Characterisation of E. coli Lipopolysaccharide Adherence to Platelet Receptors

Introduction

The interaction of bacteria with platelets is vital in the pathogenesis of sepsis and other fatal infections. Sepsis is an inflammatory response to bloodstream infections with high mortality rates and Escherichia coli (E.coli) is the most frequently isolated gram-negative organism in these infections. Platelets are a part of our innate immune system and interact with invading pathogens in our blood stream. Recently, it has been shown that E.coli interacts directly with platelets and it is believed that Lipopolysaccharide (LPS) plays a role in this interaction. Understanding this adhesive process and identification of the platelet receptors involved in bacterial adherence is vital in explaining the underlying mechanisms utilized in infections.

Methods

• Competition assays assessing E.coli adherence to platelets in the presence of antibodies to platelet surface receptors and soluble recombinant forms of these receptors were conducted.

• Competition assays were used to assess E.coli binding to platelets in the presence of E.coli K12 LPS and H.pylori LPS.

• Differences between the affinity of human and murine receptors to E.coli were investigated by assessing E.coli binding to recombinant human and murine CD36.

• E.coli strains expressing LPS structural variations were assessed to identify the LPS moiety that promotes binding to platelets and CD36.

Results

This study demonstrates for the first time that the scavenger receptor CD36 and the Toll-like receptors; TLR2 and TLR4/MD2 are platelet receptors for the direct adherence of E.coli LPS to platelets. LPS is a large molecule found on the outer membrane of gram-negative bacteria that elicits a strong immune response in humans and animals. It comprises of three main components; the O-antigen, a core polysaccharide and the lipid A region. Our work reveals that the minimum LPS components required for E.coli binding to platelets and CD36 must contain the Lipid A and inner core regions of LPS. Identifying this interaction to host receptors is important if we want to design effective anti-sepsis drugs.

In addition to this, analysis of the adherence of epidemic strains of pathogenic antibiotic resistant strains of E.coli to platelets and recombinant platelet receptors showed that the O-antigen portion of LPS may conceal LPS from recognition by these receptors as these strains bind significantly less to CD36 compared to an E. coli strain that does not express an O-antigen. This information helps explain how these pathogenic E.coli strains can evade immune recognition and therefore, continue to infect patients.

Interestingly, murine CD36 has a significantly reduced affinity for E.coli compared to human CD36. This may help to explain why the human and mouse immune response to E.coli infection is different and perhaps explain the failure of anti-sepsis drugs developed in mouse models of infection like Eritoran in human clinical trials. This evidence suggests that mouse models may not be optimal models for E. coli septicemia.

Conclusion

This research provides a new insight into the mechanisms of direct E.coli platelet interactions that can cause life-threatening diseases. We have identified the LPS components involved in the direct interaction of E.coli with platelets and that pathogenic strains can utilize the O-antigen region of their LPS structure to evade immune recognition so they can continue to infect patients. Understanding how these strains interact with our immune cells or evade them will be invaluable in designing effective treatments to combat infections.