The lipid A phosphate position determines differential host Toll-like receptor 4 responses to phylogenetically related symbiotic and pathogenic bacteria

Abstract

The human symbiont Bacteroides thetaiotaomicron promotes intestinal function and health, whereas the phylogenetically related pathogen Porphyromonas gingivalis is associated with the chronic oral inflammatory disease periodontitis. Although both B. thetaiotaomicron and P. gingivalis synthesize lipopolysaccharides (LPS) consisting of penta-acylated, monophosphorylated lipid A in addition to immunologically silent, nonphosphorylated lipid A, they elicit strikingly distinct Toll-like receptor 4 (TLR4) responses. We show that the phosphate position of penta-acylated, monophosphorylated lipid A is a key feature for determining the differential TLR4 responses elicited by these evolutionarily related bacteria. B. thetaiotaomicron produces TLR4-stimulatory lipid A bearing a 1-phosphate, in contrast to P. gingivalis, which produces TLR4-evasive lipid A bearing a 4'-phosphate. Confirming these observations, recombinant Escherichia coli LPS containing penta-acylated, 1-phosphorylated lipid A is more TLR4 stimulatory than LPS containing 4'-phosphorylated lipid A. The specific capacity of a Gram-negative bacterium to alert or evade the host innate immune defense system through TLR4-dependent signaling is currently recognized as a critical aspect defining the relationship between the host and the bacterium. We propose that the distinct lipid A phosphate positions observed for the B. thetaiotaomicron and P. gingivalis LPS contributes to the manifestation of these bacteria as commensal or pathogen within the human host.

FIG. 1.

B. thetaiotaomicron LPS and P. gingivalis LPS contain penta-acylated, monophosphorylated lipid A structures, as well as nonphosphorylated lipid A structures. (A) Negative-ion mode MALDI-TOF MS analysis of lipid A derived from B. thetaiotaomicron LPS. (B) Negative-ion mode MALDI-TOF MS analysis of lipid A derived from P. gingivalis 1626KO LPS. (C) Positive-ion mode MALDI-TOF MS analysis of lipid A derived from B. thetaiotaomicron LPS. (D) Positive-ion mode MALDI-TOF MS analysis of lipid A derived from P. gingivalis 1626KO LPS.

FIG. 2.

B. thetaiotaomicron and P. gingivalis produce differentially phosphorylated penta-acylated lipid A, as determined by negative-ion mode MALDI-TOF/TOF tandem MS spectra. (A and B) Analyses of a penta-acylated, 1-phosphorylated B. thetaiotaomicron lipid A (m/z 1,688) (A) and a penta-acylated, 4′-phosphorylated P. gingivalis 1626KO lipid A (m/z 1,688) (B). The inset cartoon structures illustrate the differentially located phosphate positions on the respective lipid A backbones, as determined by the presence or absence of A₂-type ion fragments.

FIG. 3.

The different abilities of B. thetaiotaomicron (Bt) and P. gingivalis (Pg) to elicit robust innate immune recognition are determined by the positions of the phosphate present on penta-acylated, monophosphorylated lipid A. (A) B. thetaiotaomicron LPS is more potent than either P. gingivalis 1626KO LPS or P. gingivalis 1587KO LPS in stimulating TLR4-dependent NF-κB activation in HEK cells. (B) Intact B. thetaiotaomicron bacteria are more potent than either intact P. gingivalis 1626KO bacteria or P. gingivalis 1587KO bacteria in stimulating TLR4-dependent NF-κB activation in HEK cells. NF-κB activation was determined by measurement of firefly luciferase activity, and the results were plotted as the mean fold induction (±standard deviation [SD]) of triplicate determinations relative to the unstimulated control. The asterisks represent significant differences between either B. thetaiotaomicron LPS and P. gingivalis 1587KO LPS or B. thetaiotaomicron and P. gingivalis 1587KO bacteria (P < 0.05; unpaired Student t tests). hTLR4, human TLR4; hMD-2, human MD-2.

FIG. 4.

E. coli msbB bacteria transformed with plasmids expressing F. novicida lipid A phosphatase (LpxE or LpxF) produce penta-acylated, monophosphorylated lipid A structures. (A) Negative-ion mode MALDI-TOF MS analysis of lipid A derived from E. coli msbB bacteria expressing LpxE. (B) Negative-ion mode MALDI-TOF MS analysis of lipid A derived from E. coli msbB bacteria expressing LpxF.

FIG. 5.

E. coli msbB bacteria expressing either LpxE or LpxF produce differentially phosphorylated penta-acylated lipid A, as determined by negative-ion mode MALDI-TOF/TOF tandem MS spectra. (A and B) Analyses of a penta-acylated, 4′-phosphorylated E. coli msbB LpxE lipid A (m/z 1,506) (A) and a penta-acylated, 1-phosphorylated E. coli msbB LpxF lipid A (m/z 1,506) (B). The inset cartoon structures illustrate the differentially located phosphate positions on the respective lipid A backbones, as determined by the presence or absence of A₂-type ion fragments.

FIG. 6.

E. coli (Ec) msbB LPS consisting of either penta-acylated, 1-phosphorylated or 4′-phosphorylated lipid A structures differentially activate the innate immune system. E. coli msbB LpxF LPS containing 1-phosphorylated lipid A is more potent than E. coli msbB LpxE LPS bearing 4′-phosphorylated lipid A in stimulating TLR4-dependent NF-κB activation in HEK cells. NF-κB activation was determined by measurement of firefly luciferase activity, and the results were plotted as the mean fold induction (±SD) of triplicate determinations relative to the unstimulated control. The asterisks represent significant differences between E. coli msbB LpxE LPS and E. coli msbB LpxF LPS (P < 0.05; unpaired Student t tests).