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

Kayan Tam,Victor J Torres,Joshua Horton,Nathaniel R Landau,Beili Wu,Tamara Reyes-Robles,Bénédicte Vanwalscappel,Francis Alonzo,Megan Schultz,Gerald B Pier,George Liu

doi:10.1128/mbio.02024-16

Abstract

ABSTRACTLeukocidin ED (LukED) is a bicomponent pore-forming toxin produced by Staphylococcus aureus that lyses host cells by targeting the chemokine receptors CC chemokine receptor type 5 (CCR5), CXCR1, CXCR2, and DARC. In addition to its role as a receptor for LukED, CCR5 is the major coreceptor for primary isolates of human immunodeficiency virus type 1 (HIV-1) and has been extensively studied. To compare how LukED and HIV-1 target CCR5, we analyzed their respective abilities to use CCR5/CCR2b chimeras to mediate cytotoxicity and virus entry. These analyses showed that the second and third extracellular loops (ECL) of CCR5 are necessary and sufficient for LukED to target the receptor and promote cell lysis. In contrast, the second ECL of CCR5 is necessary but not sufficient for HIV-1 infectivity. The analysis of CCR5 point mutations showed that glycine-163 is critical for HIV-1 infectivity, while arginine-274 and aspartic acid-276 are critical for LukED cytotoxicity. Point mutations in ECL2 diminished both HIV-1 infectivity and LukED cytotoxicity. Treatment of cells with LukED did not interfere with CCR5-tropic HIV-1 infectivity, demonstrating that LukED and the viral envelope glycoprotein use nonoverlapping sites on CCR5. Analysis of point mutations in LukE showed that amino acids 64 to 69 in the rim domain are required for CCR5 targeting and cytotoxicity. Taking the results together, this study identified the molecular basis by which LukED targets CCR5, highlighting the divergent molecular interactions evolved by HIV-1 and LukED to interact with CCR5.

Highlights

Leukocidin ED (LukED) is a bicomponent pore-forming toxin produced by Staphylococcus aureus that lyses host cells by targeting the chemokine receptors CC chemokine receptor type 5 (CCR5), CXCR1, CXCR2, and DARC
Chimeric receptors are named based on the parentage of their extracellular domains in the following order: N terminus, extracellular loop 1, extracellular loop 2, extracellular loop 3. “5” indicates the extracellular domain belonging to CCR5; “2” indicates the extracellular domain belonging to CCR2b. (B and C) Human embryonic kidney 293T cells (HEK293T) were cells transfected to express CD4 and the CCR5 chimeric receptors (B), the CCR2 chimeric receptors (C), or empty vector (e.v.) following incubation with JR-FL, ADA, and vesicular stomatitis virus G protein (VSV-G)-pseudotyped reporter virus
We identified extracellular loop 2 (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 human immunodeficiency virus type 1 (HIV-1) fusion by gp120

Summary

Introduction

Leukocidin ED (LukED) is a bicomponent pore-forming toxin produced by Staphylococcus aureus that lyses host cells by targeting the chemokine receptors CC chemokine receptor type 5 (CCR5), CXCR1, CXCR2, and DARC. To compare how LukED and HIV-1 target CCR5, we analyzed their respective abilities to use CCR5/CCR2b chimeras to mediate cytotoxicity and virus entry. These analyses showed that the second and third extracellular loops (ECL) of CCR5 are necessary and sufficient for LukED to target the receptor and promote cell lysis. HIV-1 and LukED target the same receptor, our data demonstrated that they interact with CCR5 differently, highlighting the molecular complexity of host-pathogen interactions. Statistical analyses were performed using two-way ANOVA with Dunnett’s multiple comparison. *, P Ͻ 0.05, **, P Ͻ 0.01, ***, P Ͻ 0.001, ****, P Ͻ 0.0001

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