Bradley R. Borlee

Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix

Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic‐resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic‐di‐GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c‐di‐GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two‐partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220u2003KDa protein that is predicted to be rod‐shaped protein harbouring a β‐helix structural motif. Western analysis indicates that the CdrA is produced as a 220u2003kDa proprotein and processed to 150u2003kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto‐aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co‐immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl‐specific lectin staining suggests that CdrA either cross‐links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural stability. Thus, this study identifies a key protein structural component of the P. aeruginosa EPS matrix.

The Pel Polysaccharide Can Serve a Structural and Protective Role in the Biofilm Matrix of Pseudomonas aeruginosa

Bacterial extracellular polysaccharides are a key constituent of the extracellular matrix material of biofilms. Pseudomonas aeruginosa is a model organism for biofilm studies and produces three extracellular polysaccharides that have been implicated in biofilm development, alginate, Psl and Pel. Significant work has been conducted on the roles of alginate and Psl in biofilm development, however we know little regarding Pel. In this study, we demonstrate that Pel can serve two functions in biofilms. Using a novel assay involving optical tweezers, we demonstrate that Pel is crucial for maintaining cell-to-cell interactions in a PA14 biofilm, serving as a primary structural scaffold for the community. Deletion of pelB resulted in a severe biofilm deficiency. Interestingly, this effect is strain-specific. Loss of Pel production in the laboratory strain PAO1 resulted in no difference in attachment or biofilm development; instead Psl proved to be the primary structural polysaccharide for biofilm maturity. Furthermore, we demonstrate that Pel plays a second role by enhancing resistance to aminoglycoside antibiotics. This protection occurs only in biofilm populations. We show that expression of the pel gene cluster and PelF protein levels are enhanced during biofilm growth compared to liquid cultures. Thus, we propose that Pel is capable of playing both a structural and a protective role in P. aeruginosa biofilms.

Intracellular Screen To Identify Metagenomic Clones That Induce or Inhibit a Quorum-Sensing Biosensor

ABSTRACT The goal of this study was to design and evaluate a rapid screen to identify metagenomic clones that produce biologically active small molecules. We built metagenomic libraries with DNA from soil on the floodplain of the Tanana River in Alaska. We extracted DNA directly from the soil and cloned it into fosmid and bacterial artificial chromosome vectors, constructing eight metagenomic libraries that contain 53,000 clones with inserts ranging from 1 to 190 kb. To identify clones of interest, we designed a high throughput “intracellular” screen, designated METREX, in which metagenomic DNA is in a host cell containing a biosensor for compounds that induce bacterial quorum sensing. If the metagenomic clone produces a quorum-sensing inducer, the cell produces green fluorescent protein (GFP) and can be identified by fluorescence microscopy or captured by fluorescence-activated cell sorting. Our initial screen identified 11 clones that induce and two that inhibit expression of GFP. The intracellular screen detected quorum-sensing inducers among metagenomic clones that a traditional overlay screen would not. One inducing clone carries a LuxI homologue that directs the synthesis of an N-acyl homoserine lactone quorum-sensing signal molecule. The LuxI homologue has 62% amino acid sequence identity to its closest match in GenBank, AmfI from Pseudomonas fluorescens, and is on a 78-kb insert that contains 67 open reading frames. Another inducing clone carries a gene with homology to homocitrate synthase. Our results demonstrate the power of an intracellular screen to identify functionally active clones and biologically active small molecules in metagenomic libraries.

Self-produced exopolysaccharide is a signal that stimulates biofilm formation in Pseudomonas aeruginosa

Bacteria have a tendency to attach to surfaces and grow as structured communities called biofilms. Chronic biofilm infections are a problem because they tend to resist antibiotic treatment and are difficult to eradicate. Bacterial biofilms have an extracellular matrix that is usually composed of a mixture of polysaccharides, proteins, and nucleic acids. This matrix has long been assumed to play a passive structural and protective role for resident biofilm cells. Here we show that this view is an oversimplification and that the biofilm matrix can play an active role in stimulating its own synthesis. Working with the model biofilm bacterium Pseudomonas aeruginosa, we found that Psl, a major biofilm matrix polysaccharide for this species, acts as a signal to stimulate two diguanylate cyclases, SiaD and SadC, to produce the intracellular secondary messenger molecule c-di-GMP. Elevated intracellular concentrations of c-di-GMP then lead to the increased production of Psl and other components of the biofilm. This mechanism represents a unique positive feedback regulatory circuit, where the expression of an extracellular polysaccharide promotes biofilm growth in a manner analogous to autocrine signaling in eukaryotes.

Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa.

ABSTRACT The increased tolerance toward the host immune system and antibiotics displayed by biofilm-forming Pseudomonas aeruginosa and other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP in P. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsive cdrA promoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP in P. aeruginosa strains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment of P. aeruginosa with the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology of P. aeruginosa.

Signal Mimics Derived from a Metagenomic Analysis of the Gypsy Moth Gut Microbiota

ABSTRACT Bacterial signaling is an important part of community life, but little is known about the signal transduction pathways of the as-yet-uncultured members of microbial communities. To address this gap, we aimed to identify genes directing the synthesis of signals in uncultured bacteria associated with the midguts of gypsy moth larvae. We constructed a metagenomic library consisting of DNA extracted directly from the midgut microbiota and analyzed it using an intracellular screen designated METREX, which detects inducers of quorum sensing. In this screen, the metagenomic DNA and a biosensor reside in the same cell. The biosensor consists of a quorum-sensing promoter, which requires an acylhomoserine lactone or other small molecule ligand for activation, driving the expression of the reporter gene gfp. We identified an active metagenomic clone encoding a monooxygenase homologue that mediates a pathway of indole oxidation that leads to the production of a quorum-sensing inducing compound. The signal from this clone induces the activities of LuxR from Vibrio fischeri and CviR from Chromobacterium violaceum. This study is the first to identify a new structural class of quorum-sensing inducer from uncultured bacteria.

Identification of Synthetic Inducers and Inhibitors of the Quorum-Sensing Regulator LasR in Pseudomonas aeruginosa by High-Throughput Screening

ABSTRACT We report the screening of 16,000 synthetic compounds for induction and inhibition of quorum sensing in a Pseudomonas putida N-acylated l-homoserine lactone (AHL) sensor strain engineered with the LasR transcriptional activator. LasR controls virulence gene expression in the opportunistic pathogen Pseudomonas aeruginosa and is of significant interest as a therapeutic target. Nine compounds that inhibit and 14 compounds that induce LasR activity were identified in our high-throughput screen.

Quorum-sensing signals in the microbial community of the cabbage white butterfly larval midgut

The overall goal of this study was to examine the role of quorum-sensing (QS) signals in a multispecies microbial community. Toward this aim, we studied QS signals produced by an indigenous member and an invading pathogen of the microbial community of the cabbage white butterfly (CWB) larval midgut (Pieris rapae). As an initial step, we characterized the QS system in Pantoea CWB304, which was isolated from the larval midgut. A luxI homolog, designated panI, is necessary for the production of N-acyl-L-homoserine lactones (AHLs) by Pantoea CWB304. To determine whether AHL signals are exchanged in the alkaline environment of the midgut, we constructed AHL-sensing bioluminescent reporter strains in Pantoea CWB304 and a panI mutant of this strain. In the gut of the CWB larvae, the reporter in an AHL-deficient Pantoea CWB304 detected AHLs when coinoculated with the wild type. To study the role of AHL signals produced by a community invader, we examined pathogenesis of Pseudomonas aeruginosa PAO1 in CWB larvae. Mortality induced by P. aeruginosa PAO1 was significantly reduced when signaling was interrupted by either a potent chemical inhibitor of QS or mutations in the lasI and rhlI AHL synthases of P. aeruginosa PAO1. These results show that AHLs are exchanged among bacteria in the alkaline gut of CWB larvae and contribute to disease caused by P. aeruginosa PAO1.

Different methods for culturing biofilms in vitro

The field of biofilm microbiology, while by no means new, has been experiencing significant “growing pains” as more and more researchers become involved. One of the underlying reasons is the lack of standardized methods for culturing biofilm communities. Many times, the culturing format will be unique to the study in question, resulting in difficulties when other labs attempt to confirm results produced by another lab. Another issue has been the limited utility of different culturing methods for the specific research questions being asked. For example, culturing formats designed to be accessible to microscopy are not always suited for other types of analyses, such as harvesting biofilm biomass for biochemical measurements.

Spatial transcriptomes within the Pseudomonas aeruginosa biofilm architecture: P. aeruginosa spatial transcriptomes

Bacterial cooperative associations and dynamics in biofilm microenvironments are of special interest in recent years. Knowledge of localized gene‐expression and corresponding bacterial behaviors within the biofilm architecture at a global scale has been limited, due to a lack of robust technology to study limited number of cells in stratified layers of biofilms. With our recent pioneering developments in single bacterial cell transcriptomic analysis technology, we generated herein an unprecedented spatial transcriptome map of the mature in vitro Pseudomonas aeruginosa biofilm model, revealing contemporaneous yet altered bacterial behaviors at different layers within the biofilm architecture (i.e., surface, middle and interior of the biofilm). Many genes encoding unknown functions were highly expressed at the biofilm‐solid interphase, exposing a critical gap in the knowledge of their activities that may be unique to this interior niche. Several genes of unknown functions are critical for biofilm formation. The in vivo importance of these unknown proteins was validated in invertebrate (fruit fly) and vertebrate (mouse) models. We envisage the future value of this report to the community, in aiding the further pathophysiological understanding of P. aeruginosa biofilms. Our approach will open doors to the study of bacterial functional genomics of different species in numerous settings.