Steven D. Bowden

Quorum Sensing in Gram-Negative Bacteria: Small-Molecule Modulation of AHL and AI-2 Quorum Sensing Pathways

Numerous species of bacteria employ a mechanism of intercellular communication known as quorum sensing. This signaling process allows the cells comprising a bacterial colony to coordinate their gene expression in a cell-density dependent manner.1-3 Quorum sensing is mediated by small diffusible molecules termed autoinducers that are synthesized intracellularly (throughout the growth of the bacteria) and released into the surrounding milieu. As the number of cells in a bacterial colony increases, so does the extracellular concentration of the autoinducer. Once a threshold concentration is reached (at which point the population is considered to be “quorate”), productive binding of the autoinducer to cognate receptors within the bacterial cells occurs, triggering a signal transduction cascade that results in population-wide changes in gene expression.4-6 Thus, quorum sensing enables the cells within a bacterial colony to act cooperatively, facilitating population-dependent adaptive behavior.6 Quorum sensing has been shown to play a critical role in both pathogenic and symbiotic bacteria-host interactions.5 In symbionts, significant quorum sensing phenotypes include bioluminescence and root nodulation.7-11 Several clinically relevant pathogens use quorum sensing systems to regulate processes associated with virulence; this enhances the survival prospects of the bacteria because a coordinated attack on the host is only made when the bacterial population reaches a high population density, increasing the likelihood that the hosts defenses will be successfully overwhelmed.12,13 For example, in Pseudomonas aeruginosa, quorum sensing is involved in the formation of biofilms and their tolerance to antimicrobial agents14-17 and the innate host immune * To whom correspondence should be addressed. Tel.: +44 (0)1223 336498. Fax: +44 (0)1223 336362. E-mail: spring@ch.cam.ac.uk. † Department of Chemistry. ‡ Department of Biochemistry. Chem. Rev. 2011, 111, 28–67 28

Applications of small molecule activators and inhibitors of quorum sensing in Gram-negative bacteria

Quorum sensing is a form of intercellular communication used by many species of bacteria that facilitates concerted interactions between the cells comprising a population. The phenotypes regulated by quorum sensing are extremely diverse, with many having a significant impact upon healthcare, agriculture, and the environment. Consequently there has been significant interest in developing methods to manipulate this signalling process and recent years have witnessed significant theoretical and practical developments. A wide range of small molecule modulators of quorum sensing systems has been discovered, providing an expansive chemical toolbox for the study and modulation of this signalling mechanism. In this review, a selection of recent case studies which illustrate the value of both activators and inhibitors of quorum sensing in Gram-negative bacteria are discussed.

Quorum sensing, virulence and secondary metabolite production in plant soft-rotting bacteria

Quorum sensing describes the ability of bacteria to sense their population density and respond by modulating gene expression. In the plant soft-rotting bacteria, such as Erwinia, an arsenal of plant cell wall-degrading enzymes is produced in a cell density-dependent manner, which causes maceration of plant tissue. However, quorum sensing is central not only to controlling the production of such destructive enzymes, but also to the control of a number of other virulence determinants and secondary metabolites. Erwinia synthesizes both N-acylhomoserine lactone (AHL) and autoinducer-2 types of quorum sensing signal, which both play a role in regulating gene expression in the phytopathogen. We review the models for AHL-based regulation of carbapenem antibiotic production in Erwinia. We also discuss the importance of quorum sensing in the production and secretion of virulence determinants by Erwinia, and its interplay with other regulatory systems.

Glucose and glycolysis are required for the successful infection of macrophages and mice by Salmonella enterica serovar Typhimurium

ABSTRACT Salmonella is a widespread zoonotic enteropathogen that causes gastroenteritis and fatal typhoidal disease in mammals. During systemic infection of mice, Salmonella enterica serovar Typhimurium resides and replicates in macrophages within the “Salmonella-containing vacuole” (SCV). It is surprising that the substrates and metabolic pathways necessary for growth of S. Typhimurium within the SCV of macrophages have not been identified yet. To determine whether S. Typhimurium utilized sugars within the SCV, we constructed a series of S. Typhimurium mutants that lacked genes involved in sugar transport and catabolism and tested them for replication in mice and macrophages. These mutants included a mutant with a mutation in the pfkAB-encoded phosphofructokinase, which catalyzes a key committing step in glycolysis. We discovered that a pfkAB mutant is severely attenuated for replication and survival within RAW 264.7 macrophages. We also show that disruption of the phosphoenolpyruvate:carbohydrate phosphotransferase system by deletion of the ptsHI and crr genes reduces S. Typhimurium replication within RAW 264.7 macrophages. We discovered that mutants unable to catabolize glucose due to deletion of ptsHI, crr, and glk or deletion of ptsG, manXYZ, and glk showed reduced replication within RAW 264.7 macrophages. This study proves that S. Typhimurium requires glycolysis for infection of mice and macrophages and that transport of glucose is required for replication within macrophages.

Structure-Activity Analysis of the Pseudomonas Quinolone Signal Molecule

We synthesized a range of PQS (Pseudomonas quinolone signal; 2-heptyl-3-hydroxy-4(1H)-quinolone) analogues and tested them for their ability to stimulate MvfR-dependent pqsA transcription, MvfR-independent pyoverdine production, and membrane vesicle production. The structure-activity profile of the PQS analogues was different for each of these phenotypes. Certain inactive PQS analogues were also found to strongly synergize PQS-dependent pyoverdine production.

An incomplete TCA cycle increases survival of Salmonella Typhimurium during infection of resting and activated murine macrophages.

Background In comparison to the comprehensive analyses performed on virulence gene expression, regulation and action, the intracellular metabolism of Salmonella during infection is a relatively under-studied area. We investigated the role of the tricarboxylic acid (TCA) cycle in the intracellular replication of Salmonella Typhimurium in resting and activated macrophages, epithelial cells, and during infection of mice. Methodology/Principal Findings We constructed deletion mutations of 5 TCA cycle genes in S. Typhimurium including gltA, mdh, sdhCDAB, sucAB, and sucCD. We found that the mutants exhibited increased net intracellular replication in resting and activated murine macrophages compared to the wild-type. In contrast, an epithelial cell infection model showed that the S. Typhimurium ΔsucCD and ΔgltA strains had reduced net intracellular replication compared to the wild-type. The glyoxylate shunt was not responsible for the net increased replication of the TCA cycle mutants within resting macrophages. We also confirmed that, in a murine infection model, the S. Typhimurium ΔsucAB and ΔsucCD strains are attenuated for virulence. Conclusions/Significance Our results suggest that disruption of the TCA cycle increases the ability of S. Typhimurium to survive within resting and activated murine macrophages. In contrast, epithelial cells are non-phagocytic cells and unlike macrophages cannot mount an oxidative and nitrosative defence response against pathogens; our results show that in HeLa cells the S. Typhimurium TCA cycle mutant strains show reduced or no change in intracellular levels compared to the wild-type [1]. The attenuation of the S. Typhimurium ΔsucAB and ΔsucCD mutants in mice, compared to their increased net intracellular replication in resting and activated macrophages suggest that Salmonella may encounter environments within the host where a complete TCA cycle is advantageous.

Virulence in Pectobacterium atrosepticum is regulated by a coincidence circuit involving quorum sensing and the stress alarmone, (p)ppGpp

Pectobacterium atrosepticum (Pca) is a Gram‐negative phytopathogen which causes disease by secreting plant cell wall degrading exoenzymes (PCWDEs). Previous studies have shown that PCWDE production is regulated by (i) the intercellular quorum sensing (QS) signal molecule, 3‐oxo‐hexanoyl‐l‐homoserine lactone (OHHL), and (ii) the intracellular ‘alarmone’, (p)ppGpp, which reports on nutrient limitation. Here we show that these two signals form an integrated coincidence circuit which ensures that metabolically costly PCWDE synthesis does not occur unless the population is simultaneously quorate and nutrient limited. A (p)ppGpp null ΔrelAΔspoT mutant was defective in both OHHL and PCWDE production, and nutritional supplementation of wild type cultures (which suppresses (p)ppGpp production) also suppressed OHHL and PCWDE production. There was a substantial overlap in the transcriptome of a (p)ppGpp deficient relA mutant and of a QS defective expI (OHHL synthase) mutant, especially with regards to virulence‐associated genes. Random transposon mutagenesis revealed that disruption of rsmA was sufficient to restore PCWDE production in the (p)ppGpp null strain. We found that the ratio of RsmA protein to its RNA antagonist, rsmB, was modulated independently by (p)ppGpp and QS. While QS predominantly controlled virulence by modulating RsmA levels, (p)ppGpp exerted regulation through the modulation of the RsmA antagonist, rsmB.

Intra-Species Bacterial Quorum Sensing Studied at Single Cell Level in a Double Droplet Trapping System

In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP) from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets.

Nutritional and metabolic requirements for the infection of HeLa cells by Salmonella enterica serovar Typhimurium

Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food - and water - borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic genes, pfkA and pfkB to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is a major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells.

Novel and Efficient Copper‐Catalysed Synthesis of Nitrogen‐Linked Medium‐Ring Biaryls

Herein, a new copper-catalysed strategy for the synthesis of rare nitrogen-linked seven-, eight- and nine-membered biaryl ring systems is described. It is proposed that the reaction proceeds through a highly activated intramolecularly co-ordinated copper catalyst. The process is technically simple, proceeds under relatively mild conditions, displays a broad substrate scope and forms biologically valuable products that are difficult to synthesise by other methods. We envisage that this methodology will prove useful in a wide synthetic context, with possible applications in both target-oriented and diversity-oriented synthesis.