The Scavenger Receptor SSc5D Physically Interacts with Bacteria through the SRCR-Containing N-Terminal Domain.

Catarina Bessa Pereira,Jiří Homola,Rita F Santos,Alexandre M Carmo,Ana Mafalda Santos,Markéta Bocková,Mafalda Martins De Araújo,Liliana Oliveira

doi:10.3389/fimmu.2016.00416

Abstract

The scavenger receptor cysteine-rich (SRCR) family comprises a group of membrane-attached or secreted proteins that contain one or more modules/domains structurally similar to the membrane distal domain of type I macrophage scavenger receptor. Although no all-inclusive biological function has been ascribed to the SRCR family, some of these receptors have been shown to recognize pathogen-associated molecular patterns (PAMP) of bacteria, fungi, or other microbes. SSc5D is a recently described soluble SRCR receptor produced by monocytes/macrophages and T lymphocytes, consisting of an N-terminal portion, which contains five SRCR modules, and a large C-terminal mucin-like domain. Toward establishing a global common role for SRCR domains, we interrogated whether the set of five SRCR domains of SSc5D displayed pattern recognition receptor (PRR) properties. For that purpose, we have expressed in a mammalian expression system the N-terminal SRCR-containing moiety of SSc5D (N-SSc5D), thus excluding the mucin-like domain likely by nature to bind microorganisms, and tested the capacity of the SRCR functional groups to physically interact with bacteria. Using conventional protein–bacteria binding assays, we showed that N-SSc5D had a superior capacity to bind to Escherichia coli strains RS218 and IHE3034 compared with that of the extracellular domains of the SRCR proteins CD5 and CD6 (sCD5 and sCD6, respectively), and similar E. coli-binding properties as Spα, a proven PRR of the SRCR family. We have further designed a more sensitive, real-time, and label-free surface plasmon resonance (SPR)-based assay and examined the capacity of N-SSc5D, Spα, sCD5, and sCD6 to bind to different bacteria. We demonstrated that N-SSc5D compares with Spα in the capacity to bind to E. coli and Listeria monocytogenes, and further that it can distinguish between pathogenic E. coli RS218 and IHE3034 strains and the non-pathogenic laboratory E. coli strain BL21(DE3). Our work thus advocates the utility of SPR-based assays as sensitive tools for the rapid screening of interactions between immune-related receptors and PAMP-bearing microbes. The analysis of our results suggests that SRCR domains of different members of the family have a differential capacity to interact with bacteria, and further that the same receptor can discriminate between different bacteria strains and species.

Highlights

Pattern recognition receptors (PRR) are membrane-bound or cytosolic receptors of plants and animals that are capable of interacting with pathogen-associated molecular patterns (PAMP), including lipopolysaccharide (LPS) of Gram-negative bacteria, the Gram-positive bacteria lipotheicoic acid (LTA) and peptydoglycan (PGN), as well as the fungi polysaccharides Zymosan or β-glucan, providing a first line of immune defense against microbes or their secreted toxins
We first assessed the binding of the scavenger receptor cysteine-rich (SRCR)-containing extracellular domains of Spα, SSc5D, CD6, and CD5 to E. coli strains BL21(DE3), IHE3034, and RS218, and to L. monocytogenes strain EGD-e, using conventional bacteria-protein binding assays
We considered the following as references for the binding spectra: (a) the positive interaction of Spα with neuropathogenic E. coli K1 RS218 and with L. monocytogenes EGD-e and (b) the null interaction of sCD5 with both bacteria species (Figure 2A)

Summary

Introduction

Pattern recognition receptors (PRR) are membrane-bound or cytosolic receptors of plants and animals that are capable of interacting with pathogen-associated molecular patterns (PAMP), including lipopolysaccharide (LPS) of Gram-negative bacteria, the Gram-positive bacteria lipotheicoic acid (LTA) and peptydoglycan (PGN), as well as the fungi polysaccharides Zymosan or β-glucan, providing a first line of immune defense against microbes or their secreted toxins. Receptors belonging to yet another group, the scavenger receptor cysteine-rich superfamily (SRCR-SF), are seldom referred to as pathogen-recognition molecules, despite the fact that several SRCR receptors have been shown to bind to and clear bacteria, fungi, or viruses from infected hosts [2]. A subfamily (group B) of the SRCR-SF consists of members present only in vertebrates [4], and four of the nine receptors described in humans have been shown to bind to bacteria or bacterial components. Shown not to bind to either Gram-positive or -negative bacteria, the T cell surface SRCR protein CD5 is reported to interact with conserved fungal components and to aggregate fungal cells [10]

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Results

Conclusion