Lipopolysaccharide is transported to the cell surface by a membrane-to-membrane protein bridge

Abstract

Gram-negative bacteria have an outer membrane that serves as a barrier to noxious agents in the environment. This protective function is dependent on lipopolysaccharide, a large glycolipid located in the outer leaflet of the outer membrane. Lipopolysaccharide is synthesized at the cytoplasmic membrane and must be transported to the cell surface. To understand this transport process, we reconstituted membrane-to-membrane movement of lipopolysaccharide by incorporating purified inner and outer membrane transport complexes into separate proteoliposomes. Transport involved stable association between the inner and outer membrane proteoliposomes. Our results support a model in which lipopolysaccharide molecules are pushed one after the other in a PEZ dispenser-like manner across a protein bridge that connects the inner and outer membranes.

Fig 1. Energy-dependent LPS transport to LptA is stimulated by LptC

(A) Bridge model of LPS biogenesis and chemical structure of Escherichia coli LPS. The E. coli lipopolysaccharide transport proteins and E. coli LPS were used for all experiments presented here. LptBFG extracts LPS from the inner membrane and transports it to LptC using energy from ATP hydrolysis. Additional energy from ATP hydrolysis is harnessed to push LPS from LptC to LptA. Kdo, 3-deoxy-D-manno-octo-2-ulosonic acid; Hep, L-glycero-D-manno-heptose; Glu, D-glucose; Gal, D-galactose. (B) LPS photocrosslinks to LptC in an ATP- and time-dependent manner. Assays were initiated by adding 5 mM ATP or buffer (“- ATP”) to proteoliposomes containing LPS and LptBFGC-T47pBPA. (C) Time-dependence of LPS release to LptA. Assays were initiated by adding 5 mM ATP to proteoliposomes containing LPS and LptBFG, LptB-E163Q-LptFGC, or LptBFGC mixed with soluble LptA-I36pBPA. In (B) and (C), aliquots were taken at the indicated time points and UV-irradiated. Crosslinking was detected by immunoblotting. Cartoons show experimental designs of the reconstituted systems. Proteins and LPS can be inserted into liposomes in either orientation, but only the productive orientation is shown for simplicity. The yellow star denotes the photo-crosslinking amino acid.

Fig 2. Reconstitution of membrane-to-membrane LPS transport

(A) Seven Lpt proteins and ATP are necessary and sufficient to observe LPS crosslinking to LptD. Proteoliposomes containing LptD-Y112pBPA/LptE and associated LptA were incubated with LPS-containing liposomes with or without LptBFGC. Assays were initiated with 5 mM ATP (or buffer). (B) LPS transport to LptD depends on time and ATP concentration. Assays were conducted as in (A), initiating with either 0.5 mM ATP or 5 mM ATP. (C) LPS simultaneously crosslinks to LptC and LptD. Proteoliposomes containing purified LptD-Y112pBPA/LptE with LptA were incubated with proteoliposomes containing LPS and LptBFGC-T47pBPA. In (A)–(C), aliquots were taken at the indicated time points and UV-irradiated. Crosslinking was detected by immunoblotting. Cartoons show experimental designs of the reconstituted systems. Proteins and LPS can be inserted into liposomes in either orientation, but only the productive orientation is shown for simplicity. The yellow star denotes the photo-crosslinking amino acid.

Fig 3. LptA induces the physical association of inner membrane (IM) and outer membrane (OM) proteoliposomes

(A) Schematic of predicted proteoliposome states in the presence or absence of LptA. Proteoliposomes containing LptD-Y112pBPA/LptE were labeled with Atto-488 fluorophore and proteoliposomes containing LptBFGC and LPS (not shown for simplicity) were labeled with Atto-565 fluorophore. (B) Flow cytometric analysis of reaction mixtures containing fluorescent proteoliposomes. Atto-488-labeled proteoliposomes containing LptD-Y112pBPA/LptE with or without pre-incubation with LptA were incubated with Atto-565-labeled proteoliposomes containing LptBFGC and LPS. An N-terminal truncated variant of LptD and an N-terminal blocked LptA were used in separate experiments as controls to substitute the corresponding Lpt components in the reaction mixtures. Samples were incubated as described for crosslinking experiments, initiating with buffer instead of ATP. After incubation, samples were diluted ten-fold and analyzed on a BD FACSAria flow cytometer. Equivalent particle distributions were observed in the presence of ATP. (C) Distribution of particle counts in gated populations shown in (B). Data were normalized such that percentages of counts represent the portion of the total number of counts in all gated sub-populations. Data represent the averages and standard deviation of triplicate experiments.

Fig 4. Observation of a long-lived, protein-mediated bridge using confocal microscopy

(A) Representative confocal microscope images of Population IM and Population OM sorted particles. Atto-488-labeled proteoliposomes containing LptD with associated LptA were incubated with Atto-565-labeled proteoliposomes containing LptBFGC and LPS and were sorted by gating based on fluorescence thresholds using a BD FACSAria flow cytometer, and imaged at 100× magnification. Scale bar: 10 µm. (B) Representative confocal microscope images of Population A and Population. Atto-488-labeled proteoliposomes containing with associated LptA were incubated with Atto-565-labeled proteoliposomes containing LptBFGC and LPS and sorted by gating based on fluorescence thresholds using a BD FACSAria flow cytometer, and imaged at 100× magnification. Scale bar: 10 µm.