Staphylococcus aureus Leukocidin LukED and HIV-1 gp120 Target Different Sequence Determinants on CCR5.

FIG 7 

Residues 64 to 69 of LukE are required for LukED cytotoxicity on CCR5-expressing cells. (A) Ribbon diagram of the LukE crystal structure (PDB 3ROH) (51) (in green) overlaid with the LukS-PV crystal structure (PDB 1T5R) (in yellow) (27). Blue, divergent region 1 (DR1) (residues 57 to 75); pink, DR2 (residues 140 to 150); red, DR3 (residues 164 to 178), gray, DR4 (residues 182 to 196); brown, residues 64 to 69. (B) Viability of SupT1 cells expressing WT CCR5 following incubation with increasing concentrations of WT LukED or LukED mutants. Cell viability was determined by CellTiter assay. n = 3; data represent mean percentages of dead cells ± SEM. (C) Viability of HEK293T cells expressing WT CCR5 following incubation with increasing concentrations of LukED or LukED mutants. Cell viability was determined by membrane permeability using eFluor450 fixable viability dye. n = 3; data represent mean percentages of dead cells ± SEM. (D) Membrane association of DyLight488-LukE (595 nM) in the presence of increasing concentrations of unlabeled WT or mutant LukE and a constant concentration of LukD^DN (565 nM). n = 3; data represent MFI of DyLight488-LukE ± SEM. Statistical analyses were performed using two-way ANOVA with Dunnett’s multiple comparison. **, P < 0.01. LukE/LukS^DR1, LukE toxin containing DR1 from LukS-PV; LukE/LukS^DR2, LukE toxin containing DR2 from LukS-PV; LukE/LukS^DR3, LukE toxin containing DR3 from LukS-PV; LukE/LukS^DR4, LukE toxin containing DR4 from LukS-PV; LukE Δ64-69, LukE containing in-frame deletion of residues 64 to 69.

DR1 is the most variable loop between the rim domains of LukE and LukS-PV (Fig. S3A) (24). In particular, LukE contains an extended amino acid sequence, KGSGYE (residues 64 to 69 of the mature, secreted toxin) (Fig. S3A). To investigate the role of these amino acids in targeting CCR5, we generated an in-frame deletion mutant lacking these residues (LukE Δ64–69). The mutated LukE was produced and purified (Fig. S3B), and its activity was evaluated as described above. These experiments revealed that deletion of amino acids KGSGYE phenocopied the lack of activity exhibited by the chimeric toxin LukE/LukS^DR1 (Fig. 7B and ​andCC).

To further evaluate the role of the LukE DR1 and the KGSGYE domain in targeting CCR5, we performed a competition assay to measure the binding of DyLight488-LukE to CCR5-positive cells in the presence of unlabeled LukE or LukE mutants. Strong competition was observed in WT LukE, LukE/LukS^DR2, LukE/LukS^DR3, and LukE/LukS^DR4, whereas LukE/LukS^DR1 and LukE Δ64–69 showed impairment in this assay (Fig. 7D). These findings demonstrated that residues 64 to 69 in DR1 are critical for LukE targeting of CCR5.

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DISCUSSION

In this study, we used CCR5/CCR2b chimeric receptors to elucidate the molecular basis of the interaction between LukED and CCR5. We identified ECL2 and ECL3 of CCR5 as necessary and sufficient for LukED-mediated cytotoxicity, while analysis of the chimeric receptors showed that ECL2 of CCR5 is necessary but not sufficient to mediate CCR5-tropic HIV-1 fusion by gp120. Using CCR5 point mutants, we identified amino acids on ECL2 and helix VII that are required for LukED cytotoxicity on CCR5-expressing cells. Moreover, using LukE/LukS-PV chimeric toxins, we determined that LukE uses residues 64 to 69 in DR1 for binding to CCR5. Taken together, our findings highlight the fact that LukED and gp120 both target CCR5 but have evolved to use different determinants on the receptor.

CCR5 is expressed by natural killer cells, CD4⁺ T cells, CD8⁺ T cells, regulatory T cells, macrophages, and dendritic cells (30), all of which contribute to the host defense against microbes. Therefore, it is not surprising that CCR5 is targeted by several different microbes, including Leishmania major (31) and poxvirus (32), to gain entry to the host. CCR5 was shown to be beneficial for the host immune responses to West Nile virus (33, 34), tickborne encephalitis virus (35), and Toxoplasma gondii (36), highlighting its multifaceted role in host defense and pathogenesis. The complex role of CCR5 in host-pathogen interactions is still not fully understood.

MVC, the small-molecule entry inhibitor of CCR5-tropic HIV-1, was demonstrated to block both HIV infection (22) and LukED intoxication (5). MVC blockade of CCR5-tropic HIV-1 entry occurs through noncompetitive allosteric binding of MVC to CCR5 (37, 38). Our study showed that while LukED and HIV shared some characteristics in binding to CCR5 (e.g., the common usage of ECL2), LukED does not prevent HIV infectivity, thus demonstrating that they interact with CCR5 differently. Based on these observations, LukED seems to bind to CCR5 by recognizing structural determinants on ECL2 and ECL3. We speculate that MVC-treated CCR5-expressing cells are protected from LukED intoxication because, upon binding to CCR5, MVC changes the receptor to a conformation that is unrecognizable by LukED via a mechanism that is similar to the allosteric inhibition of gp120 entry observed in CCR5-tropic HIV-1. However, the mode of action by which MVC acts on LukED remains to be tested.

Six leukocidins have been identified in S. aureus: LukED, LukSF-PV (or Panton-Valentine leukocidin [PVL]), HlgAB, HlgCB, LukAB, and LukMF′ (39). They target and kill host immune cells and thus are thought to play crucial roles in promoting bacterial survival in vivo. Five of the leukocidins target G protein-coupled receptors (5, 24,–26, 40, 41), while LukAB targets the integrin component CD11b, to kill host immune cells (42). However, S. aureus leukocidin and host receptor interactions are only beginning to be characterized. Previous studies have revealed that LukS-PV and HlgC target C5aR and C5L2 through the recognition of specific extracellular domains on the receptors (43), LukAB targets the I-domain of CD11b (42), and LukMF′ targets ECL2 and ECL3 of bovine CCR1 (41). Together, these studies explain some of the toxin-receptor specificity observed in vivo.

The leukocidins share high protein sequence identity, and yet they are specific for disparate receptors and kill different immune cell types (44). For LukED, DR4 was shown to be required for LukE binding and recognition of CXCR1 and CXCR2 (24), and in this study, we demonstrated that DR1 is critical for the binding and recognition of CCR5. Thus, specific LukE DR loops are responsible for receptor recognition. Consistent with this notion, tyrosine-184 in DR4 and tyrosine-250 in DR5 of LukS are important for the binding and cytotoxic activity of LukSF-PV with respect to the C5a receptor (45). In another leukocidin, LukA, glutamic acid-323 is required for receptor binding and toxin activity (46). The interactions between the leukocidins and their receptors are analogous to a lock and a key—the leukocidins require specific domains for the binding to and recognition of specific receptors, while specific regions on the receptors are essential for optimal toxin binding.

The leukocidins are critical virulence factors for S. aureus pathogenesis (24, 47,–50), and we are only now beginning to understand the molecular basis on which they interact with their receptors. Elucidating how these leukocidins interact with host receptors could provide a foundation for the development of novel inhibitors to combat this important human pathogen.

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MATERIALS AND METHODS

 

Generation of the chemokine receptors for transfection and transduction.

CCR5/CCR2b chimeric receptors were generated by overlapping PCR using CCR5 and CCR2b cDNAs followed by cloning into the pcDNA3.1(+) vector. Primers used for chimeric receptor generation are listed in Table S1 in the supplemental material. All constructs were confirmed by Sanger sequencing (Genewiz).

 

Cell culture.

Human embryonic kidney 293T cells (HEK293T; ATCC CRL-3216) and SupT1 cells (ATCC CRL-1942) were maintained at 37°C with 5% CO₂ in RPMI 1640 medium (Cellgro) supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (Pen; 100 U/ml), and streptomycin (Strep; 0.1 mg/ml). Transduced SupT1 cells expressing CCR5 and CCR5 mutants were maintained in the same media supplemented with 1 μg/ml puromycin. All experiments were conducted within approximately 1 month of thawing of frozen cell stocks.

 

Virus preparation.

To generate pLenti viruses containing WT CCR5, CCR5 mutants, or CCR5/CCR2b chimeric receptors, HEK293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and 1× Pen/Strep. HEK293T cells were cotransfected with pRSV-Rev, pMDL-gagpol, pcVSVg, pLenti construct, and pcDNA6. pcVSV-G expresses a cytomegalovirus (CMV)-driven vesicular stomatitis virus (VSV) envelope glycoprotein (G), and pRSV-Rev expresses a Rous sarcoma virus (RSV) long terminal repeat (LTR)-driven HIV-1 Rev. Viruses were harvested 48 h posttransfection, filtered through 0.45-µm-pore-size filters, and frozen at −80°C in DMEM supplemented with 10% FBS and 1× Pen/Strep.

HIV-1 luciferase reporter viruses pseudotyped with a CCR5-tropic HIV envelope were generated by cotransfecting HEK293T cells with pNL43 luc3 E-R⁻ and HIV envelope (pSV-JRFL or pSV-ADA). As a control, HIV-1 luciferase reporter virus pseudotyped with a CXCR4-tropic HIV envelope was generated by cotransfecting HEK293T cells with pNL43 luc3 E-R⁻ and HIV Srα envelope. Viruses were harvested 48 h posttransfection, filtered through 0.45-µm-pore-size filters, and pelleted through 20% sucrose at 30,000 rpm for 90 min at 4°C in an ultracentrifuge. Virus pellets were resuspended in RPMI medium supplemented with 10% FBS and 1× Pen/Strep and frozen at −80°C. Luciferase reporter viruses were normalized by infection of 5 × 10⁴ SupT1 CCR5 cells using various amounts of virus.

 

Transfection of 293T.

HEK293T cells were seeded at 25,500 cells/cm² in a 10-cm² dish overnight, followed by transfection with expression plasmids for the CCR5/CCR2b chimeras the next day. For transfection, 30 μg of plasmid DNA was added to 30 μl of Lipofectamine 2000 (Invitrogen) and incubated for 30 min at room temperature (RT). The complexes were added to HEK293T cells, incubated at 37°C with 5% CO₂ for 6 to 7 h, and then replaced with fresh media. All experiments were conducted the following day.

 

Transduction of SupT1.

SupT1 cells were seeded at 200,000/well in a 6-well plate and spinnoculated with 2 ml of pLenti lentiviral vector stock for vectors expressing WT or mutated CCR5 in the presence of Polybrene (Millipore) (10 mg/ml) at 2,500 rpm for 2 h at 30°C. The virus was then removed, and fresh medium was added. Cells were selected 3 days later in RPMI media containing puromycin (1 µg/ml). To detect transduced CCR5, cells were stained with mouse anti-hemagglutinin (anti-HA) antibody (clone 16B12; Covance) (1:1,000) in fluorescence-activated cell sorting (FACS) buffer (1× phosphate-buffered saline [PBS]–2% heat-inactivated FBS) for 1 h at RT. Then, cells were washed twice with FACS buffer and incubated with phycoerythrin-conjugated goat anti-mouse IgG (BioLegend) (1:100) in FACS buffer for 1 h at RT. Cells were washed twice and resuspended in FACS buffer to analyze expression of transduced CCR5 using flow cytometry (LSRII with FACSDiva software; BD).

 

Chimera and SupT1 HIV infection.

SupT1 stable cell lines were seeded at 50,000/well in a 96-well plate and spinnoculated with 1.8 ng of p24 from CCR5-tropic luciferase reporter viruses in the presence of Polybrene (8 µg/ml) at 2,500 rpm and 30°C for 2 h. The medium was then changed, and infectivity was determined 3 days later by luciferase assay and read on an Envision 2103 multilabel reader (PerkinElmer).

 

Toxin purification from S. aureus.

WT LukE and chimeric LukE, LukD, and LukD^DN toxins were generated as indicated previously (19, 24). To generate lukE Δ64–69, primers VJT629 and VJT684 and primers VJT683 and VJT1114 were used to generate an in-frame deletion of amino acids 64 to 69 of lukE by overlapping extension PCR. The final PCR product was amplified using primers VJT629 and VJT1114 and then cloned into the pOS1-P_lukAB-lukA^ss-6×His plasmid using BamHI and PstI restriction sites as described previously (19). The purified plasmid was transformed into Escherichia coli DH5α competent cells, selected by ampicillin resistance (100 µg/ml), and confirmed by colony PCR and sequencing (Genewiz). The plasmid from a positive clone was purified and electroporated into S. aureus RN4220 and selected for by chloramphenicol (10 µg/ml) resistance, and then the plasmid from RN4220 was purified and electroporated into S. aureus Newman ΔlukED hlgACB::tet lukAB::spec hla::ermC (ΔΔΔΔ) and selected for by chloramphenicol (10 µg/ml) resistance.

Toxins were purified as described previously (19, 24). Briefly, S. aureus Newman ΔΔΔΔ strains harboring plasmids with the respective leukocidin sequences were grown overnight in 5 ml tryptic soy broth (TSB; Fisher) supplemented with chloramphenicol (10 μg/ml) at 37°C with shaking at 180 rpm and then subcultured the following day at a 1:100 dilution in TSB supplemented with chloramphenicol (10 μg/ml) and incubated for 5 h at 37°C with shaking at 180 rpm. The cultures were centrifuged for 15 min at 6,000 rpm and 4°C and the supernatants filter sterilized through a 0.22-μm-pore-size filter (Corning). The filtrates were incubated in the presence of a final concentration of 10 mM imidazole and nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (Qiagen) equilibrated with 10 mM imidazole (Fisher) in 1× Tris-buffered saline (TBS; Cellgro) for 30 min at 4°C while nutating. The filtrates were passed through a glass column by gravity filtration, and then Ni-NTA-bound toxins were washed with 25 mM imidazole, followed by a secondary wash with 1× TBS. The Ni-NTA-bound toxins were eluted using 500 mM imidazole. The eluted toxins were dialyzed into 10% glycerol–1× TBS for storage at −80°C. When required, the toxins were concentrated using concentrator columns (Ultra-15 centrifugal filter units; EMD Millipore Amicon) (10,000 nominal molecular weight limit [NMWL], 15-ml capacity) before measurement of the protein concentration was performed using absorbance at 280 nm with a NanoDrop spectrophotometer (Thermo Scientific) and the Beer-Lambert’s equation. Two micrograms of the purified proteins was separated by SDS-PAGE at 90 V for 120 min, followed by Coomassie blue staining to visualize proteins to confirm purity.

 

Cytotoxicity assays.

To evaluate the viability of HEK293T and SupT1 cells after intoxication by the leukocidins in vitro, cells were seeded at 1 × 10⁵ cells/well in RPMI 1640 without phenol red (Gibco) and supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gemini Bio-Products) in the presence of the indicated concentrations of leukocidins for 2 h at 37°C and 5% CO₂.

To measure cell viability by FACS analysis, the cells were washed in FACS buffer (1× PBS–2% heat-inactivated FBS–0.05% sodium azide) after intoxication and then incubated with mouse anti-HA antibody (clone 16B12; Covance) (1:1,000) for 30 min on ice. The cells were washed twice on ice in FACS buffer and centrifuged for 5 min at 1,500 rpm and 4°C, followed by incubation of phycoerythrin goat anti-mouse IgG (BioLegend) (1:200) for 30 min on ice. The cells were washed twice on ice in 1× PBS and centrifuged for 5 min at 1,500 rpm and 4°C, followed by incubation with eFluor450 fixable viability dye (eBiosciences) (1:1,000) for 30 min on ice. The cells were fixed in 50 μl FACS fixation buffer (FACS buffer–2% paraformaldehyde). Cell viability was analyzed using flow cytometry (LSRII with FACSDiva software; BD); data are shown as percentages of the total receptor-positive cells that were permeable.

Cell viability was also measured using the CellTiter 96 AQueous ONE solution cell proliferation assay (Promega) when indicated. CellTiter was added to cells at a final concentration of 10% per well and incubated for 2 h at 37°C and 5% CO₂. Absorbance at 492 nm was measured using an EnVision 2103 multilabel reader (PerkinElmer).

 

Binding assays.

For binding assays, HEK293T cells expressing WT CCR5 or chimeric receptors were seeded at 1 × 10⁵ cells/well followed by incubation of mouse anti-HA antibody (clone 16B12; Covance) (1:1,000) for 30 min on ice. The cells were washed twice on ice in FACS buffer and centrifuged for 5 min at 1,500 rpm and 4°C, followed by incubation with allophycocyanin anti-mouse IgG (BioLegend) (1:200) for 30 min on ice. The cells were washed twice on ice in FACS buffer and centrifuged for 5 min at 1,500 rpm and 4°C. The cells were then resuspended in increasing concentrations of DyLight488-LukE in complex with either WT LukD or LukD^DN and incubated for 30 min on ice. The cells were centrifuged for 5 min at 1,500 rpm and 4°C, washed on ice in FACS buffer, and centrifuged again for 5 min at 1,500 rpm and 4°C, followed by fixing in 50 μl FACS fixation buffer. Fluorescence of cell-bound toxins was analyzed using flow cytometry (LSRII with FACSDiva software; BD); data are shown as median fluorescence intensity (MFI) of receptor-positive cells.

 

Competition assays—LukE interaction with target cells.

For competition assays, HEK293T cells expressing WT CCR5 were seeded at 1 × 10⁵ cells/well followed by incubation of mouse anti-HA antibody (clone 16B12; Covance) (1:1,000) for 30 min on ice, and the cells were washed twice on ice in FACS buffer and centrifuged for 5 min at 1,500 rpm and 4°C, followed by incubation with allophycocyanin goat anti-mouse IgG (BioLegend) for 30 min on ice. The cells were washed twice on ice in FACS buffer and centrifuged as before. The cells were then resuspended in a constant concentration of DyLight488-labeled WT LukE and the pore-formation defective LukD mutant and increasing concentrations of WT LukE or mutant LukE, and LukD^DN. The cells were incubated for 30 min on ice. Cells were centrifuged for 5 min at 1,500 rpm and 4°C, washed on ice in FACS buffer, and centrifuged again for 5 min at 1,500 rpm and 4°C, followed by fixing in 50 μl FACS fixation buffer. Fluorescence of cell-bound toxins was analyzed using flow cytometry (LSRII with FACSDiva software; BD), where data are shown as median fluorescent intensity (MFI) of receptor-positive cells.

 

Competition assay with LukED and blockade of HIV infectivity.

SupT1-CCR5 cells were seeded at 50,000/well on a round-bottom 96-well plate in RPMI media supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and 1 μg/ml puromycin. Cells were treated with 282 nM LukED^DN, 19.5 μM maraviroc (MVC), or PBS for 30 min. Then, cells were spin-infected with 100 µl of JRFL-, ADA-, or NL43SRα-pseudotyped luciferase reporter viruses at 2,200 rpm and 30°C for 2 h. The medium was changed, and the infectivity was determined 3 days later by luciferase assay using an Envision 2103 multilabel reader (PerkinElmer).

 

Graphical and statistical analyses.

Analyses of flow cytometric data were performed using FlowJo (Tree Star Software). Statistical significance was determined using Prism 7.0 (GraphPad Software, Inc.), with two-way analysis of variance (ANOVA) performed with Dunnett’s multiple comparison.

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SUPPLEMENTAL MATERIAL

Figure S1 

Surface staining of HEK293T cells transfected to overexpress (A) CCR5 chimeric receptors and (B) CCR2 chimeric receptors. Surface receptor expressions were detected using an anti-HA monoclonal antibody against the HA tag at the N terminus of the chimeric receptors. The histogram depicts transfections from a representative experiment; n = 3. Download

Figure S1, TIF file, 2.7 MB^{(2.7M, tif)}

Figure S2 

Surface staining of SupT1 cells expressing CCR5 mutants. Surface receptor expression was detected using an anti-HA monoclonal antibody against the HA tag at the N terminus of the CCR5 mutant receptors. The histogram depicts transfections from a representative experiment; n = 3. Download

Figure S2, TIF file, 1.4 MB^{(1.3M, tif)}

Figure S3 

(A) Multiple-sequence alignments of LukE and LukS by DNAStar using Clustal W algorithm. Blue indicates DR1, pink indicates DR2, red indicates DR3, gray indicates DR4, and brown indicates residues 64 to 69. (B) Two micrograms per lane of purified chimeric toxins visualized by Coomassie blue staining on a 12% SDS-PAGE gel. Download

Figure S3, TIF file, 21.9 MB^{(22M, tif)}

Table S1 

List of primers used for constructing CCR5/CCR2b chimeric receptors, CCR5 mutants, and LukE Δ64–69.

Table S1, XLSX file, 0.03 MB^{(27K, xlsx)}

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ACKNOWLEDGMENTS

Research reported in this publication was supported in part by the United States National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH-NIAID) under award numbers AI007180; and AI112290 to T.R.-R., AI105129 to V.J.T., and AI067059, AI22390 and AI073237 to N.R.L. V.J.T. is a Burroughs Wellcome Fund Investigator in the pathogenesis of Infectious Diseases. The content is solely our responsibility and does not necessarily represent the official views of the National Institutes of Health.

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FUNDING STATEMENT

This work, including the efforts of Tamara Reyes-Robles, was funded by HHS|NIH| National Institute of Allergy and Infectious Diseases (NIAID) (AI007180 and AI112290). This work, including the efforts of Victor J. Torres, was funded by HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) (AI105129). This work, including the efforts of Nathaniel Roy Landau, was funded byHHS| NIH | National Institute of Allergy and Infectious Diseases (NIAID) (AI067059, AI22390, AI073237).

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FOOTNOTES

 

Citation Tam K, Schultz M, Reyes-Robles T, Vanwalscappel B, Horton J, Alonzo F, Wu B, Landau NR, Torres VJ. 2016. Staphylococcus aureus leukocidin LukED and HIV-1 gp120 target different sequence determinants on CCR5. mBio 7(6):e02024-16. doi:10.1128/mBio.02024-16.

 

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REFERENCES

1. Dantes R, Mu Y, Belflower R, Aragon D, Dumyati G, Harrison LH, Lessa FC, Lynfield R, Nadle J, Petit S, Ray SM, Schaffner W, Townes J, Fridkin S, Emerging Infections Program-Active Bacterial Core Surveillance MSI . 2013. National burden of invasive methicillin-resistant Staphylococcus aureus infections, United States, 2011. JAMA Intern Med 173:1970–1978. doi:10.1001/jamainternmed.2013.10423. [PubMed] [CrossRef] [Google Scholar]
2. David MZ, Daum RS. 2010. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev 23:616–687. doi:10.1128/CMR.00081-09. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
3. CDC 2015. Prevalence of diagnosed and undiagnosed HIV infection—United States, 2008–2012. MMWR Morb Mortal Wkly Rep 64:657–662. [PMC free article] [PubMed] [Google Scholar]
4. UNAIDS 2016, Global AIDS update. UNAIDS report. [Google Scholar]
5. Alonzo F III, Kozhaya L, Rawlings SA, Reyes-Robles T, DuMont AL, Myszka DG, Landau NR, Unutmaz D, Torres VJ. 2013. CCR5 is a receptor for Staphylococcus aureus leukotoxin ED. Nature 493:51–55. doi:10.1038/nature11724. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
6. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, Di Marzio P, Marmon S, Sutton RE, Hill CM, Davis CB, Peiper SC, Schall TJ, Littman DR, Landau NR. 1996. Identification of a major co-receptor for primary isolates of HIV-1. Nature 381:661–666. doi:10.1038/381661a0. [PubMed] [CrossRef] [Google Scholar]
7. Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM, Berger EA. 1996. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955–1958. [PubMed] [Google Scholar]
8. Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, Wu L, Mackay CR, LaRosa G, Newman W, Gerard N, Gerard C, Sodroski J. 1996. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135–1148. doi:10.1016/S0092-8674(00)81313-6. [PubMed] [CrossRef] [Google Scholar]
9. Doranz BJ, Rucker J, Yi Y, Smyth RJ, Samson M, Peiper SC, Parmentier M, Collman RG, Doms RW. 1996. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149–1158. doi:10.1016/S0092-8674(00)81314-8. [PubMed] [CrossRef] [Google Scholar]
10. Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, Cayanan C, Maddon PJ, Koup RA, Moore JP, Paxton WA. 1996. HIV-1 entry into CD4⁺cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667–673. doi:10.1038/381667a0. [PubMed] [CrossRef] [Google Scholar]
11. Miller MA, Cloyd MW, Liebmann J, Rinaldo CR Jr, Islam KR, Wang SZ, Mietzner TA, Montelaro RC. 1993. Alterations in cell membrane permeability by the lentivirus lytic peptide (LLP-1) of HIV-1 transmembrane protein. Virology 196:89–100. doi:10.1006/viro.1993.1457. [PubMed] [CrossRef] [Google Scholar]
12. Srinivas SK, Srinivas RV, Anantharamaiah GM, Segrest JP, Compans RW. 1992. Membrane interactions of synthetic peptides corresponding to amphipathic helical segments of the human immunodeficiency virus type-1 envelope glycoprotein. J Biol Chem 267:7121–7127. [PubMed] [Google Scholar]
13. Rizzuto CD, Wyatt R, Hernández-Ramos N, Sun Y, Kwong PD, Hendrickson WA, Sodroski J. 1998. A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science 280:1949–1953. doi:10.1126/science.280.5371.1949. [PubMed] [CrossRef] [Google Scholar]
14. Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, Saragosti S, Lapoumeroulie C, Cognaux J, Forceille C, Muyldermans G, Verhofstede C, Burtonboy G, Georges M, Imai T, Rana S, Yi Y, Smyth RJ, Collman RG, Doms RW, Vassart G, Parmentier M. 1996. Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 382:722–725. doi:10.1038/382722a0. [PubMed] [CrossRef] [Google Scholar]
15. Rucker J, Samson M, Doranz BJ, Libert F, Berson JF, Yi Y, Smyth RJ, Collman RG, Broder CC, Vassart G, Doms RW, Parmentier M. 1996. Regions in beta-chemokine receptors CCR5 and CCR2b that determine HIV-1 cofactor specificity. Cell 87:437–446. doi:10.1016/S0092-8674(00)81364-1. [PubMed] [CrossRef] [Google Scholar]
16. Samson M, LaRosa G, Libert F, Paindavoine P, Detheux M, Vassart G, Parmentier M. 1997. The second extracellular loop of CCR5 is the major determinant of ligand specificity. J Biol Chem 272:24934–24941. doi:10.1074/jbc.272.40.24934. [PubMed] [CrossRef] [Google Scholar]
17. Blanpain C, Doranz BJ, Bondue A, Govaerts C, De Leener A, Vassart G, Doms RW, Proudfoot A, Parmentier M. 2003. The core domain of chemokines binds CCR5 extracellular domains while their amino terminus interacts with the transmembrane helix bundle. J Biol Chem 278:5179–5187. doi:10.1074/jbc.M205684200. [PubMed] [CrossRef] [Google Scholar]
18. Maeda K, Das D, Ogata-Aoki H, Nakata H, Miyakawa T, Tojo Y, Norman R, Takaoka Y, Ding J, Arnold GF, Arnold E, Mitsuya H. 2006. Structural and molecular interactions of CCR5 inhibitors with CCR5. J Biol Chem 281:12688–12698. doi:10.1074/jbc.M512688200. [PubMed] [CrossRef] [Google Scholar]
19. Reyes-Robles T, Lubkin A, Alonzo F III, Lacy DB, Torres VJ. 2016. Exploiting dominant-negative toxins to combat Staphylococcus aureus pathogenesis. EMBO Rep 17:428–440. doi:10.15252/embr.201540994. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
20. Maeda K, Das D, Yin PD, Tsuchiya K, Ogata-Aoki H, Nakata H, Norman RB, Hackney LA, Takaoka Y, Mitsuya H. 2008. Involvement of the second extracellular loop and transmembrane residues of CCR5 in inhibitor binding and HIV-1 fusion: insights into the mechanism of allosteric inhibition. J Mol Biol 381:956–974. doi:10.1016/j.jmb.2008.06.041. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
21. Königs C, Pustowka A, Irving J, Kessel C, Klich K, Wegner V, Rowley MJ, Mackay IR, Kreuz W, Griesinger C, Dietrich U. 2007. Peptide mimotopes selected with HIV-1-blocking monoclonal antibodies against CCR5 represent motifs specific for HIV-1 entry. Immunol Cell Biol 85:511–517. doi:10.1038/sj.icb.7100077. [PubMed] [CrossRef] [Google Scholar]
22. Dorr P, Westby M, Dobbs S, Griffin P, Irvine B, Macartney M, Mori J, Rickett G, Smith-Burchnell C, Napier C, Webster R, Armour D, Price D, Stammen B, Wood A, Perros M. 2005. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity. Antimicrob Agents Chemother 49:4721–4732. doi:10.1128/AAC.49.11.4721-4732.2005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
23. Tan Q, Zhu Y, Li J, Chen Z, Han GW, Kufareva I, Li T, Ma L, Fenalti G, Li J, Zhang W, Xie X, Yang H, Jiang H, Cherezov V, Liu H, Stevens RC, Zhao Q, Wu B. 2013. Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex. Science 341:1387–1390. doi:10.1126/science.1241475. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
24. Reyes-Robles T, Alonzo F III, Kozhaya L, Lacy DB, Unutmaz D, Torres VJ. 2013. Staphylococcus aureus leukotoxin ED targets the chemokine receptors CXCR1 and CXCR2 to kill leukocytes and promote infection. Cell Host Microbe 14:453–459. doi:10.1016/j.chom.2013.09.005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
25. Spaan AN, Reyes-Robles T, Badiou C, Cochet S, Boguslawski KM, Yoong P, Day CJ, de Haas CJ, van Kessel KP, Vandenesch F, Jennings MP, Le Van Kim C, Colin Y, van Strijp JA, Henry T, Torres VJ. 2015. Staphylococcus aureustargets the Duffy antigen receptor for chemokines (DARC) to lyse erythrocytes. Cell Host Microbe 18:363–370. doi:10.1016/j.chom.2015.08.001. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
26. Spaan AN, Henry T, van Rooijen WJ, Perret M, Badiou C, Aerts PC, Kemmink J, de Haas CJ, van Kessel KP, Vandenesch F, Lina G, van Strijp JA. 2013. The staphylococcal toxin Panton-Valentine leukocidin targets human C5a receptors. Cell Host Microbe 13:584–594. doi:10.1016/j.chom.2013.04.006. [PubMed] [CrossRef] [Google Scholar]
27. Guillet V, Roblin P, Werner S, Coraiola M, Menestrina G, Monteil H, Prévost G, Mourey L. 2004. Crystal structure of leucotoxin S component: new insight into the staphylococcal beta-barrel pore-forming toxins. J Biol Chem 279:41028–41037. doi:10.1074/jbc.M406904200. [PubMed] [CrossRef] [Google Scholar]
28. Olson R, Nariya H, Yokota K, Kamio Y, Gouaux E. 1999. Crystal structure of staphylococcal LukF delineates conformational changes accompanying formation of a transmembrane channel. Nat Struct Biol 6:134–140. doi:10.1038/5821. [PubMed] [CrossRef] [Google Scholar]
29. Song L, Hobaugh MR, Shustak C, Cheley S, Bayley H, Gouaux JE. 1996. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274:1859–1866. doi:10.1126/science.274.5294.1859. [PubMed] [CrossRef] [Google Scholar]
30. Griffith JW, Sokol CL, Luster AD. 2014. Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol 32:659–702. doi:10.1146/annurev-immunol-032713-120145. [PubMed] [CrossRef] [Google Scholar]
31. Yurchenko E, Tritt M, Hay V, Shevach EM, Belkaid Y, Piccirillo CA. 2006. CCR5-dependent homing of naturally occurring CD4⁺regulatory T cells to sites of Leishmania major infection favors pathogen persistence. J Exp Med 203:2451–2460. doi:10.1084/jem.20060956. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
32. Lalani AS, Masters J, Zeng W, Barrett J, Pannu R, Everett H, Arendt CW, McFadden G. 1999. Use of chemokine receptors by poxviruses. Science 286:1968–1971. doi:10.1126/science.286.5446.1968. [PubMed] [CrossRef] [Google Scholar]
33. Glass WG, Lim JK, Cholera R, Pletnev AG, Gao JL, Murphy PM. 2005. Chemokine receptor CCR5 promotes leukocyte trafficking to the brain and survival in West Nile virus infection. J Exp Med 202:1087–1098. doi:10.1084/jem.20042530. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
34. Glass WG, McDermott DH, Lim JK, Lekhong S, Yu SF, Frank WA, Pape J, Cheshier RC, Murphy PM. 2006. CCR5 deficiency increases risk of symptomatic West Nile virus infection. J Exp Med 203:35–40. doi:10.1084/jem.20051970. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
35. Kindberg E, Mickiene A, Ax C, Akerlind B, Vene S, Lindquist L, Lundkvist A, Svensson L. 2008. A deletion in the chemokine receptor 5 (CCR5) gene is associated with tickborne encephalitis. J Infect Dis 197:266–269. doi:10.1086/524709. [PubMed] [CrossRef] [Google Scholar]
36. Khan IA, Thomas SY, Moretto MM, Lee FS, Islam SA, Combe C, Schwartzman JD, Luster AD. 2006. CCR5 is essential for NK cell trafficking and host survival following Toxoplasma gondii infection. PLoS Pathog 2:e49. doi:10.1371/journal.ppat.0020049. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
37. Garcia-Perez J, Rueda P, Staropoli I, Kellenberger E, Alcami J, Arenzana-Seisdedos F, Lagane B. 2011. New insights into the mechanisms whereby low molecular weight CCR5 ligands inhibit HIV-1 infection. J Biol Chem 286:4978–4990. doi:10.1074/jbc.M110.168955. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
38. Watson C, Jenkinson S, Kazmierski W, Kenakin T. 2005. The CCR5 receptor-based mechanism of action of 873140, a potent allosteric noncompetitive HIV entry inhibitor. Mol Pharmacol 67:1268–1282. doi:10.1124/mol.104.008565. [PubMed] [CrossRef] [Google Scholar]
39. Alonzo F III, Torres VJ. 2014. The bicomponent pore-forming leucocidins of Staphylococcus aureus. Microbiol Mol Biol Rev 78:199–230. doi:10.1128/MMBR.00055-13. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
40. Spaan AN, Vrieling M, Wallet P, Badiou C, Reyes-Robles T, Ohneck EA, Benito Y, de Haas CJ, Day CJ, Jennings MP, Lina G, Vandenesch F, van Kessel KP, Torres VJ, van Strijp JA, Henry T. 2014. The staphylococcal toxins gamma-haemolysin AB and CB differentially target phagocytes by employing specific chemokine receptors. Nat Commun 5:5438. doi:10.1038/ncomms6438. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
41. Vrieling M, Koymans KJ, Heesterbeek DA, Aerts PC, Rutten VP, de Haas CJ, van Kessel KP, Koets AP, Nijland R, van Strijp JA. 2015. Bovine Staphylococcus aureus secretes the leukocidin LukMF’ to kill migrating neutrophils through CCR1. mBio 6:e00335. doi:10.1128/mBio.00335-15. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
42. DuMont AL, Yoong P, Day CJ, Alonzo F III, McDonald WH, Jennings MP, Torres VJ. 2013. Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1. Proc Natl Acad Sci U S A 110:10794–10799. doi:10.1073/pnas.1305121110. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
43. Spaan AN, Schiepers A, de Haas CJ, van Hooijdonk DD, Badiou C, Contamin H, Vandenesch F, Lina G, Gerard NP, Gerard C, van Kessel KP, Henry T, van Strijp JA. 2015. Differential interaction of the staphylococcal toxins Panton-Valentine leukocidin and gamma-hemolysin CB with human C5a receptors. J Immunol 195:1034–1043. doi:10.4049/jimmunol.1500604. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
44. DuMont AL, Torres VJ. 2014. Cell targeting by the Staphylococcus aureus pore-forming toxins: it’s not just about lipids. Trends Microbiol 22:21–27. doi:10.1016/j.tim.2013.10.004. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
45. Laventie BJ, Guérin F, Mourey L, Tawk MY, Jover E, Maveyraud L, Prévost G. 2014. Residues essential for Panton-Valentine leukocidin S component binding to its cell receptor suggest both plasticity and adaptability in its interaction surface. PLoS One 9:e92094. doi:10.1371/journal.pone.0092094. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
46. DuMont AL, Yoong P, Liu X, Day CJ, Chumbler NM, James DB, Alonzo F III, Bode NJ, Lacy DB, Jennings MP, Torres VJ. 2014. Identification of a crucial residue required for Staphylococcus aureus LukAB cytotoxicity and receptor recognition. Infect Immun 82:1268–1276. doi:10.1128/IAI.01444-13. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
47. Alonzo F III, Benson MA, Chen J, Novick RP, Shopsin B, Torres VJ. 2012. Staphylococcus aureus leucocidin ED contributes to systemic infection by targeting neutrophils and promoting bacterial growth in vivo. Mol Microbiol 83:423–435. doi:10.1111/j.1365-2958.2011.07942.x. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
48. Bubeck Wardenburg J, Bae T, Otto M, Deleo FR, Schneewind O. 2007. Poring over pores: alpha-hemolysin and Panton-Valentine leukocidin in Staphylococcus aureus pneumonia. Nat Med 13:1405–1406. doi:10.1038/nm1207-1405. [PubMed] [CrossRef] [Google Scholar]
49. DuMont AL, Yoong P, Surewaard BG, Benson MA, Nijland R, van Strijp JA, Torres VJ. 2013. Staphylococcus aureus elaborates leukocidin AB to mediate escape from within human neutrophils. Infect Immun 81:1830–1841. doi:10.1128/IAI.00095-13. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
50. Labandeira-Rey M, Couzon F, Boisset S, Brown EL, Bes M, Benito Y, Barbu EM, Vazquez V, Höök M, Etienne J, Vandenesch F, Bowden MG. 2007. Staphylococcus aureus Panton-Valentine leukocidin causes necrotizing pneumonia. Science 315:1130–1133. doi:10.1126/science.1137165. [PubMed] [CrossRef] [Google Scholar]
51. Nocadello S, Minasov G, Shuvalova L, Dubrovska I, Sabini E, Bagnoli F, Grandi G, Anderson WF. 2016. Crystal structures of the components of the Staphylococcus aureus leukotoxin ED. Acta Crystallogr D Struct Biol 72:113–120. doi:10.1107/S2059798315023207. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

 

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