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Transcription factors (TFs) are proteins that respond to a specific chemical signal and bind to DNA. In some bacteria, TFs control transition metal ion homeostasis by specifically binding with a particular metal ion or ions and then interacting with DNA. Although most first row metal ions are required as micronutrients for life, many of them can cause cellular damage or death if their concentrations are too high; this makes these TFs and their biological interactions excellent targets for drug development. The bacteria, Streptococcus pneumoniae is a pathogenic microorganism responsible for a range of diseases that target the young and the old, including pneumonia, meningitis, and bacteremia. Herein, we describe our efforts to study the Streptococcus pneumoniae TF responsible for copper(II) ion homeostasis. This thesis describes the classical biochemical techniques used to over-express and purify CopR. It also describes a series of preliminary characterization data associated with this novel copper(II)-dependent TF.

The Streptococcus Anginosus/Milleri group (SMG) colonize mucosal surfaces in humans but are also associated with numerous respiratory and invasive infections. These infections are often polymicrobial in nature, with obligate anaerobes often being isolated. The group consists of three species, S. anginosus, S. constellatus and S. intermedius. SMG are considered to be lactic acid bacteria, producing acids such as lactate, formate and acetate as byproducts of their metabolism. Their genomes have been recently sequenced but little is known about their metabolism. Understanding the basis of their metabolism is beneficial in determining optimal growth conditions and mechanisms associated with their pathogenicity. The isolation of obligate anaerobes from SMG polymicrobial infections suggests that they have anoxic microenvironments. There is also some evidence for synergy between SMG species and anaerobes. While cooperation might be occurring with certain anaerobes, streptococci also produce inhibitors such as hydrogen peroxide and short peptides called bacteriocins. These give streptococci a competitive advantage in polymicrobial commensal communities such as the oral cavity. The Streptococcus invasion locus controls bacteriocin production in Group A streptococci and has been identified in SMG species as well. It is unknown if SMG have mechanisms to compete with closely related streptococci. The goal of my thesis is to characterize the cooperative and competitive interactions of S. intermedius with other species. In chapter 2, we characterized the in vitro metabolism of S. intermedius under aerobic (5% CO2) and anaerobic conditions. Using a transcriptomic and metabolomic approach, we mapped the pathways involved in S. intermedius B196 metabolism. We found that there was a minimal upregulation of core pathways including carbohydrate metabolism under anaerobic conditions. Under aerobic conditions, oxidative stress genes were induced. An increased growth rate was also observed anaerobically. In chapter 3, I demonstrated that Streptococcus strains, including S. intermedius, can deplete oxygen and create an anaerobic environment. Certain strains could support the viability of the obligate anaerobe Prevotella melaninogenica in broth cultures under hypoxic conditions, while others inhibited Prevotella by producing hydrogen peroxide. S. intermedius B196 has an alkylhydroperoxidase system (ahpCF), which is thought to endogenously detoxify peroxides. An S. intermedius ahpCF mutant produced hydrogen peroxide and inhibited P. melaninogenica in coculture. Complementation in S. intermedius restored P. melaninogenica viability in coculture. I demonstrated that the ahpCF peroxide detoxification system directly protects S. intermedius from peroxides and indirectly affects a polymicrobial community. In chapter 4, we used a subcutaneous abscess model in BALB/c mice to demonstrate that S. intermedius promotes P. melaninogenica survival during co-infection in comparison to a P. melaninogenica mono-infection. S. intermedius induced abscesses appeared to induce apoptosis, necrosis and NETosis in neutrophils that infiltrated the site of infection. Our results demonstrate the complexity of SMG infections. In chapter 5, I demonstrated that S. intermedius B196 produces inhibitors of other SMG in response to stimulation with the pheromone peptide SilCR. This is the first case of S. intermedius inhibiting a closely related SMG strain. A bioinformatic analysis was done on the sil system in SMG. The system is associated with a genetically heterogeneous bacteriocin cluster which can carry any combination of sixteen putative open reading frames, six of which are putative bacteriocins. Together, my thesis outlines that S. intermedius has specific mechanisms of cooperation and competition. These allow it to cooperate with obligate anaerobes such as P. melaninogenica and inhibit other SMG species. Oxygen depletion, hydrogen peroxide production and bacteriocin production are only three factors addressed in this thesis. However, there are many factors involved in shaping a polymicrobial environment with SMG species. More research in SMG polymicrobial interactions is required to fully understand SMG pathogenicity. / Thesis / Doctor of Philosophy (PhD)

Streptococcus suis est un agent pathogène porcin causant des pneumonies, des septicémies et des méningites. Il est aussi un agent de zoonose responsable de plusieurs éclosions en Asie. Les souches de S. suis sont classifiées en 35 sérotypes basés sur la composition de leur capsule polysaccharidique. S. suis sérotype 2 cause la majorité des infections sévères et est sous-divisé en séquence types (ST). Le ST1 est associé avec des souches hautement virulentes. En Amérique du Nord, les souches communément isolées appartiennent aux ST25 et ST28, respectivement modérément et faiblement virulentes dans un modèle animal. La présence de S. suis sous forme de bioaérosols dans l’air des bâtiments porcins a été démontré. Le but de ce projet est d’étudier l’aérosolisation préférentielle de différentes souches de S. suis en utilisant une chambre expérimentale et un nébuliseur développés pour ce projet. Bien qu’un nombre supérieur de souches doivent être étudiées, les résultats du projet suggèrent que les souches hautement virulentes du sérotype 2 de ST1 semblent être préférentiellement aérosolisées et que l’aérosolisation préférentielle de S. suis semble être un processus souche dépendant. Cette étude est une preuve de concept et améliore nos connaissances sur la potentielle transmission de S. suis via les bioaérosols. / Streptococcus suis is a swine pathogen that causes pneumonia, septicaemia and meningitis. It is also a zoonotic agent responsible for outbreaks in Asia. S. suis strains are classified into 35 serotypes based on the composition of their polysaccharide capsule. S. suis serotype 2 causes the majority of severe infections and it is subdivided into sequence types (STs). The ST1 is associated with highly virulent strains. In North America, the strains most commonly isolated belong to ST25 and ST28, which are respectively moderately and weakly virulent in animal model. The presence of S. suis bioaerosols in the air of swine confinement buildings has been demonstrated. The aim of this study was to better understand the aerosolization behaviour of S. suis by investigating preferential aerosolization of different S. suis strains using in-house developed environmental chamber and nebulizer. Although more strains should be studied, the results suggest that the highly virulent serotype 2 ST1 strains seem to be preferentially aerosolized and that the S. suis preferential aerosolization is a strain-dependant process. This study is a proof of concept and increases our knowledge on the potential aerosol transmission of S. suis.

Face au développement incessant de la résistance aux antibiotiques, la découverte de nouvelles substances antibactériennes devient urgente. Les bactériocines, peptides bactériens à activité antibiotique synthétisés par les ribosomes, représentent une alternative. Les mutacines sont des bactériocines produites par Streptococcus mutans, une espèce bactérienne indigène de la cavité buccale. Quatre types de mutacines ont été caractérisés à ce jour: les lantibiotiques et non-lantibiotiques, monopeptidiques ou dipeptidiques. Des analyses bio-informatiques des génomes de S. mutans UA159 et NN2025 ont révélé la diversité des gènes codant potentiellement pour des bactériocines. L'étude des mutacines s'est toujours confrontée à la difficulté de les produire en milieu liquide. Deux mutacines (F-59.1 et I-T9) ont été produites et isolées d'un surnageant de culture d'un milieu élaboré à partir de perméat de lactosérum. La mutacine D-123.1 a été isolée à partir d'un milieu de culture de nature semi-solide. Les analyses biochimiques et moléculaires des mutacines révèlent que la mutacine F-59.1 est apparentée aux bactériocines de la famille des pédiocines alors que la mutacine D-123.1 appartient aux lantibiotiques. Les concentrations minimales inhibitrices (CMI) et bactéricides (CMB) des mutacines D-123.1 et F-59.1 ont été déterminées contre plusieurs pathogènes humains. La mutacine D-123.1 a montré son efficacité contre les pathogènes du domaine médical et alimentaire avec des CMI comprises entre 0.25 et 4 ug/mL. La mutacine F-59.1 inhibe les pathogènes alimentaires avec des CMI comprises entre 3.2 et 12.8 ug/mL. De façon à détecter simplement et rapidement des mutacines apparentées aux pédiocines, les 24 souches type productrices de mutacines ont été testées par un test d'antagonisme différé, pour leur capacité à produire une mutacine capable de cibler le complexe IIC des transporteurs membranaires du mannose pour exercer leur activité inihibitrice. Streptococcus salivarius et des mutants défectueux dans leur transporteur du mannose ont été utilisés comme souches indicatrices. Deux souches de S. mutans (T9 et 3B) ont démontré une activité différente contre la souche mère et ces mutants, indiquant potentiellement la production de mutacine ciblant le transporteur du mannose par ces deux souches.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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<p>The Streptococcus Milleri/Anginosus group (SMG) includes Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius. The SMG is found in healthy individuals but these bacteria are most known clinically for being associated with invasive disease and more recently, airway infections including cystic fibrosis (CF). The SMG like many other streptococci are naturally competent, being able to actively bind, uptake and integrate extracellular DNA. Competence regulation involves a competence-stimulating peptide (CSP) derived from the ComC precursor and a two- component signaling system (a histidine kinase ComD and its response regulator ComE). In this study, I examined the distribution of CSP/ComD sequences and competence in 170 SMG clinical isolates from CF airways and invasive disease. Five predicted CSP sequences were observed; one represented a newly predicted CSP and two arose from frameshift mutations in comC and appeared to be non-functional. The three CSPs fall into two functional groups that do not cross-activate due to receptor specificity. In addition, I observed that the Streptococcus constellatus subspecies pharyngis strains could not be transformed. However, I demonstrated that the pharyngis strains possess a functional ComCDE pathway, suggesting that the CSP regulates genes other than those involved in natural transformation. For many strains, I observed high endogenous competence levels that were only marginally induced by added peptide. These strains appear to be constitutively competent during exponential growth. The high basal level of expression and the heterogeneity in the SMG competence systems could impact how the SMG evolve during colonization and infections and specifically acquire antibiotic resistance and virulence genes.</p> / Master of Science (MSc)

The Streptococcus Anginosus Group (SAG) is a group of Gram-positive cocci which require carbon dioxide to grow. They are commensal members of the healthy upper respiratory, gastrointestinal and female urogenital tract; however, they are most commonly known as major pathogens in brain and liver abscesses, forming both mono- and polymicrobial infections. The Streptococcus invasion locus (sil), first identified as a virulence factor in Group A Streptococcus (GAS), has recently been identified in the SAG. The sil locus in GAS is a two component quorum-sensing system composed of three operons: silAB, coding for a two component system; silE/D/CR, coding for an ABC transporter and a signal peptide, and silC, which overlaps silCR on the opposite strand. The presence of exogenous SilCR activates SilA, which in turn upregulates the transcription of the silE/D/CR operon. In the SAG, however, silCR and silED have distinct promoters, and the SAG sil system lacks the silC gene. In this study, I examined the transcriptional dynamics of the sil system in S. intermedius B196. I determined that SilA is the major regulator of the genes in the sil system, being one of the first genes of the system to be expressed, and likely upregulates its own transcription. I also found evidence suggesting that, despite having its own promoter, silCR transcription may still be driven by the silED promoter. I also found evidence that suggests silED may be responsible for the export and/or processing of bacteriocins targeting closely related species or strains. / Thesis / Master of Science (MSc) / The Streptococcus are a group of bacteria known for causing diseases ranging from strep throat to flesh-eating disease; however, many species of Streptococcus are usually non-pathogenic, and live in our bodies without causing harm. One group of these bacteria, the Streptococcus Anginosus Group (SAG), is commonly found in our mouths and gut and usually cause no harm; however, in some cases it can cause infections. How these organisms switch from being nonpathogenic to pathogenic is unknown, but recently a gene network that appears to play a role in infection, the Streptococcus invasion locus (sil), was identified in the SAG. This gene network senses the signals released by other bacteria with the network, and only turns on when enough signal is present. The goal of this study is to examine how the system works in the SAG in order to determine how these bacteria coordinate sil gene expression.

Les progrès technologiques dans l'industrie de la viande ont des répercussions considérables sur les agents pathogènes de ces environnements. Parmi ceux-ci, Streptococcus suis occupe une place prédominante dans l’industrie porcine. En effet, S. suis, colonisateur naturel des voies respiratoires et digestives du porc, peut infecter son hôte en provoquant des méningites, septicémies, endocardites, arthrites ou pneumonies. De surcroît, S. suis peut également infecter l’humain en provoquant majoritairement des méningites et septicémies, et a notamment été la cause de deux épidémies en Chine en 1998 et 2005. La pathogenèse des infections à S. suis demeure partiellement connue à l’heure actuelle, rendant difficile le contrôle des infections. Il est par conséquent essentiel de caractériser les facteurs de virulence chez S. suis puisqu'ils pourraient représenter des cibles d’intérêt pour des applications préventives ou thérapeutiques. Ce projet de doctorat consiste donc en la caractérisation fonctionnelle de facteurs de virulence chez S. suis. Dans un premier temps, la capacité de S. suis à moduler son potentiel pro-inflammatoire en présence de concentrations sous-inhibitrices d'amoxicilline a été mise en évidence. Dans un second temps, la caractérisation plus avancée de la hyaluronate lyase de S. suis a permis de démontrer que son activité ne contribue pas à la virulence de la bactérie étant donné son absence au sein de souches les plus virulentes, mais que les interactions avec l'acide hyaluronique pourraient moduler la virulence de S. suis. Par la suite, l'étude fonctionnelle d’une DNase de S. suis a permis de démontrer son implication comme facteur de virulence et suggère son intérêt dans le développement de vaccins. Finalement, le dernier objectif du projet a permis la mise en évidence de la production de microvésicules fortement immunogéniques par S. suis. La présence de facteurs de virulence dans leur contenu protéique représente un élément encourageant dans le développement de vaccins contre l'agent pathogène. Ce projet a donc permis d'élargir les connaissances sur le potentiel néfaste de l'utilisation des antibiotiques à faible concentration dans l'industrie porcine, sur le rôle des activités hyaluronate lyase et DNase dans la virulence de S. suis, et de découvrir un nouveau mécanisme impliqué dans la virulence de la bactérie par le biais des microvésicules. / Technological progress in the meat industry has a substantial impact on pathogens within these environments. Among these pathogens, Streptococcus suis is of utmost importance in the swine industry. S. suis, natural colonizer of the respiratory and digestive tracts of porks, can infect its host causing mainly meningitis and septicemia as well as endocarditis, arthritis and pneumonia. Furthermore, S. suis can infect humans causing mainly meningitis and septicemia, and was the cause of two major outbreaks in China in 1998 and 2005. The pathogenesis of S. suis infections remains partially understood, making the control of infections challenging. Consequently, it is of utmost importance to characterize virulence factors that could represent targets of interest for preventive or therapeutic applications. This project focused on the functional charaterization of virulence factors produced by S. suis. First, the ability of S. suis to modulate its pro-inflammatory potential in the presence of sub-inhibitory concentrations of amoxicillin was demonstrated. Then, a further characterization of S. suis hyaluronate lyase brought evidence that this activity does not contribute to the bacterium's virulence since it is absent in most virulent strains. However, interactions with hyaluronic acid could modulate S. suis virulence. The functional study of S. suis DNase showed its implication as a virulence factor and suggested its interest in vaccine development. Finally, the last objective of this project lead to the discovery of the production of highly immunogenic microvesicles by S. suis. The presence of major virulence factors associated with these structures also represents an exciting fact for the development of vaccines against S. suis. This project allowed to expand the knowledge on the noxious potential of the use of low concentrations of antibiotics in the swine industry, on the role of hyaluronate lyase and DNase activities in S. suis virulence as well as on the production of microvesicles by S. suis that represents a new virulence mechanism.