Peptidoglycan-Binding Anchor Is a Pseudomonas aeruginosa OmpA Family Lipoprotein With Importance for Outer Membrane Vesicles, Biofilms, and the Periplasmic Shape.

Magnus Paulsson,Birendra Singh,Thomas Bjarnsholt,Linda Sandblad,Kristian Riesbeck,Yu-Ching Su,Kasper Nørskov Kragh

doi:10.3389/fmicb.2021.639582

Abstract

The outer membrane protein A (OmpA) family contains an evolutionary conserved domain that links the outer membrane in Gram-negative bacteria to the semi-rigid peptidoglycan (PG) layer. The clinically significant pathogen Pseudomonas aeruginosa carries several OmpA family proteins (OprF, OprL, PA0833, and PA1048) that share the PG-binding domain. These proteins are important for cell morphology, membrane stability, and biofilm and outer membrane vesicle (OMV) formation. In addition to other OmpAs, in silico analysis revealed that the putative outer membrane protein (OMP) with gene locus PA1041 is a lipoprotein with an OmpA domain and, hence, is a potential virulence factor. This study aimed to evaluate PA1041 as a PG-binding protein and describe its effect on the phenotype. Clinical strains were confirmed to contain the lipoprotein resulting from PA1041 expression with Western blot, and PG binding was verified in enzyme-linked immunosorbent assay (ELISA). By using a Sepharose bead-based ELISA, we found that the lipoprotein binds to meso-diaminopimelic acid (mDAP), an amino acid in the pentapeptide portion of PGs. The reference strain PAO1 and the corresponding transposon mutant PW2884 devoid of the lipoprotein were examined for phenotypic changes. Transmission electron microscopy revealed enlarged periplasm spaces near the cellular poles in the mutant. In addition, we observed an increased release of OMV, which could be confirmed by nanoparticle tracking analysis. Importantly, mutants without the lipoprotein produced a thick, but loose and unorganized, biofilm in flow cells. In conclusion, the lipoprotein from gene locus PA1041 tethers the outer membrane to the PG layer, and mutants are viable, but display severe phenotypic changes including disordered biofilm formation. Based upon the phenotype of the P. aeruginosa PW2884 mutant and the function of the protein, we designate the lipoprotein with locus tag PA1041 as “peptidoglycan-binding anchor” (Pba).

Highlights

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that can adapt to a wide range of environments by means of its unusually large genome (Stover et al, 2000)
peptidoglycan-binding anchor” (Pba) Is Expressed in Clinical Strains and Localized in the Outer Membrane In P. aeruginosa, Pba is expressed and translated from gene locus PA1041 as a 210-amino acid polypeptide, forming a 21.7-kDa lipoprotein with a predicted localization in the outer membrane based on the topology prediction software PSORTb V3.0
We present evidence that Pba is a lipoprotein in the outer membrane of P. aeruginosa encoded by the gene locus PA1041

Summary

Introduction

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that can adapt to a wide range of environments by means of its unusually large genome (Stover et al, 2000). It can cause various human infections, including acute and chronic infections of the lungs, keratitis, wound infections, urinary tract infections, and bacteremia. P. aeruginosa infections are often difficult to treat because of the wide-ranging antibiotic resistance and the formation of biofilms. The biofilm protects bacteria from antibiotic drugs by including bacteria at different growth stages and obstructing drug diffusion into the inner cells of the matrix (Evans et al, 1991; Walters et al, 2003)

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