The dangerous liaisons in innate immunity involving recombinant proteins and endotoxins: examples from the literature and the Leptospira field

Endotoxins, also known as lipopolysaccharides (LPS), are essential components of cell walls of diderm bacteria such as Escherichia coli (E. coli). LPS are microbe-associated molecular patterns (MAMPs) that can activate pattern recognition receptors (PRRs). While trying to investigate the interactions between proteins and host innate immunity, some studies using recombinant proteins expressed in E. coli reported interaction and activation of immune cells. Here, we set out to provide information on endotoxins, that are highly toxic to humans and bind to numerous molecules, including recombinant proteins. We begin by outlining the history of the discovery of endotoxins, their receptors and the associated signaling pathways that confer extreme sensitivity to immune cells, acting alone or in synergy with other MAMPs. We list the various places where endotoxins have been found. Additionaly, we warn against the risk of data misinterpretation due to endotoxin contamination in recombinant proteins, which is difficult to estimate with the limulus amebocyte lysate (LAL) assay, and cannot be completely neutralized (e.g. treatment with polymyxin B, or heating). We further illustrate our point with examples of recombinant heat-shock proteins and viral proteins from SARS-COV2, Dengue and HIV, for which endotoxin contamination has eventually been shown to be responsible for the inflammatory roles previously ascribed. We also critically appraised studies on recombinant Leptospira proteins regarding their putative inflammatory roles. Finally, to avoid these issues, we propose alternatives to express recombinant proteins in non-microbial systems. Microbiologists wishing to undertake innate immunity studies with their favorite pathogens should be aware of these difficulties.