Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

Abstract

Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation.

Figure 1

The assembly of the type I pilus. The periplasmic protein FimC binds secreted pilus subunits, from the SecYEG translocon based in the internal membrane, to the periplasm. A process of accelerated subunit folding by FimC (periplasmic chaperone) occurs, followed by delivery to the usher outer assembly platform FimD, also performed by FimC. These FimC-subunit complexes are recognized and bind to the N-terminal domain of the usher: FimD_N. Uncomplexed FimC is then released to the periplasm when subunits are assembled into the pilus. The tip of the pilus (fibrillum) consists of the protein adhesins FimF, FimG, and FimH, with FimA forming the bulk of the pilus rod. Adapted from Capitani, 2006 [37].

Figure 2

Structure of Escherichia coli Type P pili encompassing the PapG unit containing galabiose specific receptors (Gal(α1–4)Gal-) for attachment to urinary tract tissue. The pilus is anchored to the membrane by PapH, whose location is yet to be characterized fully but has been hypothesized by Verger and colleagues to terminate the pilus structure at the base as shown, allowing anchoring to the membrane [46]. Type P pili subunits enter the periplasm by the Sec transport system. In the presence of PapD, stable chaperone-subunit complexes are formed via attachment to the hydrophobic C-terminus of pili subunits [47]. PapD acts as the chaperone to assemble and deliver pili subunits to the outer membrane usher PapC. PapC is a pore forming protein that facilitates pilus assembly by creating a narrow channel across the outer membrane. Assembly of subunits from the outer membrane PapC occurs through a donor strand exchange mechanism. PapA forms a tightly wound helix fiber on the external cell and provides a driving force for the translocation of pili subunits across the outer membrane, facilitating outward pilus growth [48]. Adapted from Mu and Bullitt, 2006 [48] and Mu, 2005 [49].

Figure 3

The synthesis and polymerization mechanism involved in the production of Pseudomonas aeruginosa alginate. The letters A–X and numbers 8 and 44 correlate to alginate biosynthetic enzymes that are preceded by Alg (for example A = AlgA). AlgA, AlgC, and AlgD control the production of the alginate precursor GDP-mannuronic acid. Both Alg8 and Alg44 transport this molecule for polymerization in the periplasm. Alginate lysase (AlgL) produces short oligomers that prime polymerization. AlgG interacts with AlgK and AlgX protecting the production of the alginate polymer by forming a scaffold in the periplasm. Epimerization of polymerized mannuronate residues is also controlled by AlgG, a C-5-epimerase. Acetylation of some mannuronate residues occurs via the enzymes AlgF, AlgJ, and AlgI at O2 and/or O3 positions, with AlgE transporting the formed alginate out of the cell. Adapted from Franklin and Ohman 2002 [122], Ramsey 2005 [125], and Gimmestad, 2003 [126].

Figure 4

The las, pqs, and rhl interlinked quorum sensing systems in Pseudomonas aeruginosa. The las system consists of the proteins LasR (transcriptional activator) and LasI (synthase enzyme) coded for by lasR and lasI respectively. The rhl system consists of RhlR (transcriptional activator) and RhlI (synthase enzyme) coded for by rhlR and rhlI. Adapted from Raina, 2009 [177].

Figure 5

Summary of the LuxS Quorum sensing system of Escherichia coli. Autoinducer-II, represented by pentagons, is formed from a LuxS catalyzed cleavage reaction of S-ribosylhomocysteine to 4,5-dihydroxy 2,3-pentanedione and homocysteine [213]. Key: AIP-II: Autoinducer-II: DPD: 4,5-dihydroxy-2,3-pentanedione. Adapted from Li, 2007 [184].