Molecular basis of in vivo biofilm formation by bacterial pathogens

Abstract

Bacterial biofilms are involved in a multitude of serious chronic infections. In recent years, modeling of biofilm infection in vitro has led to the identification of microbial determinants that govern biofilm development. However, we lack information as to whether the biofilm formation mechanisms identified in vitro have relevance for biofilm-associated infection. Here, we discuss the molecular basis of biofilm formation. Staphylococci and Pseudomonas aeruginosa are used to illustrate key points because their biofilm development process has been well studied. We focus on in vivo findings, such as obtained in animal infection models, and critically evaluate the in vivo relevance of in vitro findings. Although conflicting results about the role of quorum sensing in biofilm formation have been obtained, we argue that integration of in vitro and in vivo studies allows a differentiated view of this mechanism as it relates to biofilm infection.

Fig. 1. Phases of in-vivo biofilm development

Biofilms develop via initial attachment, which depends on transport of the bacteria to a surface, which is passive in the case of non-motile bacteria such as staphylococci (yellow), and active in the case of motile bacteria such as P. aeruginosa (red). Attachment itself is governed by specific protein-protein interactions of bacterial surface with human matrix proteins. Attachment to an abiotic surface such as a catheter depends on bacterial surface hydrophobicity, but this mechanism is believed to have minor importance in vivo. Subsequent steps do not differ in principle between motile and non-motile bacteria. They involve proliferation, embedding in an extracellular matrix, and maturation. The latter depends on cell-cell disruptive factors, recently identified to be primarily surfactants. Strong production of surfactants, which are controlled by QS, leads to biofilm detachment (dispersal). In the case of motile bacteria, up-regulation of motility, starting in the center of biofilm “mushroom caps” assists dispersal.

Fig. 2. Biofilm exopolysaccharides in P. aeruginosa and staphylococci

The major biofilm exopolysaccharide of staphylococci (and some other bacteria) is PIA (or PNAG), a homopolymer of beta-1,6-linked N-acetyl-glucosamine residues, of which about a quarter become de-acetylated after export. De-acetylation creates free amino groups, which at neutral or acid pH give the molecule a cationic character (shown in blue). Major exopolysaccharides of P. aeruginosa are the mannuronic acid/guluronic acid-based, negatively charged alginate (negative charges, red) and the mannose-rich neutral Psl. GlcNAc, N-acetyl-glucosamine; ManUA, mannuronic acid; GulUA, guluronic acid; Manp, mannopyranose; Rhap; rhamnopyranose; Glcp, glucopryranose.

Fig. 3. Quorum-sensing in staphylococci

QS in staphylococci is exerted by the agr locus, which contains the agrA, agrC, agrD, and agrB genes (RNAII transcript) and RNAIII, the intracellular effector of the system, which also contains the hld gene for the PSM δ-toxin. AgrD is a pre-pheromone, which is exported and modified by AgrB, resulting in a characteristic thiolactone-containing autoinducing peptide (AIP). Activation of the AgrC/AgrA two-component system by AIP binding leads to transcription of RNAIII and RNAII, the latter leading to auto feedback and fast up-regulation of agr and agr target expression at a certain threshold of cell density. Agr-regulated biofilm-relevant genes are first and foremost PSMs, which are regulated by direct binding of AgrA to their promoters, rather than via RNAIII. In contrast, many MSCRAMMs are negatively regulated by RNAIII, reflecting that tissue attachment is a mechanism no longer needed during later stages of infection.

Fig. 4. Quorum-sensing in P. aeruginosa

P. aeruginosa uses at least 3 QS systems, which are arranged in hierarchical order. The rhl system is under control of the las system; both use an acyl homoserine lactone (AHL) signal that is produced by the LasI or RhlI AHL synthetases, respectively. Target genes are under control of the DNA-binding regulators LasR, RhlR, and QscR, defining the respective QS regulons. QS-characteristic auto feedback loops are established by the fact that the AHL synthetase genes are under control of the corresponding regulator proteins. The qsc system responds to, but does not produce AHLs. The QscR DNA-binding protein, in addition to controlling production of the qsc’s systems target genes, inhibits expression of the AHL-producing LasI and RhlI enzymes.

Fig. 5. Role of quorum-sensing in biofilm-associated infection

QS systems (such as the staphylococcal Agr shown here) contribute to maturation and dispersal of biofilms. Accordingly, biofilms of an Agr QS wild-type strain, as shown by CLSM in the middle, contain channels between cellular agglomerations. Active expression of the QS system (as shown on the top right in green, using an agr promoter gfp fusion construct) leads to dispersal. During prolonged chronic infection, the QS system in biofilms cells may be irreversibly inactivated by mutation, leading to excessive growth of compact biofilms, which likely have lost the capacity to disperse and disseminate. The phenotype of a surfactant mutant, in which all psm genes controlled by Agr have been inactivated (bottom right), has the same phenotype as the agr QS mutant (bottom left), underlining the importance of surfactants in QS-mediated control of biofilm maturation and detachment.