residues associated with biological activity of bound LcrV. LcrV of Y. pestis promoted both TLR2/CD14-dependent and TLR2/CD14-independent amplification of IL-10 and concomitant downregulation of TNF-R in human target cells. The ability of LcrV to utilize human IFN-γ (a major inflammatory effector of innate immunity) to minimize inflammation is insidious and may account in part for the severe symptoms of plague in man. Keywords: plague • Yersinia pestis • virulence antigen LcrV • Toll-like receptor TLR2 • interferon-γ

Introduction Bubonic plague caused by Yersinia pestis is the most devastating bacterial infection known to man. This organism very recently evolved from Yersinia pseudotuberculosis, which diverged from Yersinia enterocolitica about 50 million years ago.1 All three species share an ∼70-kb plasmid (termed pCD in Y. pestis and pYV in enteropathogenic Y. pseudotuberculosis and Y. enterocolitica) that encodes the virulence antigen (Vantigen, LcrV), yersiniae outer proteins (Yops), and an attendant * Correspondence should be addressed to: Robert R. Brubaker, Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824. Phone: (517) 355-6463. Fax: (517) 353-8957. E-mail: [email protected]. ‡ Institute of Immunological Engineering. # University of Texas Medical Branch. § The Gamaleya Institute of Epidemiology and Microbiology. ¶ Michigan State University.

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type III secretion system (T3SS). These functions are activated by pCD/pYV-encoded LcrF (VirF in enteropathogenic yersiniae)2 at 37 °C3 and expressed upon host cell contact4 or residence in Ca2+-deficient medium5 and host fluids.6 Plague bacilli do not overwhelm innate immunity but rather prevent its expression as first shown for pCD-dependent downregulation of proinflammatory cytokines.7 This strategy also involves production of noninflammatory lipopolysaccharide,8 degradation of those Yops that fail to undergo translocation into host cell cytoplasm,9 and inhibition of host mitogen-activated protein kinase (MAPK) and NF-κB pathways, especially by YopJ (YopP in Y. enterocolitica), resulting in apoptosis of professional phagocytes.10 Finally, LcrV upregulates the powerful antiinflammatory cytokine interleukin (IL)-1011 that utilizes signal transducer and activator of transcription-3 (STAT3) to block expression of proinflammatory cytokines and numerous other mediators of inflammation.12,13 10.1021/pr070036r CCC: $37.00

 2007 American Chemical Society

research articles

Yersinia LcrV Binds to Human TLR2 and IFN-γ Receptor

Figure 1. Amino acid sequences of LcrV and target peptides: (A) primary structure of LcrV from Y. pestis KIM48 where the amino acids of the construct (LcrV68-326) first used to prove ability to raise protective antibodies26 and demonstrate amplification of IL-1011 are in red. Amino acid sequences duplicated as synthetic peptides are in bold, and putative TLR2 binding sites containing adjacent glutamic acid residues are underlined (note the presence of DKN motifs removed from each binding site by five amino acids). (B) Synthetic peptides from LcrV where putative TLR2 binding sites (in bold red) are underlined, and putative CD14 binding sites DKN (in bold black) are underlined; (C) synthetic peptide from IL-2; (D) synthetic peptides from extracellular domains of human (hTLR2) and mouse (mTLR2) where putative LcrV binding sites (in bold red) are underlined; (E) alignment of amino acid sequences of carboxyl-termini of human IFN-γ (hIFN-γ), truncated hIFN-γ (h∆IFN-γ), and mouse IFN-γ (mIFN-γ) where identical amino acids are underlined and the putative site for binding LcrV is in bold red; and (F), synthetic peptides from human IFN-γ where the putative LcrV binding site (in bold red) is underlined.

Amplification of IL-10 by released LcrV requires coexpression of Toll-like receptor-2 (TLR-2) associated with the differentiation factor CD14 and fails to occur in IL-10-/- knockout mice, which are resistant to infection.14-16 LcrV is encoded within an lcrGVH-yopBD operon17,18 and was reported to bind to LcrG,19 LcrH (SycD in Y. enterocolitica),20 TLR2,15 and YscF.21 Little is known about the avidity or molecular basis of these interactions. Indeed, association of LcrV and LcrH was disputed,22,23 until formation of a 1:1 complex was detected following stabilization by chemical cross-linking.24 LcrV serves as a major regulator and effector of virulence.10,25 This 37.5-kDa protein is also the predominate protective antigen against plague due, at least in part, to immunity provided by one or more internal epitopes located between amino acids 168-275.20,26 It is therefore essential that investigators fully resolve the interactions between LcrV and its various receptors. Hill et al.27 reported that an N-terminal region of LcrV (amino acids 2-135) promotes modest immunity and confirmed that one or more epitopes comprising amino acids 135-245 promote significant protection. Nevertheless, Sing et al.15 reported that the ability of LcrV to downregulate proinflammatory

cytokines was mediated by the N-terminus, although this region was absent in the truncated LcrV first used to demonstrate upregulation of IL-10.11 The purpose of this investigation was to obtain information regarding the specificity and avidity of the interaction between LcrV and TLR2. We show here that LcrV possesses two noncooperative binding domains (LEEL32-35 and DEEI203-205) capable of recognizing both free TLR-2 as well as human (but not mouse) IFN-γ bound to its receptor (IFN-γRIFN-γ). In addition, we demonstrate that both binding domains of LcrV can upregulate IL-10 and downregulate LPS-induced TNF-R, although only the site unique to amino acids 168-275 may function within the native molecule.

Materials and Methods Recombinant Proteins. LcrV (defined in Figure 1A) was produced using lcrV encoded within the lcrGVH-yopBD operon of pCD1 from Y. pestis strain KIM.28 After amplification with PCR using sites for EcoRI and BamHI, lcrV was inserted into the vector pRSET A (Invitrogen, Carlesbad, CA) opened with BamHI and EcoRI. This construct, expressed in Escherichia coli Journal of Proteome Research • Vol. 6, No. 6, 2007 2223

research articles BL21(DE3), encoded N-terminal hexahistidine, an enterokinase cleavage site, and then LcrV in its entirety. Similar engineering of E. coli BL21(DE3) transformed with pVHB62 encoding LcrV68-326 has been described.20 LcrV and LcrV68-326 encoded by these constructs were induced by IPTG, purified to nearhomogeneity by Ni-affinity chromatography, and then freed of hexahistidine by treatment with enterokinase.20 Homogeneous dimers of LcrV and LcrV68-326 were purified by gelfiltration on Sepharose CL-4B (Sigma Chemical Co., St. Louis, MO). Endotoxin was removed from preparations of purified protein using polymyxin B-agarose (Sigma). Endotoxin-depleted preparations contained 0.16 ( 0.03 pg (mean ( SE, n ) 5) of endotoxin/µg of protein as measured by the Limulus amebocyte lysate assay system. Preparations of recombinant human IFN-γ (antiviral activity of 1.5 × 107 U/mg) and h∆IFN-γ (antiviral activity 1.0 × 107 U/mg) lacking 6 C-terminal amino acids as defined in Figure 1E were kindly supplied by Dr. V. Fedyukin (JSC “ImmunoPharm”, Obolensk, Russia). Highly purified human TNF-R, IFN-R2, and epidermal growth factor (EGF) were purchased from PeproTech, Ltd. (London, U.K.). Purity of recombinant proteins was monitored by SDS-PAGE and silver staining.29 Synthetic Peptides and LPS. Derivatives of LcrV (Figure 1B), interleukin-2 (IL-2) (Figure 1C), extracellular domains of human and mouse TLR2 (Figure 1D), and IFN-γ (Figure 1F) were synthesized with a solid-phase model 9500 peptide synthesizer (Biosearch Technologies, Inc., Novato, CA) and purified by HPLC chromatography. The purity and structure of these peptides were confirmed by amino acid analysis and mass spectrometry. Reagent grade E. coli LPS was purchased from Calbiochem (San Diego, CA). Host Cell Lines and Primary Isolates. Human embryonic kidney (HEK) 293 cells (obtained from ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Gaithersburg, MD) containing 10% fetal calf serum and transfected with the expression plasmid pUnohTLR2 (InvivoGen, San Diego, CA) according to the manufacturer’s instructions. As shown in Figure 2, HEK 293 cells were CD14- and devoid of detectable TLR2 unless transfected with pUno-hTLR2. The VTEC2.H2 human thymic epithelial cell line transformed with SV40 virus was obtained from the Institute of Immunology (Moscow, Russia) and maintained in suspension (2 × 105 to 9 × 105 cells/mL) in RPMI 1640 medium (ICN Biomedicals, Inc., Irvine, CA) supplemented with 10% (v/v) fetal calf serum (ICN Biomedicals), HEPES buffer (15.0 mM), L-glutamine (2.0 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL) in 5% CO2 at 37 °C. Human monocytic leukemia U937 cells from the Institute of Carcinogenesis (Moscow, Russia) were similarly cultured (2 × 105 to 7 × 105 cells/mL) in the same medium. The differentiated THP-1 human myelomonocytic cell line was grown according to instructions provided by ATCC; 48 h before use in experiments, cell culture plates were supplemented with 80 nM 1R, 25-dihydroxyvitamin D3 (Calbiochem) to induce expression of CD14. Primary human monocytes were isolated from blood by centrifugation over Lympho Separation Medium (ICN Biomedicals). Interface cells (106 per mL) in RPMI 1640 medium (ICN Biomedicals) with 10% fetal calf serum were placed in wells for monolayer formation, and after 1 h, unattached cells were removed with replacement of volume with the same medium. The resulting monolayer contained over 98% viable monocytes after culture for 24 h at 37 °C with 5% CO2 and was 2224

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used to characterize expression of cytokines and binding of LcrV and its derivatives. Human alveolar macrophages were isolated from bronchoalveolar lavage (BAL) fluid of three normal (nonsmoking) individuals. Bronchoscopy was performed at the Blokchin Russian Oncology Scientific Center of RAMS according to standard guidelines. The study was approved by the Institutional Ethics Committee, and written informed consent was obtained from all volunteers. The BAL fluid was aspirated via fiberoptic bronchoscope into a siliconized glass bottle and stored on ice for no longer than 15 min until processing. After a washing atep with phosphate-buffered saline solution (500g for 10 min), recovered cells were suspended to a final concentration of 1 × 106/mL in RPMI 1640 medium supplemented with 20% human serum, L-glutamine (2 mmoles/l), penicillin (200 U/mL), and streptomycin (200 µg/mL). This suspension was added at 1 × 106 cells per well to a 24 well plastic tissue culture plate and incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 1 h to permit adherence. Nonadherent cells were then removed by three washes with the same culture medium. Purity of the human alveolar macrophages was estimated at >95% by morphology and nonspecific esterase staining. The ability of these cell lines and primary isolates to express surface-associated TLR2 and CD14 is illustrated in Figure 2. Radiolabeling and Binding of Radioactive Reagents. 125ILcrV derivatives (all 0.09 mCu/µg) as well as iodinated hIFN-γ, h∆IFN-γ, mIFN-γ, and hIFN-R2 (all 0.1 mCu/µg) were prepared with Iodo-Gen (Pierce, Rockford, IL) and Na125I30 and then separated by chromatography on Sephadex G-25. Cells were collected, washed three times with RPMI-1640 medium, and then adjusted to a concentration of 107 per mL of the same medium. Radioactive ligands were then added to individual cultures (total volume of 300 µL), which were then incubated for either 1 h at 4 °C or 15 min at 37 °C. Thereafter, 50 µL of the cell culture was layered on 250 µL of an n-dibutylphthalate/bis (2-ethylhexyl)-phthalate mixture (1:1 v/v) and centrifuged for 2 min at 14 000g. Radioactivity in the resulting precipitate was measured using a model 1275 MINI GAMMA counter (LKB WALLAC, Sweden). Nonspecific binding of radioactive ligands to cells or plastic plates was determined by incubation in 10 000-fold excess of corresponding unlabeled ligand and was equal to 25-27% of the total (specific plus nonspecific) binding. Results were expressed as the (mean ( SEM, n ) 3) in molarity, from which nonspecific binding was subtracted. An essentially identical procedure was used to determine dissociation constants of radioactive LcrV and its derivatives for synthetic fragments representing the extracellular domain of human and mouse TLR2 immobilized on PVC flat bottom 96 multi-well plates. Determination of Cytokines. Production of IL-10 was measured with an ELISA kit obtained from Biosource International (Carlsbad, CA) yielding a sensitivity of 1 pg/mL. Human TNF-R was similarly determined according to the protocol specified for a specific ELISA kit purchased from the State Research Institute of Highly Pure Biopreparations, St. Petersburg, Russia (specificity ) 1 pg/mL). Flow Cytometry Analysis. TLR2 and CD14 receptor expression on the surface of immunocompetent cells were determined by flow cytometry using FACSCalibur (BD Bioscience). The percentage of TLR2+ and CD14+ cells in these preparations is illustrated in Figure 2.

research articles

Yersinia LcrV Binds to Human TLR2 and IFN-γ Receptor

Results Radioactive LcrV31-50 and LcrV68-326 (defined in Figure 1A) avidly bound to HEK293 cells transfected with pUno-hTLR2 (TLR2+/CD14-) as opposed to nontransfected HEK293 cells (TLR2-/CD14-), which yielded Kd values of g10-3 (Figure 3A,B). As illustrated in Figure 3C, these associations were markedly inhibited by the unlabeled synthetic peptide hTLR2295-323 (defined in Figure 1D) comprising the extracellular domain of TLR2. Neither the corresponding synthetic fragment lacking the IRRL314-317 motif (defined in Figure 1D) or the synthetic fragment of hTLR2101-116 containing those leucine residues at positions 107, 112, and 115 (defined in Figure 1D) responsible for substrate recognition31 caused similar inhibition (data not shown). These observations suggested that two TLR2-interactive regions of Y. pestis LcrV can compete for a single receptor on the extracellular domain of hTLR2 and that this receptor contains the IRRL314-317 motif. Labeled LcrV31-50, LcrV193-210, and LcrV68-326 bound immobilized mTLR2295-323 (defined in Figure 1D) at low Kd values of (7.2 ( 1.0) × 10-10 M (Figure 4A), (3.5 ( 0.6) × 10-10 M (Figure 4B), and (5.5 ( 0.4) × 10-10 M (Figure 4C), respectively. Native 125I-LcrV (Figure 4D) exhibited a slightly reduced affinity for mTLR2295-323 (Kd of (3.9 ( 0.7) × 10-9 M). Nevertheless, both LcrV and LcrV68-326 successfully competed with LcrV31-50 for mTLR2295-323 (data not shown) suggesting that LcrV and LcrV68-326 share a second binding site distinct from that present on LcrV31-50. Radioactive LcrV68-326 did not bind to mTLR2295-323 or to its human analog if the IRRL314-317 motif was absent (Table 1). 125I-LcrV68-326 also failed to bind hTLR2101-116 (defined in Figure 1D); identical results were obtained with radioactive LcrV31-50 and LcrV193-210 (Kd values g10-3). These findings verified that the IRRL314-317 site of LcrV accounts for its interaction with the extracellular domains of mouse and human TLR2. Furthermore, derivatives of LcrV31-50 lacking K42 and DKN41-43 bound to hTLR2295-323 with Kd values of about 10-10 M, whereas removal of the LEEL32-35 motif prevented significant binding (Table 1). Replacement of E34 for Q34 reduced the affinity of LcrV31-50 for hTLR2295-323 (Kd ∼ 10-7), whereas removal of DEEI203-206 from the LcrV193-210 peptide eliminated binding altogether. However, deletion of DKN195-197 in the LcrV193-210 peptide did not diminish its affinity for hTLR2295-323. Control peptides LcrV152-168 and IL-252-57 (defined in Figure 1B, 1C) containing motifs similar to LEEL32-35 and DEEI203-206 also failed to bind hTLR2295-323 (data not shown). Considered together, these observations indicate that the LEEL32-35 and DEEI203-205 sites are responsible for specific interaction of LcrV with TLR2.

Figure 2. Flow cytometric analysis showing surface expression of TLR2 and CD14 on cells used in experiments. Cells were incubated at 4 °C for 1 h with or without isotype-matched mouse IgG or either anti-TLR2 or CD14 mAbs and then with FITCconjugated anti-mouse IgG. Specific mAB-stained cells are shown as bold lines, and isotype controls are shown as thin lines in histograms. Surface expression was 0% TLR2 and 0% CD14 for nontransfected HEK293 cells, 19.3 ( 2.5% TLR2 and 0% CD14 for HEK293 cells transfected with pUNO-hTLR2 expressing the human TLR2 gene, 22.6 ( 2.9% TLR2 and 1.5 ( 1.1%, CD14 for VTEC2.HS cells, 96.0 ( 3.9% TLR2 and 91.9 ( 5.4% CD14 for primary monocytes, 18.4 ( 2.4% TLR2 and 50.6 ( 3.8% CD14 for THP-1 cells, 1.0 ( 0.9% TLR2 and 3.4 ( 1.1% CD14 for U937 cells, and 2.3 ( 1.6% TLR2 and 27.4 ( 3.5% CD14 for alveolar macrophages.

Human thymic epithelial VTEC2.HS cells (TLR2+/CD14-) also bound 125I-LcrV31-50 (Figure 5A) and 125I-LcrV68-326 (Figure 5B) with low Kd values. Binding of 125I-LcrV was not detectable with VTEC2.HS cells, although unlabeled LcrV could still displace TLR2 receptor-bound 125I-LcrV31-50 but with less efficiency than did unlabeled LcrV68-326 (Figure 5C). Binding of 125I-LcrV to primary monocytes (TLR2+/CD14+) and differentiated THP-1 cells (TLR2+/CD14+) was also less avid than that detected for LcrV31-50 and LcrV68-326 (Table 2) as previously observed with immobilized mTLR2295-323. Radioactive LcrV and its derivatives exhibited high-affinity binding to human U937 monocytic leukemia cells (TLR2-/ CD14-) and human alveolar macrophages (TLR2-/ CD14-) provided that human IFN-γ was also added to the reaction (Table 3). The affinities of radioactive derivatives of LcrV for Journal of Proteome Research • Vol. 6, No. 6, 2007 2225

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Figure 3. Scatchard analyses for specific binding of (A) 125I-LcrV31-50 and (B) 125I-LcrV68-326 to transfected HEK293 cells (TLR2+/CD14-); (C) inhibition of the specific binding of 10-13 mol of 125I-LcrV31-50 (-b-) or 10-13 mol of 125I-LcrV68-326 (-O-) to transfected HEK293 cells by unlabeled hTLR2295-323. In panels A and B, molar concentrations of specifically bound radioactive LcrV derivatives (B) are plotted as the abscissa and ratios of the bound and free labeled protein (B/F) constitute the ordinate. Individual specific binding constants (Kd) are provided. Points (means ( SEM) represent duplicate determinations from each of three assays.

Figure 4. Scatchard analyses for specific binding of (A) 125I-LcrV31-50, (B) 125I-LcrV193-210, (C) 125I-LcrV68-326, and (D) 125I-LcrV to mTLR2295-323. Molar concentrations of specifically bound radioactive LcrV and derivatives (B) are plotted as the abscissa; ratios of the bound and free labeled protein (B/F) constitute the ordinate. Individual specific binding constants (Kd) are provided. Points (means ( SEM) represent duplicate determinations from each of three assays.

U937 cells with added IFN-γ were equivalent to those observed previously for TLR2+ cells in the absence of IFN-γ as shown for LcrV31-50 (Figure 6A), LcrV193-210 (Figure 6B), and LcrV68-326 (Figure 6C). Unlike the reduced affinity of whole 125I-LcrV for the various TLR2+ cells noted above, 125I-LcrV bound to U937 cells with a Kd of (0.6 ( 0.1) × 10-11 M in the presence of the 2226

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C-terminal peptide of hIFN-γ (Figure 6D), defined in Figure 1F. No binding occurred (Kd g 10-3) if this peptide lacked the GRRA138-141 motif (defined in Figure 1F). Furthermore, LcrV68-326 provided identical binding to U937 cells, and alveolar macrophages if hIFN-γ (but not hIFN-R2) was present (Table 3). These findings demonstrate that TLR2 receptors of CD14+ or

research articles

Yersinia LcrV Binds to Human TLR2 and IFN-γ Receptor

Figure 5. Scatchard analyses for specific binding of (A) 125I-LcrV31-50 and (B) 125I-LcrV68-326 to VTEC2.HS cells (TLR2+/CD14-); (C) inhibition of the specific binding of 125I-LcrV31-50 to VTEC2.HS cells by unlabeled LcrV (b) or LcrV68-326 (9). In panels A and B, molar concentrations of specifically bound radioactive LcrV derivatives (B) are plotted as the abscissa and ratios of the bound and free labeled protein (B/F) constitute the ordinate. Individual specific binding constants (Kd) are provided. Points (means ( SEM) represent duplicate determinations from each of three assays. Table 1. Molar Dissociation Constants (Kd) Defining the Affinity between Various Immobilized TLR2 Receptors of LcrV and Derivatives of LcrV and Their Analogues Immobilized target or source of probes

Immobilized peptide target sequence

Kd (M)

125I-LcrV

Specific Binding of 68-326 to Various Immobilized TLR2 Targets hTLR2 extracellular domain FRASDNDRVIDPGKVETLTIRRLHIPRFY mTLR2 extracellular domain FNPSESDVVSELGKVETVTIRRLHIPQFY hTLR2 extracellular domain FRASDNDRVIDPGKVETLT----HIPRFY mTLR2 extracellular domain FNPSESDVVSELGKVETVT----HIPQFY Specific Binding of Various 125I-LcrV31-50 Derivatives (or Analogues) to Immobilized hTLR2295-323 125I-LcrV VLEELVQLVKDKNIDISIKY 31-50 125I-LcrV VLEQLVQLVKDKNIDISIKY 31-50 125I-LcrV V----VQLVKDKNIDISIKY 31-50 125I-LcrV VLEELVQLVK---IDISIKY 31-50 125I-LcrV SKLREELAELTAELKIYS 151-168 125I-IL-2 LEEELKLEEVLNLY 52-72 Specific Binding of Various 125I-LcrV193-210 Derivatives (or Analogues) to Immobilized hTLR2295-323 125I-LcrV LMDKNLYGYTDEEIFKAS 193-210 125I-LcrV LMDKNLYGYT----FKAS 193-210 125I-LcrV LM---LYGYTDEEIFKAS 193-210 125I-LcrV SKLREELAELTAELKIYS 152-168 125I-IL-2 LEEELKLEEVLNLY 52-72 Table 2. Specific Binding of Radioactive LcrV, LcrV31-50, and LcrV68-326 to Receptors on the Surface of VTEC2HS Cells, Primary Monocytes, and Differentiated THP-1 Cells cells type, dissociation constant (Kd), [M] ligand 125

I-LcrV I-LcrV31-50 125 I-LcrV68-326 125

VTEC2.HS -3

g10 (1.3 ( 0.7) × 10-10 (8.0 ( 0.7) × 10-10

Monocytes

THP-1

(2.2 ( 0.2) × (4.7 ( 0.8) × 10-10 (2.6 ( 0.2) × 10-10

(2.4 ( 0.2) × 10-9 (7.9 ( 0.8) × 10-10 (5.2 ( 0.5) × 10-10

10-9

CD14- cells are not essential for binding of LcrV to host target cells if IFN-γR-IFN-γ complexes (or IFN-γR-IFN-γ C-terminal hIFN-γ peptide complexes) are available.

10-10 10-10 g10-3 g10-3 10-10 10-7 g 10-3 10-10 g 10-3 g 10-3 10-10 g 10-3 10-10 g 10-3 g 10-3

Additional characterization of this interaction showed that LcrV and its derivatives did not interact with free hIFN-γ in solution but rather required hIFN-γR-hIFN-γ (data not shown). Furthermore, truncated hIFN-γ (h∆IFN-γ) lacking six C-terminal amino acids (defined in Figure 1E) did not promote binding of 125I-LcrV68-326 to CD14-U937 or CD14+ alveolar macrophages (Table 3). Mouse IFN-γ similarly bound with high affinity to homologous cells (data not shown) but failed to promote attachment of 125I-LcrV68-326 to human cells (Table 3). Comparison of the primary structures of the C-termini of hIFNγ, h∆IFN-γ, and mIFN-γ (defined in Figure 1E) revealed a GRRA138-141 site on the full-sized hIFN-γ molecule that is analogous to the IRRL314-317 site of the extracellular domain of Journal of Proteome Research • Vol. 6, No. 6, 2007 2227

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Figure 6. Scatchard analyses for specific binding of (A) 125I-LcrV31-50, (B) 125I-LcrV193-210, and (C) 125I-LcrV68-326 to U937 cells (TLR2-/ CD14-) in the presence of hIFN-γ and (D) 125I-LcrV to U937 cells in the presence of the hIFN-γ C-terminal peptide SQMLFRGRRASQ. Molar concentrations of specifically bound radioactive LcrV and derivatives (B) are plotted as the abscissa; ratios of the bound and free labeled protein (B/F) constitute the ordinate. Individual specific binding constants (Kd) are provided. Points (means ( SEM) represent duplicate determinations from each of three assays. Table 3. Specific Binding of Radioactive LcrV68-326 in the Presence of Human IFN-γ, Human ∆IFN-γa, Mouse IFN-γ, and Human IFN-R2 to Human Cells with Low Surface Expression of TLR2 cells type, dissociation constant, (M) ligand 125I-LcrV

68-326 plus hIFN-γ

125I-hIFN-γ 125I-h∆IFN-γa 125I-mIFN-γ 125I-hIFN-R 125I-LcrV 125I-LcrV 125I-LcrV 125I-LcrV 125I-LcrV a

2

68-326 68-326+h∆IFN-γ

a

68-326+mIFN-γ 68-326+IFN-R2

U-937

alveolar macrophages

(8.0 ( 0.7) × 10-10

(6.0 ( 0.6) × 10-10

Controls (6.8 ( 0.8) × 10-11 (1,3 ( 0,1) × 10-10 g10-3 (3.6 ( 0.4) × 10-10 g10-3 g10-3 g10-3 g10-3 g10-3

(6.2 ( 1.5) × 10-10 (3.0 ( 0.4) × 10-10 g10-3 (1.2 ( 0.1) × 10-10 g10-3 g10-3 g10-3 g10-3 g10-3

Human IFN-γ lacking the first six C-terminal amino acids.

human and mouse TLR2 (defined in Figure 1D). This observation demonstrates that the GRRA138-141 site facilitated binding of LcrV and its derivatives to human immunocompetent cells in the presence of either hIFN-γ or its C-terminal peptide (Table 3). Discovery that hIFN-γ serves as an alternative receptor for LcrV prompted an evaluation of its ability to influence synthesis of IL-10 by human blood monocytes and differentiated THP-1 cells. As shown in Figure 7A, prompt upregulation of IL-10 in monocytes by LcrV, LcrV31-50, and LcrV68-326 was observed in 2 h with maximum production occurring by 6-8 h. Note that the value obtained for LcrV plus LPS closely approximates the sum of IL-10 observed for LcrV alone and LPS alone. As already noted, mutation of E34Q in LcrV31-50 (defined in Figure 1B) reduced its affinity by 2 orders of magnitude (Kd ∼ 10-7 M) for the synthetic immobilized peptide TLR2295-323 (Table 2). Nevertheless, this avidity remained sufficient to ensure upregulation of IL-10 (Figure 7A), an occurrence previously interpreted by Sing et al.32 as evidence supporting direct interaction of DKN 2228

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with TLR2. At the same time, deletion of LEEL32-35 in LcrV31-50 eliminated binding with TLR2295-323 (Table 1) and led to complete loss of the ability to up-regulate IL-10 (Figure 7A). LcrV and its derivatives without added hIFN-γ did not induce early upregulation of IL-10 in U937 cells or in alveolar macrophages during 2-8 h of incubation (data not shown), although LcrV and LcrV68-326 caused prompt expression of IL-10 in THP-1 cells (Figure 7B). In this case, upregulation occurred by 2 h with maximum yields at 4 h as opposed to delayed but, nevertheless, significant synthesis with added LPS alone. Inclusion of hIFN-γ to LPS blocked subsequent appearance of IL-10, although this inhibition was cancelled by LcrV or LcrV68-326, which induced the delayed pattern of expression. The combination of LPS plus h∆IFN-γ also blocked IL-10, but in this case, addition of LcrV or LcrV68-326 restored the pattern of prompt expression typical of LcrV alone. These findings are consistent with exploitation of the TLR2/CD14 receptor system by LcrV for early synthesis of IL-10.

Yersinia LcrV Binds to Human TLR2 and IFN-γ Receptor

Figure 7. Production by primary human monocytes (TLR2+/ CD14+) of IL-10 (A) in cultures (2 × 105/mL) without addition (O) or with addition of LPS (1.0 µg/mL) provided 1 h before treatment with 120 nM LcrV (9); also shown is production after addition of 120 nM LcrV alone (2), 120 nM LcrV68-326 alone (1), 120 nM LcrV31-50 alone ([), 120 nM LcrV(31-50)E34Q alone (0), 120 nM LcrV(31-50)∆(32-35) alone (]), and LPS (1.0 µg/mL) alone (b). Shown in panel B is cancellation of the inhibitory effect of exogenous hIFN-γ in the presence of LcrV or LcrV68-326 on production IL-10 by differentiated human THP-1 cells (TLR2+/CD14+) in supernatants of cultures (2 × 105/mL); results without addition (O) or with addition of LPS (1.0 µg/mL) alone (9), LPS (1.0 µg/mL) plus 0.7 nM hIFN-γ ([), or LPS (1.0 µg/mL) plus 0.7 nM h∆IFN-γ (]), 120 nM LcrV alone (2), 120 nM LcrV68-326 alone (1), LPS (1.0 µg/mL) plus 0.7 nM hIFN-γ and 120 nM LcrV (4), LPS (1.0 µg/mL) plus 0.7 nM hIFN-γ and 120 nM LcrV68-326 (3), LPS (1.0 µg/mL) plus 0.7 nM h∆IFN-γ and 120 nM LcrV (\), and LPS (1.0 µg/mL) plus 0.7 nM h∆IFN-γ and 120 nM LcrV68-326 (×). Error bars represent the standard deviation of triplicate determinations.

Human primary monocytes exposed to LPS and resident human alveolar macrophages primed with IFN-γ were used to evaluate downregulation of TNF-R by LcrV and LcrV68-326. TNF-R was expressed by monocytes after activation with LPS (100 ng/mL), although addition of 120 nM LcrV or 120 nM LcrV68-326 after 10 min of activation markedly inhibited synthesis (Figure 8A). Results of preliminary experiments demonstrated that addition of catalytic levels hIFN-γ amplified priming of human alveolar macrophages by enabling marked expression of TNF-R after addition of an otherwise ineffective concentration of LPS. Macrophages were subjected to this procedure by amplification with hIFN-γ (400 U/mL) 4 h before addition of LPS 5 (ng/mL) and then monitored for expression

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Figure 8. Inhibition of TNF-R synthesis by cultures (2 × 105/mL) of (A) human primary monocytes (TLR2+/CD14+) either untreated (O) or treated with either LPS (100 ng/mL) alone (9), LcrV (120 nM) alone (4), LcrV68-326 (120 nM) alone (3), with LcrV after 10 min of exposure to LPS (100 ng/mL) (2), or with LcrV68-326 after 10 min of exposure to LPS (100 ng/mL) (1). Shown in panel B is similar inhibition in cultures (2 × 105/mL) of alveolar macrophages (TLR2-/CD14+) either untreated (O), treated with LcrV (120 nM) alone (4), treated with LcrV68-326 (120 nM) alone (3), primed with hIFN-γ (400 U/mL) without added LPS (]), stimulated with LPS (5 ng/mL) without added hIFN-γ (0), primed with hIFN-γ and then stimulated 4 h later with LPS (5 ng/mL) (b), primed with hIFN-γ plus 120 nm LcrV and then stimulated 4 h later with LPS (5 ng/mL) (2), and primed with hIFN-γ plus 120 nm LcrV68-326 and then stimulated 4 h later with LPS (5 ng/mL) (1). This experiment was repeated with alveolar macrophages in panel C showing cultures either untreated (O), treated with LcrV (120 nM) alone (4), treated with LcrV68-326 (120 nM) alone (3), primed with h∆IFN-γ (400 U/mL) without added LPS (]), stimulated with LPS (5 ng/mL) without added h∆IFN-γ (0), primed with h∆IFN-γ and then stimulated 4 h later with LPS (5 ng/mL) (b), primed with h∆IFN-γ plus 120 nm LcrV and then stimulated 4 h later with LPS (5 ng/mL) (2), and primed with h∆IFN-γ plus 120 nm LcrV68-326 and then stimulated 4 h later with LPS (5 ng/ mL) (1). Error bars represent the standard deviation of triplicate determinations.

of TNF-R either alone or in the presence of LcrV and LcrV68-326. As shown in Figure 8B, inclusion of LcrV or LcrV68-326 negated upregulation of TNF-R, indicating that the macrophages were Journal of Proteome Research • Vol. 6, No. 6, 2007 2229

research articles not activated. However, similar addition of h∆IFN-γ did not downregulate synthesis of TNF-R (Figure 8C) further establishing the critical nature of the GRRA138-141 motif of hIFN-γ in promoting association with LcrV.

Discussion It is now generally recognized that terminal murine plague is an anti-inflammatory disease7 and that LcrV contributes to this process33 by upregulating IL-10,11,15 a powerful antiinflammatory cytokine that prevents expression of a variety of host inflammatory factors.13,34 Results presented here regarding anti-inflammatory activity support previous observations including prompt LcrV-dependent induction of IL-10 as opposed to delayed synthesis after exposure to LPS, where peak expression occurs at about 24 h.35 Also included are new findings showing that LcrV of Y. pestis possesses two active TLR2/CD14 binding regions for LcrV and is also capable of upregulating IL-10 after binding to human but not mouse IFN-γ. The active N-terminal TLR2/CD14 recognition complex of Y. pestis LcrV contains adjacent LEEL32-35 and DKN41-43 sites, and the active internal TLR2/CD14 recognition complex contains analogous DEEI203-206 and DKN195-197 sites. The LEEL32-35 and DEEI203-206 sites are responsible for the observed highaffinity binding with the IRRL314-317 site on the extracellular domain of TLR2 as well as the GRRA138-141 site on cell-bound hIFN-γ. The DKN41-43 and DKN195-197 sites account for the binding of LcrV to CD14 after interaction with TLR2. As first shown by Sing et al.,15 the ability of LcrV to recognize TLR2/ CD14 receptors is essential for the biological activity of LcrV. These workers later demonstrated that a K42Q mutation in LcrV of Y. enterocolitica abolished biological activity as determined by an NF-κB reporter assay, whereas an E34Q mutation had no effect.15 This observation is consistent with our finding that the Kd of the E34Q mutant (∼10-7) was still sufficient to ensure binding to the TLR2/CD14 receptor complex (Table 2). It is of interest that the K42Q mutation did not influence T3SS-related effects of LcrV.32 Further study will be required to determine if the LEEL32-35 and DEEI203-206 binding sites described here are involved in the T3SS of pathogenic Yersinia. The primary structure of Y. enterocolitica LcrV differs from that of Y. pestis and Y. pseudotuberculosis in that it contains an insertion of 9 amino acid residues (ELHEVGVIA228-236) located between two β-structures (β4202-228-β5229-233) forming a β-hairpin conformation that protrudes from the main body of the protein into aqueous solvent.36 This insert prolongs the β-hairpin of Y. enterocolitica LcrV thereby screening its internal TLR2/CD14 active region containing the DKN195-197 site located on the R8194-199 helix and DEEI203-206 site located on the R9203-209 helix. This occlusion likely causes loss of TLR2/CD14 binding activity within the internal region of LcrV193-210 in Y. enterocolitica. In LcrV of Y. pestis, the LEEL32-35 site is located within sterically inaccessible R-helix 1 whereas the DEEI203-206 and LcrV68-326 sites reside within exposed R-helix 9 and are movably connected to the molecular surface via two unstructured hydrophobic sequences.36 These results are consistent with the hypothesis that the observed binding of TLR2 and IFN-γ occurs at the LEEL site of LcrV31-50 and at the DEEI203-206 site of LcrV193-210 and LcrV68-326. Furthermore, as judged by Kd values and steric constraints, binding is not cooperative and only the DEEI203-206 site shared by LcrV193-210 and LcrV68-326 functions in native LcrV. This concept explains why LcrV of Y. pestis lacking the N-terminal LEEL32-35 site and attendant DKN residue possessed full ability to upregulate IL-10, while this induction was diminished upon removal of the second DEEI site unique to 2230

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Abramov et al.

LcrV193-210 and LcrV68-326.37 However, the latter mutant protein still provided excellent protection as opposed to LcrV containing a more C-terminal deletion at amino acids 241-270. Evidence is also provided indicating that LcrV can utilize TLR2 as well as receptor-bound IFN-γ to upregulate IL-10 (with downregulation of TNF-R) in human cell systems, but that the mouse IFN-γ receptor is ineffective. This distinction is in accord with the observation that both the N- and the C-terminal regions of IFN-γ are important for its biologic activity.38,39 Depending upon the species, however, receptor binding for IFN-γ involves the KRKRS motif (residues 131-135),39 the loop connecting the A and Β helices (residues 20-23 of the SDVA motif),40 and H111.41 The SDVA motif and H111 are absent in mIFN-γ, thereby accounting for the lack of immunological cross-reactivity between the two molecules.40 In addition, hIFN-γ (unlike mIFN-γ) has an additional 9 amino acid residue at the C-terminus containing the GRRA138-141 motif42 shown here to be responsible for interaction of IFN-γ receptor-bound hIFN-γ with LcrV. Additional work will be required to define the significance of the inactive N-terminal binding site of native LcrV of Y. pestis and what role, if any, naturally occurring proteolysis43 plays in exposing this site during infection. As already noted, LcrV is a regulator, essential T3SS element, and effector of virulence as well as a protective antigen. Specific antibody might therefore promote immunity by serving as an opsonin,44,45 preventing translocation of Yops or LcrV itself,21,46 or blocking amplification of IL-10.37 We favor the hypothesis that anti-LcrV protects against disease by directly or indirectly restoring normal inflammatory potential because specifically immunized mice express proinflammatory cytokines after infection7 and contain invading organisms within protective granulomas.33,47 hIFN-γ is a major inflammatory effector of innate immunity; the ability of LcrV to utilize this molecule to minimize inflammation further underscores the penchant of plague bacilli to practice stealth as a primary stratagem for causing acute disease.

Acknowledgment. This work was supported by ISTC Grant No. 2069 from the USA (Department of Defense) and European Union; and by the Region V ‘Great Lakes’ RCE (NIH award 1-U54-AI-057153) (to R.R.B.). Note Added after ASAP Publication. This article was published ASAP on April 19, 2007. Changes have been made to Table 1. The correct version was published on April 23, 2007.

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