Modification of the structure and activity of lipid A in Yersinia pestis lipopolysaccharide by growth temperature

Abstract

Yersinia pestis strain Yreka was grown at 27 or 37 degrees C, and the lipid A structures (lipid A-27 degrees C and lipid A-37 degrees C) of the respective lipopolysaccharides (LPS) were investigated by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Lipid A-27 degrees C consisted of a mixture of tri-acyl, tetra-acyl, penta-acyl, and hexa-acyl lipid A's, of which tetra-acyl lipid A was most abundant. Lipid A-37 degrees C consisted predominantly of tri- and tetra-acylated molecules, with only small amounts of penta-acyl lipid A; no hexa-acyl lipid A was detected. Furthermore, the amount of 4-amino-arabinose was substantially higher in lipid A-27 degrees C than in lipid A-37 degrees C. By use of mouse and human macrophage cell lines, the biological activities of the LPS and lipid A preparations were measured via their abilities to induce production of tumor necrosis factor alpha (TNF-alpha). In both cell lines the LPS and the lipid A from bacteria grown at 27 degrees C were stronger inducers of TNF-alpha than those from bacteria grown at 37 degrees C. However, the difference in activity was more prominent in human macrophage cells. These results suggest that in order to reduce the activation of human macrophages, it may be more advantageous for Y. pestis to produce less-acylated lipid A at 37 degrees C.

FIG. 1.

SDS-PAGE of LPS from Y. pestis strain Yreka grown at 27 and 37°C. Lane 1, Salmonella serovar Abortus-equi (1 μg); lane 2, Salmonella serovar Minnesota R60 (0.2 μg); lane 3, Salmonella serovar Minnesota R595 (0.2 μg); lane 4, Y. pestis grown at 27°C (0.2 μg); lane 5, Y. pestis grown at 37°C (0.2 μg).

FIG. 2.

MALDI-TOF mass spectra of lipid A from Y. pestis strain Yreka grown at 27°C. (Top) Negative-ion mode. (Bottom) Positive-ion mode.

FIG. 3.

MALDI-TOF mass spectra of lipid A from Y. pestis strain Yreka grown at 37°C. (Top) Negative-ion mode. (Bottom) Positive-ion mode.

FIG. 4.

Structural modification of Y. pestis lipid A by growth temperature. In this figure the structure of the lipid A backbone is shown in analogy to other published structures (2, 39), although it was not precisely determined in this study. The positions of C_16:1 and C_12:0 may be interchanged. The second Ara4N detected by MALDI-TOF mass spectrometry as a minor component is not shown here.

FIG. 5.

TNF-inducing activities of lipid A and LPS from Y. pestis strain Yreka in RAW 264.7 murine macrophages. (A) RAW 264.7 cells were cultured in the presence of the indicated concentrations of synthetic lipid A 506 (▴), Y. pestis lipid A grown at 27°C (□), or Y. pestis lipid A grown at 37°C (•). (B) RAW 264.7 cells were cultured in the presence of the indicated concentrations of Salmonella serovar Abortus-equi LPS (▴), LPS of Y. pestis grown at 27°C (□), and LPS of Y. pestis grown at 37°C (•). Culture supernatants obtained at 24 h were assayed for determination of TNF-α activity. Data are means ± standard errors of the means from triplicate samples. Results of one experiment representative of three independent experiments are shown.

FIG. 6.

TNF-inducing activities of lipid A and LPS of Y. pestis strain Yreka in U937 human macrophages. U937 cells were cultured in the presence of the indicated concentrations of lipid A's (A) or LPSs (B). Symbols in panels A and B are as explained for the corresponding panels of Fig. 5. Culture supernatants were obtained at 4 h and assayed for TNF-α activity. Data are means ± standard errors of the means from triplicate samples. Results of one experiment representative of three independent experiments are shown.