Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms

Jules D P Valentin,Jeremy S Webb,Christian H Ahrens,Marion Lemare,Franziska Pietsch,Henny C Van Der Mei,Frank Schreiber,Qun Ren,Hervé Straub

doi:10.1038/s41396-021-01157-9

Abstract

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.

Highlights

Infections caused by the opportunistic pathogen Pseudomonas aeruginosa are among the most prevalent and difficult to treat [1, 2]
This study shows that repression of the flgE gene in biofilms and likely in cystic fibrosis (CF) lung infections alters the structure of P. aeruginosa biofilms, promoting bacterial antibiotic tolerance
The transposon mutants fiuA and arnB were selected as reference strains for biofilm formation instead of P. aeruginosa MPAO1 wild type (WT), to eliminate the bias caused by the presence of the Tn5 transposon [37]

Summary

Introduction

Infections caused by the opportunistic pathogen Pseudomonas aeruginosa are among the most prevalent and difficult to treat [1, 2]. Biofilms are known to promote the survival of bacteria under harsh environmental conditions, and to protect bacterial cells from immune responses and antibiotic treatment [4, 5]. Known to be influenced by chemotaxis [14], bacterial motility [15], and environmental parameters [12, 16, 17], the stalks of mushroom-like structures were shown to be initiated by proliferation of non-motile bacteria, while the caps were formed after climbing and aggregation of a motile subpopulation [15]. Among the repressed genes involved in motility, the flgE (PA1080) gene encodes for the flagellar hook, a curved structure at the basis of the flagellum [25]. FlgE is known to be proinflammatory [29, 30] and has been found to be absent from the membranes of P. aeruginosa PAK cells grown in mucus [31]

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Conclusion