Rational design of ‘controller cells’ to manipulate protein and phenotype expression

Coordination between cell populations via prevailing metabolic cues has been noted as a promising approach to connect synthetic devices and drive phenotypic or product outcomes. However, there has been little progress in developing ‘controller cells’ to modulate metabolic cues and guide these systems. In this work, we developed ‘controller cells’ that manipulate the molecular connection between cells by modulating the bacterial signal molecule, autoinducer-2, that is secreted as a quorum sensing (QS) signal by many bacterial species. Specifically, we have engineered Escherichia coli to overexpress components responsible for autoinducer uptake (lsrACDB), phosphorylation (lsrK), and degradation (lsrFG), thereby attenuating cell–cell communication among populations. Further, we developed a simple mathematical model that recapitulates experimental data and characterizes the dynamic balance among the various uptake mechanisms. This study revealed two controller ‘knobs’ that serve to increase AI-2 uptake: overexpression of the AI-2 transporter, LsrACDB, which controls removal of extracellular AI-2, and overexpression of the AI-2 kinase, LsrK, which increases the net uptake rate by limiting secretion of AI-2 back into the extracellular environment. We find that the overexpression of lsrACDBFG results in an extraordinarily high AI-2 uptake rate that is capable of completely silencing QS-mediated gene expression among wild-type cells. We demonstrate utility by modulating naturally occurring processes of chemotaxis and biofilm formation. We envision that ‘controller cells’ that modulate bacterial behavior by manipulating molecular communication, will find use in a variety of applications, particularly those employing natural or synthetic bacterial consortia.     

细菌群体感应及其在食品变质中的作用

食品相关细菌引起的生物被膜形成和食品变质是食品工业中的重大问题。研究表明细菌群体感应(Quorum sensing,QS)与被膜形成、食品腐败变质密切相关。重点对细菌产生的各种QS信号分子及其在被膜形成的作用和被膜在食品工业中的重要性做了介绍。QS信号分子与食品变质密切相关,故对QS抑制剂作为新型食品防腐剂以延长储存期限及加强食品安全的前景进行了概述。     

群体感应信号分子及其抑制剂快速检测方法的建立

细菌能自发产生、释放一些特定的信号分子,并能感知其浓度变化,调节微生物的群体行为,这一调控系统称为群体感应。细菌群体感应参与包括人类、动植物病原菌致病力在内的多种生物学功能的调节,群体感应抑制剂成为抗感染药物开发的靶点。利用紫色色杆菌(Chromobacterium violaceum)和根癌农杆菌(Agrobacterium tumefaciens)作为指示菌,建立检测高丝氨酸内酯(AHLs)及其抑制剂的简便方法。结果表明,通过平板交叉划线接种,使用指示菌能够有效地检测AHLs,并且通过薄层层析(TLC)与细菌生物感应器相结合的方法可以快速、方便地鉴定AHLs的种类;通过双层平板法观察指示菌色素产生情况,能够有效地检测群体感应信号分子AHLs抑制剂,且该方法简单易行。     

Isolation and identification of quorum quenching bacteria from environmental samples

A large number of Gram-negative pathogens produce N-acylhomoserine lactones (AHLs) as signal molecules for quorum sensing (QS). This cell-cell communication system allows them to coordinate gene expression and regulate virulence. Therefore, strategies to inhibit QS are promising for the control of infectious diseases. The aim of the present study was to develop a high-throughput method for the isolation and identification of AHL-degrading bacteria from environmental samples. Samples were cultured in a microtitre plate in a minimal medium containing 1 mM N-(3-oxo-dodecanoyl)-l-homoserine lactone and 2 mM N-(3-oxo-hexanoyl)-l-homoserine lactone as the sole sources of carbon and nitrogen. Isolates growing on this minimal medium were subcultured and identified by partial 16S rRNA gene sequencing. Subsequently, the AHL-degrading capacity of each isolate was evaluated in the Pseudomonas aeruginosa QSIS2 biosensor assay, as such or after treatment with heat or proteinase K. The 16 samples tested yielded a total of 59 isolates which are, either alone or as part of a consortium, able to use AHL signal molecules as sole sources of carbon and nitrogen. Follow-up experiments have shown that in each sample there is at least one isolate with quorum quenching (QQ) activity, and that for all samples combined, 41 isolates have QQ activity. Furthermore, heat treatment did not fully inhibit QQ activity in all isolates. In some isolates, QQ activity was lost after proteinase K treatment, while others remained able to quench QS. Therefore, it is likely that some isolates produce and secrete (a) heat-stable, low molecular weight inhibitory compound(s).     

铜绿假单胞菌群体感应系统研究进展

群体感应系统是一种细胞密度依赖的基因表达系统,其广泛存在于细菌性病原体中,是细菌细胞通讯方式的一种。群体感应系统可利用细菌释放的信号分子不断监控周围细菌的密度。当细菌密度达到阈值时,群体感应系统网络将启动,参与调控生物被膜、细菌毒力等特定基因的表达,从而使临床抗感染治疗失败。而通过抑制群体感应系统,可一定程度上治疗铜绿假单胞菌引起的感染。本文通过查阅近年国内外相关文献,对铜绿假单胞菌群体感应系统研究进展进行总结,为临床铜绿假单胞菌治疗提供新的方向,即群体感应系统抑制剂有可能成为治疗铜绿假单胞菌感染的新策略。     

A computational model of chemotaxis-based cell aggregation

We present a computational model that successfully captures the cell behaviors that play important roles in 2-D cell aggregation. A virtual cell in our model is designed as an independent, discrete unit with a set of parameters and actions. Each cell is defined by its location, size, rates of chemoattractant emission and response, age, life cycle stage, proliferation rate and number of attached cells. All cells are capable of emitting and sensing a chemoattractant chemical, moving, attaching to other cells, dividing, aging and dying. We validated and fine-tuned our in silico model by comparing simulated 24-h aggregation experiments with data derived from in vitro experiments using PC12 pheochromocytoma cells. Quantitative comparisons of the aggregate size distributions from the two experiments are produced using the Earth Mover's Distance (EMD) metric. We compared the two size distributions produced after 24 h of in vitro cell aggregation and the corresponding computer simulated process. Iteratively modifying the model's parameter values and measuring the difference between the in silico and in vitro results allow us to determine the optimal values that produce simulated aggregation outcomes closely matched to the PC12 experiments. Simulation results demonstrate the ability of the model to recreate large-scale aggregation behaviors seen in live cell experiments.     

Developing next generation antimicrobials by intercepting AI-2 mediated quorum sensing

Bacteria have been evolving antibiotic resistance since their discovery in the early twentieth century. Most new antibiotics are derivatives of older generations and there are now bacteria that are virtually resistant to almost all antibiotics. This poses a global threat to human health and has been classified as a clinical “super-challenge”, which has necessitated research into new antimicrobials that inhibit bacterial virulence while minimizing selective pressures that lead to the emergence of resistant strains. Quorum sensing (QS), the process of population dependent bacterial cell-cell signaling, can accelerate bacterial virulence and is an increasingly interesting target for developing next generation antimicrobials. Most QS inhibitors target species-specific signals, such as acylhomoserine lactones (AHLs) and oligopeptides. Methodologies for intercepting the cross-species signal, autoinducer-2 (AI-2), have only recently emerged. We review these strategies to prevent the relay of the AI-2 signal amongst pathogens, including Escherichia coli, Salmonella enterica serovar Typhimurium, Vibrio cholerae and Pseudomonas aeruginosa. Inhibition mechanisms are categorized based on the target (i.e., enzymes for signal generation, the signal molecule itself, or the various components of the signal transduction process). The universal nature of the AI-2 signal imparts on its inhibitors the potential for broad spectrum use.     

On a simplified model for pattern formation in honey bee colonies

We present a simplified version of a previously presented model (Camazine et al. (1990)) that generates the characteristic pattern of honey, pollen and brood which develops on combs in honey bee colonies. We demonstrate that the formation of a band of pollen surrounding the brood area is dependent on the assumed form of the honey and pollen removal terms, and that a significant pollen band arises as the parameter controlling the rate of pollen input passes through a bifurcation value. The persistence of the pollen band after a temporary increase in pollen input can be predicted from the model. We also determine conditions on the parameters which ensure the accumulation of honey in the periphery and demonstrate that, although there is an important qualitative difference between the simplified and complete models, an analysis of the simplified version helps us understand many biological aspects of the more complex complete model. Corresponding author     

„Small Talk“

The silent communication of bacteria Bacteria communicate via small diffusible molecules, a process that microbiologists refer to as quorum sensing. These language molecules are released by the bacteria in the environment and are then sensed by their neighbours via specific receptors. Thus, the community can arrange and adapt specific phenotypes in dependence on the cell count termed quorum. Due to the different structures and modifications of the communication molecules bacteria have evolved different languages and dialects, which can in addition give information about time and venue. Moreover, bacteria have small talk with their hosts such as animals, plants and yet humans. Since communication is a prerequisite for the infection of hosts by pathogenic bacteria, the molecular components of the bacterial communication are promising candidates as targets for badly needed new antimicrobial drugs.     

Quorum sensing intervened bacterial signaling: Pursuit of its cognizance and repression

Bacteria communicate within a system by means of a density dependent mechanism known as quorum sensing which regulate the metabolic and behavioral activities of a bacterial community. This sort of interaction occurs through a dialect of chemical signals called as autoinducers synthesized by bacteria. Bacterial quorum sensing occurs through various complex pathways depending upon specious diversity. Therefore the cognizance of quorum sensing mechanism will enable the regulation and thereby constrain bacterial communication. Inhibition strategies of quorum sensing are collectively called as quorum quenching; through which bacteria are incapacitated of its interaction with each other. Many virulence mechanism such as sporulation, biofilm formation, toxin production can be blocked by quorum quenching. Usually quorum quenching mechanisms can be broadly classified into enzymatic methods and non-enzymatic methods. Substantial understanding of bacterial communication and its inhibition enhances the development of novel antibacterial therapeutic drugs. In this review we have discussed the types and mechanisms of quorum sensing and various methods to inhibit and regulate density dependent bacterial communication.     

N-acylhomoserine lactone-degrading bacteria isolated from hatchery bivalve larval cultures

Quorum sensing (QS) systems, which depend on N-acylhomoserine lactone (AHL) signal molecules, mediate the production of virulence factors in many pathogenic microorganisms. One hundred and forty-six bacterial strains, isolated from a bivalve hatchery, were screened for their capacity to degrade five synthetic AHLs [N-butyryl-dl-homoserine lactone (C₄-HSL), N-hexanoyl-dl-homoserine lactone (C₆-HSL), N-octanoyl-dl-homoserine lactone (C₈-HSL), N-decanoyl-dl-homoserine lactone (C₁₀-HSL) and N-dodecanoyl-dl-homoserine lactone (C₁₂-HSL)] using well diffusion agar-plate assays with three biosensors, Chromobacterium violaceum CV026, C. violaceum VIR07 and Agrobacterium tumefaciens NTL4 (pZLR4). The results of these assays led to our choosing four strains (PP2-67, PP2-459, PP2-644 and PP2-663) that were able to degrade all five synthetic AHLs, thus showing a wide spectrum of quorum quenching (QQ) activity. We subsequently confirmed and measured the QQ activity of the four strains by high-performance liquid chromatography plus mass-spectrometry analysis (HPLC–MS). One of the strains which showed the highest AHL-degrading activity, PP2-459, identified as being a member of the genus Thalassomonas was chosen for further study. Finally, using thin-layer chromatography (TLC), we went on to confirm this strain's capacity to degrade the AHLs produced by other non-pathogenic and pathogenic bacteria not taxonomically related.     

Quorum sensing in biofilms--how to destroy the bacterial citadels or their cohesion/power?

Biofilms or microbial communities formed by adherent and cohesive cells on cellular or inert substrata (like medical devices), are involved in ∼60% of all infections and characterized by moderate intensity symptoms, chronic evolution and resistance to antibiotics. Biofilms’ pathogenicity, even of those formed by opportunistic microorganisms, is amplified by two major biofilm characteristics: 1) the increased resistance to antimicrobials; 2) the protection of cells against the host’s defence mechanisms. The studies at the molecular level shown that the biofilms formation is controlled by cell-to-cell signalling mechanisms and the gene regulation during biofilm growth is due to the accumulation of signal molecules. In this regard, quorum sensing mechanism (QS) is defined as a cell-density dependent bacterial intercellular communication, involved in gene expression (e.g. virulence genes for exoenzymes, exopolysaccharides) and the consequent changed behaviour of biofilm’s cells, including the resistance to stress conditions; this resistance is different of well known antibioresistance, being named phenotypical resistance or tolerance. Considering the differences in physiology and susceptibility to antibiotics of biofilm embedded bacteria, as well as their increased power against the host defence responses, there are necessary new strategies for prevention and therapy of biofilm associated infections. The dental plaque is a typical example of biofilm, involved in the ethiology of cariogenesis and periodontal diseases associated with local chronic inflammation and cytokines production. The genetical and phenotypical versatility of the biofilm’s cells represent a challenge for discovering new methods of treatment and prevention of biofilm associated infections. A novel class of antibiofilm and antipathogenic therapeutics which are interfering with a new target – the QS pathway, not based on growth inhibition and called QS inhibitors, natural, with different origins or artificial, are now developing as an alternative to antibiotherapy.     

A comparative study of non-native N-acyl l-homoserine lactone analogs in two Pseudomonas aeruginosa quorum sensing receptors that share a common native ligand yet inversely regulate virulence

Certain bacteria can coordinate group behaviors via a chemical communication system known as quorum sensing (QS). Gram-negative bacteria typically use N-acyl l-homoserine lactone (AHL) signals and their cognate intracellular LuxR-type receptors for QS. The opportunistic pathogen Pseudomonas aeruginosa has a relatively complex QS circuit in which two of its LuxR-type receptors, LasR and QscR, are activated by the same natural signal, N-(3-oxo)-dodecanoyl l-homoserine lactone. Intriguingly, once active, LasR activates virulence pathways in P. aeruginosa, while activated QscR can inactivate LasR and thus repress virulence. We have a limited understanding of the structural features of AHLs that engender either agonistic activity in both receptors or receptor-selective activity. Compounds with the latter activity profile could prove especially useful tools to tease out the roles of these two receptors in virulence regulation. A small collection of AHL analogs was assembled and screened in cell-based reporter assays for activity in both LasR and QscR. We identified several structural motifs that bias ligand activation towards each of the two receptors. These findings will inform the development of new synthetic ligands for LasR and QscR with improved potencies and selectivities.     

细菌群体感应系统研究进展及其应用

细菌能自发产生、释放一些特定的信号分子,并能感知其浓度变化,调节微生物的群体行为,这一调控系统称为群体感应。细菌群体感应参与包括人类、动植物病原菌致病力在内的多种生物学功能的调节。本文综述了细菌群体感应的最新研究进展,并阐述了其在生物技术领域的应用前景。     

革兰氏阴性细菌LuxR/I型群体感应抑制剂研究进展

摘要：细菌群体感应（Quorum sensing, QS）被视为对抗细菌感染与解决细菌耐药性问题的新靶点。以AHLs为信号分子的LuxR/I型群体感应系统广泛存在于革兰氏阴性菌包括多种临床致病菌中,因此寻找LuxR/I型群体感应抑制剂（Quorum sensing inhibitors, QSIs）是研发抗革兰氏阴性致病菌药物的重要途径。迄今为止,已知的LuxR/I型小分子QSIs来源包括化学合成、天然产物与已知药物库的化合物,大分子则包括群体感应淬灭酶与群体感应淬灭抗体。本文总结了近年来LuxR/I型QSIs研究进展,为新型抗菌药物研发提供理论依据。