Metabolome-based studies of virulence factors in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen and an important causative agent of potentially life-threatening nosocomial infections in predisposed patients. The Gram-negative bacterium produces a large and diverse repertoire of small-molecule secondary metabolites that serve as regulators and effectors of its virulence. In this study, a range of mass spectrometry-based bacterial metabolomics approaches was used to investigate these small-molecule virulence factors and their interplay with pseudomonal metabolism as well as with phenotypic traits related to virulence. The groundwork was laid by exploring the metabolite inventory of P. aeruginosa and improving the coverage of its metabolome by the application of a custom software named CluMSID, that clusters analytes based on similarities of their MS² spectra. CluMSID led to the annotation of, i.a., 27 novel members of the class of alkylquinolone quorum sensing signalling molecules, which represent crucial players in the highly complex network that regulates pseudomonal virulence. The tool was developed towards a versatile and user-friendly R package hosted on Bioconductor, whose functionalities and benefits are described in detail. The new findings on the alkylquinolone chemodiversity led to further studies with a mechanistic focus that probed the substrate specificity of the enzyme complex PqsBC. It was demonstrated that PqsBC accepts different medium-chain acyl-coenzyme A substrates for the condensation with 2-aminobenzoylacetate and thereby produces alkylquinolones with various side chain lengths, whose distribution is a function of substrate specificity and substrate availability. Moreover, it was shown that PqsBC also synthesises alkylquinolones with unsaturated side chains. The focus was further broadened from metabolite and pathway-centred questions to a more global perspective on pseudomonal virulence and metabolism, which directed attention at PrmC, an enzyme with a partially unknown function indispensable for in vivo virulence. An untargeted metabolomics experiment yielded insights into the role of PrmC and its influence on the pseudomonal endo- and exometabolome. Finally, clinical P. aeruginosa strains with different virulence phenotypes were examined by untargeted metabolomics in order to disclose metabolic variation and interconnections between virulence and metabolism. The analysis resulted in the discovery of a putative virulence biomarker and enabled the construction of a random forest classification model for certain virulence phenotypes based only on metabolomics data. In summary, this study demonstrated the potential of metabolomics for the investigation of P. aeruginosa virulence factors and thereby contributed towards the comprehension of the complex interplay of metabolism and virulence in this important pathogen.Pseudomonas aeruginosa ist ein wichtiger opportunistischer Erreger potenziell lebensbedrohlicher nosokomialer Infektionen bei prädisponierten Patienten. Das Gram-negative Bakterium produziert ein vielfältiges Repertoire an niedermolekularen Sekundärmetaboliten, die als Regulatoren und Effektoren seiner Virulenz dienen. In dieser Studie wurde eine Reihe von Massenspektrometrie-basierten Ansätzen der bakteriellen Metabolomik verwendet, um diese niedermolekularen Virulenzfaktoren und ihre Wechselwirkungen mit dem pseudomonalen Metabolismus sowie mit virulenzassoziierten phänotypischen Merkmalen zu untersuchen. Die Grundlage bilden die Untersuchung des Metaboliteninventars von P. aeruginosa und die Verbesserung der analytischen Abdeckung des Metaboloms durch die Anwendung einer selbstentwickelten Software namens CluMSID, die MS²-Spektren nach Ähnlichkeit clustert. CluMSID führte zur Annotation von u.a. 27 neuen Mitgliedern der Klasse der Alkylchinolone, die als Quorum-Sensing-Signalmoleküle entscheidende Akteure im hochkomplexen Netzwerk der Virulenzregulation darstellen. Das Tool wurde zu einem R-Paket entwickelt, das auf Bioconductor verfügbar ist und dessen Funktionalitäten und Vorteile ausführlich beschrieben werden. Die neuen Erkenntnisse über die Chemodiversität der Alkylchinolone führten zu weiteren Studien mit mechanistischem Schwerpunkt, die die Substratspezifität des Enzymkomplexes PqsBC untersuchten. Es wurde nachgewiesen, dass PqsBC verschiedene mittelkettige Acyl-Coenzym-A-Substrate für die Kondensation mit 2-Aminobenzoylacetat akzeptiert und dadurch Alkylchinolone mit verschiedenen Seitenkettenlängen produziert, deren Verteilung eine Funktion der Substratspezifität und der Substratverfügbarkeit ist. Zudem konnte gezeigt werden, dass PqsBC auch Alkylchinolone mit ungesättigten Seitenketten synthetisiert. Im Weiteren wurde der Fokus von Metaboliten- und Stoffwechselweg-zentrierten Fragen hin zu einer globaleren Perspektive der pseudomonalen Virulenz und des Metabolismus erweitert, was die Aufmerksamkeit auf PrmC lenkte, ein Enzym mit teilweise unbekannter, für die in vivo-Virulenz unverzichtbarer Funktion. Ein globales Metabolomik-Experiment lieferte Einblicke in die Rolle von PrmC und seinen Einfluss auf das pseudomonale Endo- und Exometabolom. Schließlich wurden klinische P. aeruginosa-Stämme mit unterschiedlichen Virulenzphänotypen mittels ungerichteter Metabolomik untersucht, um metabolische Variationen und Zusammenhänge zwischen Virulenz und Metabolismus aufzudecken. Die Analyse resultierte in der Entdeckung eines putativen Virulenzbiomarkers und ermöglichte die Konstruktion eines Random-Forest-Klassifikationsmodells für bestimmte Virulenzphänotypen, das nur auf Metabolomik-Daten basiert. Zusammenfassend hat diese Studie das Potenzial der Metabolomik für die Untersuchung der Virulenzfaktoren von P. aeruginosa aufgezeigt und damit zum Verständnis des komplexen Zusammenspiels von Metabolismus und Virulenz bei diesem wichtigen Pathogen beigetragen

Consensus on Wound Antisepsis: Update 2018

Wound antisepsis has undergone a renaissance due to the introduction of highly effective wound-compatible antimicrobial agents and the spread of multidrug-resistant organisms (MDROs). However, a strict indication must be set for the application of these agents. An infected or critically colonized wound must be treated antiseptically. In addition, systemic antibiotic therapy is required in case the infection spreads. If applied preventively, the Wounds-at-Risk Score allows an assessment of the risk for infection and thus appropriateness of the indication. The content of this updated consensus recommendation still largely consists of discussing properties of octenidine dihydrochloride (OCT), polihexanide, and iodophores. The evaluations of hypochlorite, taurolidine, and silver ions have been updated. For critically colonized and infected chronic wounds as well as for burns, polihexanide is classified as the active agent of choice. The combination 0.1% OCT/phenoxyethanol (PE) solution is suitable for acute, contaminated, and traumatic wounds, including MRSA-colonized wounds due to its deep action. For chronic wounds, preparations with 0.05% OCT are preferable. For bite, stab/puncture, and gunshot wounds, polyvinylpyrrolidone (PVP)-iodine is the first choice, while polihexanide and hypochlorite are superior to PVP-iodine for the treatment of contaminated acute and chronic wounds. For the decolonization of wounds colonized or infected with MDROs, the combination of OCT/PE is preferred. For peritoneal rinsing or rinsing of other cavities with a lack of drainage potential as well as the risk of central nervous system exposure, hypochlorite is the superior active agent. Silver-sulfadiazine is classified as dispensable, while dyes, organic mercury compounds, and hydrogen peroxide alone are classified as obsolete. As promising prospects, acetic acid, the combination of negative pressure wound therapy with the instillation of antiseptics (NPWTi), and cold atmospheric plasma are also subjects of this assessment

A moonlighting enzyme imposes second messenger bistability to drive lifestyle decisions in E. coli.

Bacteria preferentially colonize surfaces and air-liquid interfaces as matrix embedded communities called biofilms. Biofilms exhibit specific physiological properties, including general stress tolerance, increased antibiotic recalcitrance and tolerance against phagocytic clearance. Together this largely accounts for increased biofilm persistence, chronic infections and infection relapses. One of the principle regulators of biofilm formation is c-di-GMP, a bacterial second messenger controlling various cellular processes. Cellular levels of c-di-GMP are controlled by two antagonistic enzyme families, diguanylate cyclases and phosphodiesterases. But despite the identification and characterization of an increasing number of components of the c-di-GMP network in different bacterial model organisms, details of c-di- GMP mediated decision-making have remained unclear. In particular, how cells shuttle between specific c-di-GMP regimes at the population and single cell level is largely unknown and moreover how these transitions are deterministically made in time and space, given that bacterial networks of diguanylate cyclases and phosphodiesterases show a high degree of complexity. Here we describe a novel mechanism regulating c-di-GMP mediated biofilm formation in E. coli. This mechanism relies on the bistable expression of a key phosphodiesterase that acts both as catalyst for c- di-GMP degradation and as a transcription factor promoting its own production. Bistability results from two interconnected positive feedback loops operating on the catalytic and gene expression level. Based on genetic, structural and biochemical analyses we postulate a simple substrate-induced switch mechanism through which this enzyme can sense changing concentration of c-di-GMP and convert this information into a bistable c-di-GMP response. This mechanism may explain how cellular heterogeneity of small signaling molecules is generated in bacteria and used as a bet hedging strategy for important lifestyle transitions

Pili: the microbes' Swiss army knifes

Surface attachment is the crucial first step for a single cell transitions from a planktonic to a surface associated state, which can lead to the development of multicellular communities called biofilms. Microbes extensively use pili for initial surface attachment. Pili are filamentous appendages that extend into the extracellular environment and can attach to a wide range of surfaces. This Thesis contributes to the understanding of how pili work and how bacteria transition from a planktonic to a surface bound life style. This will aid future development in creating new ways to prevent bacterial attachment and biofilm formation and thereby avoid the necessity for the removal of fully developed biofilms which often requires harsh physical and chemical treatments which can be impractical in a biomedical context. We used single cell studies, microfluidic methods and quantitative computational analysis to study in detail the mechanism of pili-mediated attachment in Caulobacter crescentus and Pseudomonas aeruginosa. In C. crescentus we confirm the recently described ability of pili to retract, which was previously considered not possible for this type of pili. We characterized this functionality in greater detail and our results highlight the importance of pili in reorienting cells and bringing the cell body closer to surfaces, whereby cells can promote long term attachment by secreting a glue-like substance called holdfast. We also investigated the role of the second messenger c-di-GMP during pilimediated cell attachment and biofilm formation. We show a novel role for c-di-GMP in directly regulating elongation and retraction of pili in C. crescentus and P. aeruginosa. In P aeruginosa a novel c-di-GMP effector, FimW, regulates surface attachment and walking behaviour, and how its asymmetric distribution drives surface colonization. In C. crescentus we show that c-di-GMP positively regulates attachment. We manipulated a key component of the secretion machinery, HfsK, and show that c-di-GMP not only regulates the timing of holdfast synthesis, but also its cohesion and adhesion properties. Lastly, we report a novel protein, PdeL, which is both a phosphodiesterase and a transcriptional factor that regulates the expression of biofilm related genes in Escherichia coli. In the appendixes we describe in detail the process for creating microfluidic devices, extensively used in the studies described in this thesis. Moreover, we include a manual for the use of WHISIT, a custom-made software program for the analysis of bacterial fluorescent signals in an automated and quantitative approach

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

Einbau von prosthetischem Häm in cytoplasmatische und membrangebundene Proteine

Tetrapyrroles are macromolecules involved in nearly all fundamental biologically processes and further represent essential components of many organisms metabolism. They play a central role in electron transfer-dependent, energy-generating processes like photosynthesis and respiration. Heme, an iron containing tetrapyrrole acts as a prosthetic group for e. g. hemoglobin, which mediates oxygen transport in the blood. It was investigated how heme arrives at its functionally destination in cytoplasmic or membrane-bound proteins. The aim of this study was the identification of heme-binding or transporting proteins in vivo by a Pseudomonas aeruginosa "Bacterial Adenylate Cyclase Two-Hybrid System". By means of these screenings a possible heme-transporter was isolated among many hydrophobic so far not characterized proteins. Further studies revealed this protein to be an iron-transporter, potentially responsible for iron transport to ferrochelatase. In a second project the E. coli protein HemW was to be biochemically and biophysically characterized. A Lactococcus lactis hemW mutant was known to accumulate free heme in the cell however not able to respire on supplementation with heme. An E. coli hemW mutant was successfully complemented with the hemW gene of L. lactis. The E. coli protein HemW was isolated under anaerobic conditions and chromatographically purified HemW contains a [4Fe-4S] cluster, like its L. lactis relative. Furthermore HemW dimerizes and binds heme specifically and covalently. During this binding-process an electron transfer from the [Fe-S] cluster to an as of yet to be identified bound heme takes place.Tetrapyrrole sind Makromoleküle, die an fast allen fundamentalen biologischen Prozessen beteiligt sind und darüber essentielle Komponenten des Stoffwechsels vieler Organismen auf der Erde darstellen. Sie spielen eine zentrale Rolle in elektronentransfer-abhängigen, Energie-generierenden Prozessen wie der Photosynthese und Atmung. Das Fe-haltige Tetrapyrrol Häm repräsentiert u. a. die prosthetische Gruppe von Hämoglobin, das für den Sauerstofftransport im Blut verantwortlich ist. In dieser Arbeit wurde untersucht wie das Häm an seinen funktionellen Zielort in cytoplasmatische oder membrangebundene Proteine gelangt. Ziel der Arbeit war die Identifikation Häm-bindender oder Häm-transportierender Proteine in vivo mittels des Pseudomonas aeruginosa "Bacterial Adenylate Cyclase Two-Hybrid Systems". Mit Hilfe dieses Screenings konnte neben vielen hydrophoben nicht-bisher charakterisierten Proteinen ein Kandidat isoliert werden, der sich als möglicher Häm-Transporter zeigte. In weiteren Untersuchungen stellte sich dieses Protein als potentieller Eisen-Transporter heraus, der für den Fe-Transport zur Ferrochelatase verantwortlich sein könnte. In einem zweiten Projekt sollte ein Protein, E. coli HemW, biochemisch und biophysikalisch untersucht werden. Von dem Lactobacillus Lactococcus lactis war eine hemW Mutante bekannt, die freies Häm in der Zelle akkumulierte und auch durch Supplementation von Häm nicht respirieren konnte. Es gelang zudem eine E. coli hemW Mutante mit dem L. lactis hemW-Gen zu komplementieren. Das E. coli Protein HemW wurde unter anaeroben Bedingungen isoliert und chromatographisch gereinigt. E. coli HemW verfügt wie der Lactobacillus-Verwandte über ein [4Fe-4S] Cluster. Desweiteren dimerisiert HemW und es verfügt über die Fähigkeit Häm spezifisch und kovalent zu binden. Während dieser Bindung erfolgt eine Elektronenübertragung vom [Fe-S] Cluster auf das gebundene Häm