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.

Scala, R., Di Matteo, A., Coluccia, A., Lo Sciuto, A., Federici, L., Travaglini-Allocatelli, C., et al. (2020). Mutational analysis of the essential lipopolysaccharide-transport protein LptH of Pseudomonas aeruginosa to uncover critical oligomerization sites. SCIENTIFIC REPORTS, 10(1), 11276 [10.1038/s41598-020-68054-7].

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

Lo Sciuto A.;Visca P.;Silvestri R.;Imperi F.
2020-01-01

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.
Scala, R., Di Matteo, A., Coluccia, A., Lo Sciuto, A., Federici, L., Travaglini-Allocatelli, C., et al. (2020). Mutational analysis of the essential lipopolysaccharide-transport protein LptH of Pseudomonas aeruginosa to uncover critical oligomerization sites. SCIENTIFIC REPORTS, 10(1), 11276 [10.1038/s41598-020-68054-7].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11590/373163
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