LPS remodeling is an evolved survival strategy for bacteria

Abstract

Maintenance of membrane function is essential and regulated at the genomic, transcriptional, and translational levels. Bacterial pathogens have a variety of mechanisms to adapt their membrane in response to transmission between environment, vector, and human host. Using a well-characterized model of lipid A diversification (Francisella), we demonstrate temperature-regulated membrane remodeling directed by multiple alleles of the lipid A-modifying N-acyltransferase enzyme, LpxD. Structural analysis of the lipid A at environmental and host temperatures revealed that the LpxD1 enzyme added a 3-OH C18 acyl group at 37 °C (host), whereas the LpxD2 enzyme added a 3-OH C16 acyl group at 18 °C (environment). Mutational analysis of either of the individual Francisella lpxD genes altered outer membrane (OM) permeability, antimicrobial peptide, and antibiotic susceptibility, whereas only the lpxD1-null mutant was attenuated in mice and subsequently exhibited protection against a lethal WT challenge. Additionally, growth-temperature analysis revealed transcriptional control of the lpxD genes and posttranslational control of the LpxD1 and LpxD2 enzymatic activities. These results suggest a direct mechanism for LPS/lipid A-level modifications resulting in alterations of membrane fluidity, as well as integrity and may represent a general paradigm for bacterial membrane adaptation and virulence-state adaptation.

Fig. 1.

Characterization of temperature-regulated structural modifications of Fn lipid A by negative ion MALDI-TOF MS. (A) MS from Fn grown at 37 °C; dominant lipid A at m/z 1665. (B) MS from Fn grown at 25 °C; dominant lipid A at m/z 1637. (C) MS from Fn grown at 18 °C; dominant lipid A at m/z 1609. (D) Acyl group frequency () for 2 and 2′ positions of Fn grown at 18 °C (black), 25 °C (gray), and 37 °C (white) (n = 2). (E) Percentage of lipid A species isolated at 18 °C. Red diamond, m/z 1609; blue circle, m/z 1637; green triangle, m/z 1665. (F) Percentage of lipid A species isolated at 37 °C. (G) Percentage of lipid A species isolated at 18 °C then switched to 37 °C. (H) Percentage of lipid A species isolated at 37 °C then switched to 18 °C. For all data points (E–H) (n = 3 ± SE).

Fig. 2.

Identification and temperature regulation of two N-acyltransferases LpxD1 and LpxD2 in Fn. (A) Lipid A biosynthesis pathway in Fn. (B) Conversion of [α-³²P] UDP-3-O-acyl-GlcN to [α-³²P] UDP-3-O-diacyl-GlcN by LpxD1/2 at 21 °C, 30 °C, and 37 °C. (C) Specific activity of LpxD1/2 at 21 °C, 30 °C, and 37 °C. Substrate conversion was quantified with ImageQuant software and used for the specific assay calculation. For all data points (n = 3 ± SE).

Fig. 3.

Characterization of Fn lipid A from lpxD1-null and lpxD2-null mutants by negative ion MALDI-TOF MS. MS of lipid A from Fn lpxD1-null mutant grown at 18 °C (A), 25 °C (B), and 37 °C (C), respectively. Dominant lipid A peaks were locked at m/z 1609. (D) Acyl group frequency () for 2 and 2′ positions of lpxD1-null Fn grown at 18 °C (black), 25 °C (gray), and 37 °C (white). MS of lipid A from Fn lpxD2-null mutant grown at 18 °C (E), 25 °C (F), and 37 °C (G), respectively. Dominant lipid A was locked at m/z 1665. (H) Acyl group frequency () for 2 and 2′ position of lpxD2-null Fn grown at 18 °C (black), 25 °C (gray), and 37 °C (white). Data shown are representative of two independent analyses (D and H).

Fig. 4.

Virulence assays of lpxD1-null and lpxD2-null Fn mutants in C57BL/6 mice. (A) Mice were inoculated with lpxD1-null, lpxD2-null, and WT Fn by the s.c. route. (B) Mice were inoculated with higher lpxD1-null mutant doses. (C) Mice were inoculated by s.c. injection on day −30 and subsequently challenged with WT Fn on day 0. (D) Mice were inoculated by s.c. injection on day −45 and day −14 and subsequently challenged with WT Fn on day 0. Data are representative of two independent experiments.