Identification of a domain critical for Staphylococcus aureus LukED receptor targeting and lysis of erythrocytes

Abstract

Leukocidin ED (LukED) is a pore forming toxin produced by Staphylococcus aureus which lyses host cells and promotes virulence of the bacteria. LukED enables S. aureus to acquire iron by lysing erythrocytes, which is dependent on targeting the host receptor Duffy antigen receptor for chemokines (DARC). The toxin also targets DARC on the endothelium contributing to the lethality observed during bloodstream infection in mice. LukED is comprised of two monomers, LukE and LukD. LukE binds to DARC and facilitates hemolysis, but the closely related Panton-Valentine Leukocidin S (LukS-PV) does not bind to DARC and is not hemolytic. The interaction of LukE with DARC, and the role this plays in hemolysis, are incompletely characterized. To determine the domain(s) of LukE that are critical for DARC binding, we studied the hemolytic function of LukE-LukS-PV chimeras, in which areas of sequence divergence (divergence regions or DRs) were swapped between the toxins. We found that two regions of LukE’s rim domain contribute to hemolysis, namely residues 57-75 (DR1) and residues 182-196 (DR4). Interestingly, LukE DR1 is sufficient to render LukS-PV capable of DARC binding and hemolysis. Further, LukE, by binding DARC through DR1, promotes the recruitment of LukD to erythrocytes, likely by facilitating LukED oligomer formation. Finally, we show that LukE targets murine Darc through DR1 in vivo to cause host lethality. These findings expand our biochemical understanding of the LukE-DARC interaction and the role that this toxin-receptor pair plays in S. aureus pathophysiology.

Structural insights into recognition of chemokine receptors by Staphylococcus aureus leukotoxins

“…We next sought to analyze the binding of LukE to its human chemokine receptor targets. A wealth of experimental information is available indicating that the expression of CCR5, ACKR1, CXCR1 and CXCR2 render human cells susceptible to LukED killing (Alonzo, Kozhaya et al 2013, Spaan, Reyes-Robles et al 2015, Tam, Schultz et al 2016, Vasquez, Lubkin et al 2020. Direct interaction between LukE and CCR5 or ACKR1 has also previously been characterized by surface plasmon resonance (SPR) in purified systems, indicating Kd values around 40 and 60 nM, respectively (Alonzo, Kozhaya et al 2013, Vasquez, Lubkin et al 2020.…”

“…A wealth of experimental information is available indicating that the expression of CCR5, ACKR1, CXCR1 and CXCR2 render human cells susceptible to LukED killing (Alonzo, Kozhaya et al 2013, Spaan, Reyes-Robles et al 2015, Tam, Schultz et al 2016, Vasquez, Lubkin et al 2020. Direct interaction between LukE and CCR5 or ACKR1 has also previously been characterized by surface plasmon resonance (SPR) in purified systems, indicating Kd values around 40 and 60 nM, respectively (Alonzo, Kozhaya et al 2013, Vasquez, Lubkin et al 2020. In order to complement the available experimental data and to verify that our recombinantly produced LukE is biologically active, we performed competitive binding assays in HEK293T cells using Homogenous Time Resolved FRET (TR-FRET) technology (Zwier, Roux et al 2010) (figure 2).…”

“…In order to frame these crystallographic results into a more global context, we set out to build computational models of the full-length ACKR1-LukE and CCR5-LukE complexes. A wealth of mutagenesis data is available in the literature that provides partial information about interacting residues for the ACKR1-LukE (Spaan, Reyes-Robles et al 2015, Peng, Takeshita et al 2018, Vasquez, Lubkin et al 2020) and CCR5-LukE complexes (Reyes-Robles, Alonzo et al 2013, Tam, Schultz et al 2016. The regions of LukE that are potentially implicated in receptor interaction based on previous work and on the sulfopeptide-bound structures were mapped onto the structure of LukE (figure S5A), revealing a contiguous interaction surface.…”

“…Several of these toxin-GPCR pairs have been characterized at the molecular level by swapping mutagenesis of divergent regions/loops (DRs) of the rim domain, in particular for LukE and HlgA that can target multiple chemokine receptors (Reyes-Robles, Alonzo et al 2013, Laventie, Guerin et al 2014, Tam, Schultz et al 2016, Peng, Takeshita et al 2018, Vasquez, Lubkin et al 2020). These studies highlighted the critical role of several DRs located in the rim domain in determining receptor specificity.…”

Mechanisms of GPCR hijacking byStaphylococcus aureus

“…Binding of leukotoxins to GPCRs is poorly understood at the molecular and structural level. Various residues in the loops of the rim domain of HlgA and LukE as well as a 4residue region in the cap domain of HlgA were shown to be necessary for the haemolytic activity and/or binding to erythrocytes (Nariya and Kamio, 1997;Peng et al, 2018;Vasquez et al, 2020). From the receptor side, LukE and HlgA seem to target different regions of ACKR1 N-terminal part, a highly flexible region, whereas both require sulfation of tyrosine residues in this same part of the receptor (Spaan et al, 2015).…”