Mathematical Modelling of Bacterial Quorum Sensing: A Review

Bacterial quorum sensing (QS) refers to the process of cell-to-cell bacterial communication enabled through the production and sensing of the local concentration of small molecules called autoinducers to regulate the production of gene products (e.g. enzymes or virulence factors). Through autoinducers, bacteria interact with individuals of the same species, other bacterial species, and with their host. Among QS-regulated processes mediated through autoinducers are aggregation, biofilm formation, bioluminescence, and sporulation. Autoinducers are therefore “master” regulators of bacterial lifestyles. For over 10�years, mathematical modelling of QS has sought, in parallel to experimental discoveries, to elucidate the mechanisms regulating this process. In this review, we present the progress in mathematical modelling of QS, highlighting the various theoretical approaches that have been used and discussing some of the insights that have emerged. Modelling of QS has benefited almost from the onset of the involvement of experimentalists, with many of the papers which we review, published in non-mathematical journals. This review therefore attempts to give a broad overview of the topic to the mathematical biology community, as well as the current modelling efforts and future challenges. � 2016, Society for Mathematical Biology

Development of Solution Blow Spun Nanofibers as Electrical and Whole Cell Biosensing Interfaces

Infectious pathogens place a huge burden on the US economy with more than $120 billion spent annually for direct and indirect costs for the treatment of infectious diseases. Rapid detection schemes continue to evolve in order to meet the demand of early diagnosis. In chronic wound infections, bacterial load is capable of impeding the healing process. Additionally, bacterial virulence production works coherently with bacterial load to produce toxins and molecules that prolongs the healing cycle. This work examines the use of nonwoven polymeric conductive and non-conductive nanofiber mats as synthetic biosensor scaffolds, drug delivery and biosensor interface constructs. A custom-made nanofiber platform was built to produce solution blow spun nanofibers of various polymer loading. Antimicrobial nanofiber mats were made with the use of an in-situ silver chemical reduction method. Ceria nanoparticles were incorporated to provide an additional antioxidative property. Conductivity properties were examined by using silver and multi-walled carbon nanotubes (MWCNT) as a filler material. SBS parameters were adjusted to analyze electrical conductivity properties. Nanofiber mats were used to detect bacteria concentrations in vitro. Protein adhesion to conductive nanofibers was studied using fluorescent antibodies and BCA assay. Anti-rabbit and streptavidin Alexa Flour 594 was used to examine the adsorption properties of SBS nanofiber mats. Enhancements were made to further improve interface design for specificity. SBS nanofiber electrodes were fabricated to serve as scaffold and detection site for spike protein detection. Bacteria virulence production was examined by the detection of pyocyanin and quorum sensing molecules. The opportunistic pathogen, Pseudomonas aeruginosa is a nosocomial iii pathogen found in immunocompromised patients with such as those with chronic wounds and cystic fibrosis. Pyocyanin is one of four quorum sensing molecules that the pathogen produces which can be detected electrochemically due to its inherent redox-active activity. SBS has been used to develop a sensing scheme to detect pyocyanin. This work also examines the use of a synthetic biosensor with a LasR based system capable of detecting homoserine lactone produced by P. aeruginosa and other common gram-negative pathogens. Genetic modifications were made to biosensor in order to replace a green, fluorescent reporter with a chromoprotein based reporter system for visual readout. Additionally, work related to community service and outreach regarding the encouragement of middle school students to pursue Science, Technology, Engineering and Math (STEM) was conducted. Results from outreach program showed an increase in the STEM interest among a group of middle school students. There was a general trend with STEM career knowledge, STEM self-efficacy and the level of interest in STEM careers and activities. Military research was also done with the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) to develop several assays for the detection of several highly infectious viruses and bacteria. Due to confidentiality, the work cannot be published in this manuscript

Stochastic Effects in Quorum Sensing

[cat] En aquesta tesi, estudiem els efectes de la estocàsticitat en la aparició del comportament col·lectiu en poblacions de bacteris que comuniquen per quorum sensing (QS). Ens centrem en el interruptor genètic com a paradigma dels processos de decisió cel·lulars tant en sistemes de bacteris naturals com sintètics. El nostre mètode es basa en la modelització matemàtica i en les simulacions estocàstiques, tant a nivell d'una cèl·lula individual com a nivell d'una població de cèl·lules. A nivell d'una cèl·lula individual, mostrem que el soroll afavoreix l'estabilitat del fenotip de l'estat ``baix'' de l'interruptor genètic autoactivador i que la regió de biestabilitat s'estén quan creix la intensitat de les fluctuacions, un efecte que hem anomenat estabilització estocàstica. A nivell d'una població de cèl·lules, mostrem que el procés de difusió del mecanisme de QS modifica les fluctuacions i la dinàmica de la molècula autoinductora dins de la cèl·lula i interactua amb el soroll en la expressió genètica. En el sistema canònic de QS LuxR/LuxI, mostrem que el soroll en la expressió genètica de LuxR és el principal factor que controla la variabilitat transitòria de l'activació del QS. El soroll intrínsec disminueix la precisió de la coordinació de la població i modifica la dinàmica de la transició de QS. A més, presentem un model d'una població d'interruptors genètics de toggle switch que comuniquen per l'intercanvi de dos senyals difusius de QS. Mostrem que l'increment de la velocitat de difusió, que augmenta la força de l'acoblament entre les cèl·lules, porta a una transició de fase: va des d'una fase desordenada on les cèl·lules salten de manera aleatòria entre els dos estats de l'interruptor, fins a una fase ordenada amb totes les cèl·lules bloquejades en el mateix estat estable. La mateixa transició s'ha trobat en una població de cèl·lules que creixen exponencialment en un volum tancat, amb totes les cèl·lules entrant en l'estat ordenat quan arriben a una mida crítica del sistema. Els nostres resultats suggereixen un nou mecanisme per la decisió cel·lular col·lectiva basat en el fenomen de la transició de fase.[eng] Stochastic fluctuations, or noise, are ubiquitous in biological systems and play an important role in many cellular processes. Experimental evidences have shown that noise affects the reliability of cell coordination in populations of communicating cells. In this thesis, we study the effects of stochasticity in the emergence of collective behavior in populations of bacteria communicating by QS. We focus on the genetic switch as a paradigm of cellular decision making in both natural and synthetic bacterial systems. Our approach is based on mathematical modeling and stochastic simulations, both at the level of the single cell and at the level of the cell population. We focus on four main topics. In the first topic, we analyze the interplay between intracellular noise and the diffusion process of the QS signaling mechanism. We build a model describing the expression of the signaling molecule and its diffusion in a population of cells, focusing on the situation of very low constitutive expression rate. We show that varying the diffusion rate produces a repertoire of dynamics for the signaling molecule. Our results reveal the contribution of intrinsic noise and transcriptional noise (mRNA copy number fluctuations) in the fluctuations of the signaling molecule. We observe that the total noise exhibits a maximum as a function of the diffusion rate, in contrast to previous studies. Thus, the QS communication mechanism modifies the fluctuations of the signaling molecule inside the cell and interacts with the gene expression noise. In the second topic, we study the effects of gene expression noise on the precision of the population coordination in the QS activation of the LuxR/LuxI system. We analyze the response and dynamics of a population of cells to different levels of autoinducer. Our results show that gene expression noise in LuxR is the main factor that controls the transient variability of the QS activation. This study sheds light on the relation between the single cell stochastic dynamics and the collective behavior in a population of communicating cells. In the third topic, we analyze the effects of intrinsic noise in an autoactivating switch in an isolated single cell. We show that noise promotes the stability of the low-state phenotype of the switch and that the bistable region is extended when increasing the intensity of the fluctuations, an effect that we call stochastic stabilization. Our results show that intrinsic noise modifies the epigenetic landscape as well as the switching rate, which results in complex behavior of the stochastic switching dynamics when varying the intensity of noise. Thus, at the level of a single cell, intrinsic noise contributes to the cell-to-cell variability of the genetic switch and can modify its stable states and its dynamics. In the fourth topic, we build a model of a population of toggle switches communicating by the exchange of two diffusible QS signals. We show that increasing the diffusion rate, which increases the coupling strength between the cells, leads to a phase transition from an unordered phase where the cells randomly flip between the two states of the switch, to an ordered phase with all the cells locked into the same stable state. The same transition is found in a population of cells growing exponentially in a closed volume. Moreover, the response of the cells to a varying external signal exhibits a hysteresis loop. We show that the cell-cell coupling enhances the sensitivity of the population response to the external signal and suggest that this new mechanism could be used to increase the robustness and sensitivity of biosensors. Our results suggest a new mechanism for collective cell decision making based on the phenomenon of phase transition

Modeling the Quorum Sensing Signaling Regulatory Network in \u3cem\u3eVibrio fischeri\u3c/em\u3e

Quorum sensing is a mechanism by which bacteria can sense the levels of signaling molecules and respond by controlling the expression of target genes. The marine bacterium, Vibrio fischeri, has been extensively studied as a model for the quorum sensing mechanism in Gram-negative bacteria. In order to systematically investigate the quorum sensing regulatory network in V. fischeri, a conceptual model was first established based on the existing knowledge. Next, molecular microbiology and bioinformatics techniques were employed to both qualitatively and quantitatively characterize the system. These techniques included the quantification of the 3-oxo-C6-HSL concentrations in the cell culture supernatant using a bioluminescent bioreporter strain of E. coli, the measurements of the messenger RNA levels of quorum sensing genes (luxI, luxR, ainS and litR) using the reverse transcription-polymerase chain reaction (RT-PCR), as well as the sequence analysis of the promoter regions of quorum sensing related genes. A mathematical model composed of ordinary differential equations was created to characterize the regulatory process. The simulated annealing method was used to minimize the weighted discrepancy between the modeling output and the experimental data with correlations ranging from 0.85 to 0.99. This study, mathematically modeled the comprehensive quorum sensing regulatory system, which encompasses 3-oxo-C6-HSL, lux operon (luxR and luxICDABEG), C8-HSL, ainS, ainR, luxO, and litR, and can benefit the understanding of dozens of similar quorum sensing regulatory systems

A Synthetic Biology Approach to Bacteria Mediated Tumor Targeting

Development of a drug delivery agent that selectively targets and destroys tumor cells with minimal toxicity to normal tissues is a major challenge in cancer therapy. It has been known for more than 60 years that anaerobic bacteria such as Clostridium can selectively colonize inside the necrotic core of solid tumors. Inoculation of a tumor by wild type Clostridium results in colonization of the necrotic core and consequently significant tumor destruction. This treatment strategy is hampered by the fact that the outer rim of the tumor is typically viable, and so does not present an anaerobic environment. As a result, colonization by Clostridium is unlikely to lead to complete tumor regression, since tumor regrowth occurs from the remaining outer viable rim, as evidenced by clinical trials. This project aims to address the problem of regrowth by developing a novel selectively aerotolerant strain of Clostridium that cannot colonize inside healthy tissue, but that could grow in the viable rim of an infected tumor. We have engineered a gene coding for an aerotolerance enzyme into Clostridium sporogenes. To couple the selective expression of this gene to tumor colonization, it can be placed under the control of a promoter activated by a synthetic quorum sensing circuit. This document describes the foundational work that will allow this system to be implemented. A suitable strain of C. sporogenes was selected, and a cloning technique (via conjugation with E. coli) was implemented. Expression of the aerotolerance enzyme and a synthetic quorum sensing circuit were verified in engineered colonies, and appropriate function was confirmed in both cases. Additionally, a model-based design exercise was carried out in order to better understand the system behavior and to identify key parameters for controlling the bacterial population. This analysis was based on mathematical models of the quorum-sensing circuit and of bacterial growth in the tumor environment. Sensitivity analysis reveals the design parameters that have the most significant impact on the extent and specificity of colonization of the viable rim, and thus provides insights into efficient design of the synthetic mechanism

The Role of Quorum Sensing in Bacterial Colony Dynamics

The quorum sensing (QS) signalling system allows colonies of bacteria to coordinate gene expression to optimise behaviour at low and high cell densities, giving rise to individual and group responses, respectively. The main aim of this thesis is to understand better the important roles of QS in bacterial colony dynamics. Thus a mathematical description was developed to thoroughly explore key mechanisms and parameter sensitivity. The nature of the QS system depends very much on the species. Pseudomonas aeruginosa was chosen as a model species for this study. P. aeruginosa is a Gram-negative bacterium that is responsible for a wide range of chronic infections in humans. Its QS signalling system is known to involve the las, rhl and pqs systems; this thesis focuses on the first two. The las system includes the LasR regulator and LasI synthase, which direct the synthesis of autoinducer 3O-C12-HSL. Similarly, the rhl system consists of the RhlR regulator and RhlI synthase, directing the synthesis of autoinducer C4-HSL. The mathematical model of the las system displays hysteresis phenomena and excitable dynamics. In essence, the system can have two stable steady states reflecting low and high signal molecule production, separated by one unstable steady state. This feature of the las system can give rise to excitable pulse generation with important downstream impact on the rhl system. The las system is coupled to the rhl system in two ways. First, LasR and 3O-C12-HSL activate the expression of their counterpart in the rhl system. Second, 3O-C12-HSL blocks activation of RhlR by C4-HSL. Furthermore, the las-rhl interaction provides a `quorum memory' that allows cells to trigger rhamnolipid production when they are at the edge of colony. It was demonstrated how the dynamical QS system in individual cells and with coupling between cells can affect the dynamics of the bacterial colony

Examining recombination and intra-genomic conflict dynamics in the evolution of anti-microbial resistant bacteria

The spread of antimicrobial resistance (AMR) among pathogenic bacterial species threatens to undercut much of the progress made in treating infectious diseases. AMR genes can disseminate between and within populations via horizontal gene transfer (HGT). Selfish mobile genetic elements (MGEs) can encode resistance and spread between host cells. Homologous recombination can alter the core genes of pathogens with resistant donors via HGT too. MGEs may be cured from host genomes through transformation. Hence, MGEs may be able to avoid deletion by disrupting transformation. This work aims to understand how the dynamics of these processes affect the epidemiology of AMR pathogens. To understand these dynamics, I co-developed a new version of the popular recombination detection tool Gubbins. Through simulation studies, I find this new version to be both accurate in reconstructing the relationships between isolates, and efficient in terms of its use of computational resources. I then apply Gubbins to both AMR lineages and species-wide datasets of the pathogen Streptococcus pneumoniae. I find that recombination frequently occurs around core genes involved in both drug resistance and the host immune response. Additionally, an MGE was able to successfully spread within a population by disrupting the transformation machinery, preventing its loss from the host. Finally, I investigate two recent examples of MGEs disrupting transformation in the gram-negative species Acinetobacter baumannii and Legionella pneumophila. I find that while these insertions may decrease the efficiency of transformations within cells, the observed recombination rates largely reflect the selection pressures on isolates. With MGEs only partially able to inhibit these observable transformation events. These results show how selection pressures from clinical interventions shape pathogen genomes through diverse, often interspecies, recombination events. The spread of MGEs can also be favoured by both these selection pressures, and their ability to disrupt host cell machinery.Open Acces

Calcium signaling in Pseudomonas aeruginosa

P. aeruginosa is an opportunistic pathogen and a major cause of hospital acquired infections and severe chronic infections in endocarditis and cystic fibrosis (CF) patients. During such infections, it encounters elevated levels of Ca2+. Ca2+ regulates gene expression, physiology, and production of several virulence factors, thereby, enhancing adaptability and virulence of P. aeruginosa. This indicates signaling role of Ca2+ in P. aeruginosa, however, molecular mechanisms involved in Ca2+ regulation are yet unknown. In eukaryotes, Ca2+ is a well established intracellular signal regulating essential cellular processes. We aim to characterize the molecular mechanisms involved in Ca2+ regulation and ultimately test the intracellular signaling role of Ca2+ in P. aeruginosa. Our studies established that P. aeruginosa maintains submicromolar level of intracellular Ca2+ ([Ca2+]in), which transiently increases in response to external Ca2+ . We determined that Ca2+ homeostasis is maintained by multiple transporters and putative Ca2+ binding proteins, and that P. aeruginosa responds to Ca2+ by using a two-component regulatory system CarSR, which regulates the expression of genes encoding putative Ca2+ binding proteins. These proteins contribute to the maintenance of intracellular Ca2+ homeostasis, which, in turn, plays an important role in Ca2+ regulation of physiology and virulence. Finally, we showed that Ca2+ regulates quorum sensing (QS) signaling in P. aeruginosa by affecting the transcription of QS regulatory genes. The ongoing work using Ca2+ transient defective mutant is aiming to provide direct experimental evidence for role of Ca2+ as a secondary messenger