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

Abstract

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. 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.

Structure of CC Chemokine Receptor 5 with a Potent Chemokine Antagonist Reveals Mechanisms of Chemokine Recognition and Molecular Mimicry by HIV

“…Specifically, it appears that the basic residues in the V3 stem and C4 of gp120 bind the sulfated N-terminus of CCR5 (thus mimicking the CRS1 interaction) and that the V3 crown of CCR5 reaches into the TM binding pocket of CCR5 (CRS2). This latter CRS2-mimicking interaction critically depends on the conserved P311 in the V3 crown (Cormier and Dragic, 2002; Cormier et al, 2001) (Figure S5D and S5E) as well as on CCR5 residue Y108 3.32 (Tam et al, 2016) (Garcia-Perez et al, 2011; Maeda et al, 2006) at the base of the CRS2 pocket (Figure 3A). Based on these findings, we hypothesized that gp120 P311 interacts with CCR5 Y108 3.32 in the same manner as P2 of both WT CCL5 and [5P7]CCL5 (Figure 3A, Figure 6C, and Figure S5E).…”

“…The antagonist affinity determinants included packing of the bulky hydrophobic residues 4–7 of [5P7]CCL5 in the minor subpocket of the receptor with simultaneous extension of the engineered pGlu0 into the major subpocket, both reminiscent of Maraviroc. At the same time, the polar residues in the proximal N-terminus of the chemokine (Q8 and S9, absent in WT CCL5) ensured closure of the extracellular parts of the receptor and engagement of ECL2 residues critical for gp120 interaction (like E172 ECL2 (Maeda et al, 2006; Tam et al, 2016)). Finally, immersion of the [5P7]CCL5 30s loop along with its N-terminus into the receptor binding pocket effectively filled it (Figure 7E-G) and completed the gp120 blockade.…”

Leukocidins: staphylococcal bi-component pore-forming toxins find their receptors

“…The S-components share 70–90% amino acid sequence identity (Box 2) 44 and the alignment of leukocidin amino acid sequences revealed divergent regions (DRs) in the rim of the S-components (Figure 1a) 35 . For LukED, the DR1 is involved in LukE targeting of CCR5 (Figure 1b) 45 , whereas DR4 determines LukE targeting of CXCR1 and CXCR2, but not for CCR5 35 (Figure 1b). The identification of different regions in one leukocidin that are involved in the recognition of different receptors permitted to make mutant LukED toxins that only target CCR5 or CXCR1 and CXCR2, which subsequently allowed the relative importance of the different leukocidin-receptor interactions to be assessed in vivo 35 .…”

The Expanding Therapeutic Perspective of CCR5 Blockade

“…LukED kills CCR5 + cells in vivo in mice, and animals lacking CCR5 are protected from mortality due to S. aureus infection ( 35 ). Even though both LukE and gp120 target CCR5, they use different determinants on the receptor ( 36 ). Interestingly, CCR5 antagonism by maraviroc (a small chemical HIV-1 entry inhibitor) confers mice with resistance to lethal S. aureus infection.…”