Mutational analysis of the essential lipopolysaccharide-transport protein LptH of Pseudomonas aeruginosa to uncover critical oligomerization sites

Alessandra Lo Sciuto,Francesco Imperi,Adele Di Matteo,Antonio Coluccia,Romina Scala,Romano Silvestri,Luca Federici,Carlo Travaglini-Allocatelli,Paolo Visca

doi:10.1038/s41598-020-68054-7

Abstract

Lipopolysaccharide (LPS) is a critical component of the outer membrane (OM) of many Gram-negative bacteria. LPS is translocated to the OM by the LPS transport (Lpt) system. In the human pathogen Pseudomonas aeruginosa, the periplasmic Lpt component, LptH, is essential for LPS transport, planktonic and biofilm growth, OM stability and infectivity. LptH has been proposed to oligomerize and form a protein bridge that accommodates LPS during transport. Based on the known LptH crystal structure, here we predicted by in silico modeling five different sites likely involved in LptH oligomerization. The relevance of these sites for LptH activity was verified through plasmid-mediated expression of site-specific mutant proteins in a P. aeruginosa lptH conditional mutant. Complementation and protein expression analyses provided evidence that all mutated sites are important for LptH activity in vivo. It was observed that the lptH conditional mutant overcomes the lethality of nonfunctional lptH variants through RecA-mediated homologous recombination between the wild-type lptH gene in the genome and mutated copies in the plasmid. Finally, biochemical assays on purified recombinant proteins showed that some LptH variants are indeed specifically impaired in oligomerization, while others appear to have defects in protein folding and/or stability.

Highlights

Lipopolysaccharide (LPS) is a critical component of the outer membrane (OM) of many Gramnegative bacteria
A model has been proposed in which LPS is sequentially pushed from the IM complex LptB2FG to the OM translocon LptDE through a periplasmic hydrophobic groove formed by the periplasmic domains of LptC and LptD connected through the periplasmic protein L ptA3,4,8
While this periplasmic bridge was thought to involve an LptA m ultimer[10,11,12], a recent study showed that an LptA monomeric variant lacking the C-terminal region is still able to support E. coli growth, leading to propose that LptA oligomerization might not be an essential requirement for LPS transport[13]

Summary

Introduction

Lipopolysaccharide (LPS) is a critical component of the outer membrane (OM) of many Gramnegative bacteria. LptH has been proposed to oligomerize and form a protein bridge that accommodates LPS during transport. Based on the known LptH crystal structure, here we predicted by in silico modeling five different sites likely involved in LptH oligomerization The relevance of these sites for LptH activity was verified through plasmidmediated expression of site-specific mutant proteins in a P. aeruginosa lptH conditional mutant. It has been recently demonstrated that a truncated LptA variant, that lacks the entire C-terminal region and that is not able to oligomerize, can partially support E. coli growth[13], suggesting the LptA oligomerization could not be strictly required for LPS transport

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