Humanized TLR4/MD-2 Mice Reveal LPS Recognition Differentially Impacts Susceptibility to Yersinia pestis and Salmonella enterica

Abstract

Although lipopolysaccharide (LPS) stimulation through the Toll-like receptor (TLR)-4/MD-2 receptor complex activates host defense against Gram-negative bacterial pathogens, how species-specific differences in LPS recognition impact host defense remains undefined. Herein, we establish how temperature dependent shifts in the lipid A of Yersinia pestis LPS that differentially impact recognition by mouse versus human TLR4/MD-2 dictate infection susceptibility. When grown at 37°C, Y. pestis LPS is hypo-acylated and less stimulatory to human compared with murine TLR4/MD-2. By contrast, when grown at reduced temperatures, Y. pestis LPS is more acylated, and stimulates cells equally via human and mouse TLR4/MD-2. To investigate how these temperature dependent shifts in LPS impact infection susceptibility, transgenic mice expressing human rather than mouse TLR4/MD-2 were generated. We found the increased susceptibility to Y. pestis for “humanized” TLR4/MD-2 mice directly paralleled blunted inflammatory cytokine production in response to stimulation with purified LPS. By contrast, for other Gram-negative pathogens with highly acylated lipid A including Salmonella enterica or Escherichia coli, infection susceptibility and the response after stimulation with LPS were indistinguishable between mice expressing human or mouse TLR4/MD-2. Thus, Y. pestis exploits temperature-dependent shifts in LPS acylation to selectively evade recognition by human TLR4/MD-2 uncovered with “humanized” TLR4/MD-2 transgenic mice.

Author Summary

The outer leaflet of the outer membrane of Gram-negative bacteria is mainly composed of lipopolysaccharide (LPS, endotoxin). The structure of the bioactive component of LPS, lipid A, varies between bacteria and even within the same species grown under different environmental conditions. Yersinia pestis has been associated with highly lethal bubonic plagues of the past. It alters the structure of its LPS based on temperature. When grown at ambient temperatures comparable to fleas in temperate climates, the LPS is mainly hexa-acylated. However, upon growth at 37°C, the mammalian host temperature, Y. pestis switches to synthesize a hypo-acylated LPS that is less stimulatory to the human compared with murine LPS receptor complex composed of Toll-like receptor (TLR) 4 and MD-2. To test whether the change in LPS structure associated with replication at mammalian temperature promotes Y. pestis virulence by evading recognition by the human receptor complex, we generated “humanized” mice that express human rather than mouse TLR4 and MD-2. We find that these mice are indeed more sensitive to Y. pestis infection than WT mice supporting the notion that evasion of recognition by TLR4/MD-2 promotes Y. pestis virulence in humans.