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

Abstract

Lipopolysaccharide (LpS) is a critical component of the outer membrane (oM) of many Gramnegative 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 plasmidmediated 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. The cell envelope of diderm (Gram-negative) bacteria consists of two concentric membranes, the inner (IM) and outer membrane (OM), which confine an aqueous compartment, the periplasmic space, in which a thin layer of peptidoglycan is embedded. While the IM is a typical phospholipids bilayer, the OM of most diderm bacteria is an asymmetric membrane composed of lipopolysaccharide (LPS) and phospholipids in the outer and inner leaflets, respectively 1. LPS is a negatively charged glycolipid that forms a tightly packed layer at the cell surface. The LPS layer is important for the structural stability of the OM, and provides an effective permeability barrier to the entry of potentially noxious compounds 2. LPS is synthesized in the cytoplasm, matured in the periplasm and translocated to the OM by the Lpt (Lipopolysaccharide transport) system that, in the model organism Escherichia coli, is composed of seven essential proteins (LptABCDEFG). The Lpt protein complex spans the entire cell envelope and consists of two subassemblies, LptB 2 CFG at the IM and LptDE at the OM, connected by the periplasmic protein LptA 3-5. LptB 2 FG is an ATP-binding cassette (ABC) transporter that, in association with the bitopic protein LptC, powers LPS transport to the cell surface, while the β-barrel protein LptD and the lipoprotein LptE constitute the OM translocon that inserts LPS into the outer leaflet of the OM 3-5 (Fig. 1A).

RecA and Specialized Error-Prone DNA Polymerases Are Not Required for Mutagenesis and Antibiotic Resistance Induced by Fluoroquinolones in Pseudomonas aeruginosa

“…Deletion of recA in CIP R spontaneous mutants was obtained using the pDM4 ∆recA derivative (Table S1) as described [65]. Single and double deletion mutants in recA, polB, dinB and/or imuC of the reference strain PAO1 (Table S1) were previously generated [65,66].…”

Effect of a Defective Clamp Loader Complex of DNA Polymerase III on Growth and SOS Response in Pseudomonas aeruginosa

“…To obtain the construct for the deletion mutagenesis of ruvCAB genes (pDM4∆ruvCAB), two DNA fragments corresponding to the region upstream of ruvC and the region downstream of ruvB were directionally cloned into pBluescript II KS, checked by sequencing and then subcloned into pDM4. The construct pDM4∆recA has been described elsewhere [29].…”

Virulence factors of human pathogens: an always-needed approach / Fatores de virulência de patógenos humanos: uma abordagem sempre necessária

“…Among the benefits of LPS, the participation of the structural integrity of the outer membrane and the formation of a barrier against the entry of toxic substances stand out (Auer and Weibel 2017;Scala et al 2020). In addition, LPS act to modulate virulence, innate and adaptive host immune responses, as well as bacterial sepsis, which represents one of the most important virulence factors in Gram-negative bacteria (Barakat et al 2019).…”