Innate immune recognition of Salmonella and Francisella : two model intracellular bacterial pathogens

The innate immune system is the first line of host defense against invading pathogens. In multicellular organisms, specialized innate immune cells recognize conserved pathogen-associated molecular patters (PAMPs) with germ-line encoded pattern recognition receptors (PRR). Thereby, the organism discriminates between self and non-self and engages mechanisms to eliminate the invader. Beside PAMPs, PRRs recognize mislocalized self-molecules, so called danger-associated molecular patterns (DAMPs), which are indicators of tissue or cellular damage. Upon PAMP or DAMP recognition, PRRs induce innate immune signaling pathways leading to the activation of pro-inflammatory genes and interferon production, which are important mediators of inflammation. Therefore the recognition of invading pathogen and thereby activation of innate immune signaling pathways determines the success of the immune system to eliminate the potential threat. Innate immune signaling pathways largely depend on phosphorylation cascades. Today, global phosphorylation changes are analyzed by mass spectrometry, however the number of detected phosphopeptides remains unchanged despite technical improvements. Therefore, we investigated the issue of phosphopeptide detection in mass spectrometry. The analyses of phosphopeptide-enriched samples have revealed lower signal intensities in MS1 spectra compared to total cell lysate samples, which results in poor phosphopeptide detection with mass spectrometry. Based on these observations, we hypothesized that the phosphate groups of phosphopeptides account for this poor detection. Indeed, we significantly increase the signal intensities in MS1 spectra after enzymatic removal of phosphate groups from phosphopeptides, and consequently we detect three-times more peptides in phosphatase-treated samples. Validation experiments elucidate that most of the newly detected peptides have been initially phosphorylated. Moreover, the newly detected peptides enlarge the activated signaling network upon Salmonella infection. Importantly, we identify known innate immune signaling pathways, which were missing in the analyses of phospho-enriched samples. Taken together, the phosphate groups of phosphopeptides globally suppress peptide ionization efficacy and therefore account for the low phosphopeptide detection rate by mass spectrometry. By removing the phosphate groups, we identify three times more peptides after phosphatase treatment. The newly detected peptides enlarge the network of activated innate immune signaling pathways upon Salmonella infection and include signaling pathways that are important but have not been detected in phospho-enriched samples. Therefore our findings improve the analyses of innate immune signaling pathways by mass spectrometry and consequently the understanding of innate immunity. One of the main mechanisms to eliminate invading microbes is by phagocytosis and degradation within phago-lysosomes. However, professional pathogens have developed various defense mechanisms to resist intracellular killing and can even use innate immune cells as replicative niches. For example, the bacterial pathogen Francisella tularensis causes a severe and life-threatening disease called tularemia in humans, because Francisella can survive and replicate in macrophages and dendritic cells. Critical for Francisella pathogenicity is the ability of the phagocytosed bacteria to escape from the phagosome to the host cytosol. Even though we know that genes encoded on the Francisella pathogenicity island (FPI) are essential for escaping from the phagosome, the mechanism is unknown. Homology analyses have suggested that the FPI encodes a type 6 secretion system (T6SS). However experimental evidence is missing, which show that the FPI encode a functional T6SS. Therefore, we investigated whether the FPI encodes a functional T6SS and what impact a functional T6SS has on Francisella virulence in vitro and in vivo. We show that the FPI of Francisella novicida (F. novicida) encodes a functional T6SS that assembles exclusively at bacterial poles. T6SS function depends on the unfoldase ClpB, which specifically recognizes contracted T6SS sheaths leading to their disassembly. Furthermore we have characterized FPI genes that show no homology with known T6SSs. We have identified IglF, IglG, IglI and IglJ as structural components of the T6SS and PdpC, PdpD, PdpE and AnmK as potential T6SS effector proteins. Whereas PdpE and AnmK are dispensable for phagosomal escape, AIM2 inflammasome activation and virulence in mice, pdpC- and pdpD-deficient bacteria are impaired in all aforementioned analyses. This suggests that PdpC and PdpD are bacterial effector proteins involved in phagosomal escape and thereby in the establishment of a F. novicida infection. Taken together, F. novicida uses its T6SS to deliver the effector proteins PdpC and PdpD into host cells. PdpC and PdpD are involved in phagosomal rupture and consequently in bacterial escape to the cytosol. These findings are a major breakthrough in the understanding of Francisella pathogenicity and could lead to new vaccination strategies to eradicate the life-threatening human disease Tularemia

Bio-Inspired Soft Artificial Muscles for Robotic and Healthcare Applications

Soft robotics and soft artificial muscles have emerged as prolific research areas and have gained substantial traction over the last two decades. There is a large paradigm shift of research interests in soft artificial muscles for robotic and medical applications due to their soft, flexible and compliant characteristics compared to rigid actuators. Soft artificial muscles provide safe human-machine interaction, thus promoting their implementation in medical fields such as wearable assistive devices, haptic devices, soft surgical instruments and cardiac compression devices. Depending on the structure and material composition, soft artificial muscles can be controlled with various excitation sources, including electricity, magnetic fields, temperature and pressure. Pressure-driven artificial muscles are among the most popular soft actuators due to their fast response, high exertion force and energy efficiency. Although significant progress has been made, challenges remain for a new type of artificial muscle that is easy to manufacture, flexible, multifunctional and has a high length-to-diameter ratio. Inspired by human muscles, this thesis proposes a soft, scalable, flexible, multifunctional, responsive, and high aspect ratio hydraulic filament artificial muscle (HFAM) for robotic and medical applications. The HFAM consists of a silicone tube inserted inside a coil spring, which expands longitudinally when receiving positive hydraulic pressure. This simple fabrication method enables low-cost and mass production of a wide range of product sizes and materials. This thesis investigates the characteristics of the proposed HFAM and two implementations, as a wearable soft robotic glove to aid in grasping objects, and as a smart surgical suture for perforation closure. Multiple HFAMs are also combined by twisting and braiding techniques to enhance their performance. In addition, smart textiles are created from HFAMs using traditional knitting and weaving techniques for shape-programmable structures, shape-morphing soft robots and smart compression devices for massage therapy. Finally, a proof-of-concept robotic cardiac compression device is developed by arranging HFAMs in a special configuration to assist in heart failure treatment. Overall this fundamental work contributes to the development of soft artificial muscle technologies and paves the way for future comprehensive studies to develop HFAMs for specific medical and robotic requirements

Genomic comparison of novel Staphylococcus aureus bacteriophage and their anti-biofilm properties against MRSA sequence type 22 and 36

Staphylococcus aureus (including methicillin-resistant S. aureus - MRSA) remains a leading cause of both nosocomial and community acquired infections globally and despite constant improvement efforts to patient safety within healthcare, it still remains associated with considerable rates of morbidity and mortality. S. aureus is a common cause of biofilm-associated infections observed in chronic wounds, exhibiting a reduced susceptibility to the action of conventional antimicrobial agents and are often difficult to eradicate. The acquisition of resistance to almost any antibiotic with reference to MRSA has greatly reduced the number of alternative antimicrobial agents effective in the treatment of infections. Current development pipeline for new classes of antibiotics are greatly limited, requiring new, alternative approaches for therapeutic and prophylactic intervention in attempt to effectively control and overcome this current global health threat. Bacteriophage therapy exploits the natural killing ability of lytic bacteriophage (phage) as a means of controlling multidrug-resistant pathogenic bacteria. The utility of phage and their derivatives has been shown to effectively reduced the biofilms of major MRSA clones in vitro and in vivo. Global MRSA infections are caused by highly-successful isolates from a small number of epidemic lineages (clones). ST22 and ST36 are two of the most prevalent clones with global impact and largely responsible for the national epidemic of MRSA infections within UK healthcare system throughout the mid-1990s up until the mid-2000s. Understanding the phenotypic and genotypic characteristics of these clones in relation to the ability of bacteriophage to infect and disrupt established biofilms has yet to be explored. In this study, a total of 46 novel obligately lytic phage were isolated from wastewater samples by utilising a modified Staphylococcus carnosus TM300 isolate with expressed S. aureus wall teichoic acids to aid in phage adsorption. The addition of 32 more phage from our current laboratory stocks helped to establish a collection of 78 phage that were screened against a panel of 185 genetically diverse S. aureus, consisting of major clonal groups with high prevalence within the UK and United States, including 43 ST22 and 24 ST36 strains. The majority of the members displayed a wide host range against our panel. Based on this, the four most effective (wide host-range) phage were assessed for their anti-biofilm properties in polystyrene plates biofilm assays produced using four ST22 and four ST36 isolates. Treatment of mature biofilms was shown to significantly reduce biofilm biomass and viable cell counts. However these assays selected for the emergence of phage resistant mutants. Whole genome sequencing was performed on 22 phage isolates and these were found to share a high degree of similarity to genomes of 38 previously classified Twortvirinae phages represented in GenBank. Comparisons of these 60 phage genomes found a surprisingly high level of genetic diversity. Pairwise distances resolved groups of phage in distinct clusters representing individual genera within the Twortvirinae subfamily. Pan-genome analysis identified no single gene present amongst all phage genomes, however phage displayed a core genome amongst other members of the cluster. The structural homology tool HHpred was used to predict the protein structure of genes encoding for lytic enzymes among our phage genomes. We found that all phage encode a protein that shares high structural similarity to the same CHAP domain protein, a catalytic domain of endolysins employed by phage to degrade the bacterial host cell wall in order thus, mediate cell lysis. Suggesting that the all phage most likely share the same catalytic N-terminal endopeptidase domain of endolysins which have a modular domain structure. Interestingly, endolysins have been proposed as possible candidates for the control of antibiotic resistant S. aureus infections

Bacteriophages

Bacteriophages have received attention as biological control agents since their discovery and recently their value as tools has been further emphasized in many different fields of microbiology. Particularly, in drug design and development programs, phage and prophage genomics provide the field with new insights. Bacteriophages reveals information on the organisms ranging from their biology to their applications in agriculture and medicine. Contributors address a variety of topics capturing information on advancing technologies in the field. The book starts with the biology and classification of bacteriophages with subsequent chapters addressing phage infections in industrial processes and their use as therapeutic or biocontrol agents. Microbiologists, biotechnologists, agricultural, biomedical and sanitary engineers will find Bacteriophages invaluable as a solid resource and reference book

Conformable light emitting modules

As we become increasingly aware that there is more to light than the image it forms on our retina, and as we become more environmentally aware, the value of non-image-forming light increases along with the need for various new light related appliances. In particular, some lighting related applications are emerging which demand conformability (flexibility and stretchability). Well-being, automotive or wearable electronic applications are just a few examples where these trends can be observed. We are finding that conformability could bring various benefits to both users (tactile and optical comfort, optical efficiency, multi-functionality, work/living space savings) as well as manufacturers (heterogeneous integration, light-weight, design freedom, differentiation and less stringent tolerancing). Developed by Ghent University, the SMI (Stretchable Molded Interconnect) technology attempts to address these demands and has been the main focus of this work. With the SMI technology it was possible to design highly conformable circuits using fabrication methods similar to these found in the PCB and FCB industries and standard off-the-shelf electronic components. The goal of this work was to characterize the technology materials in terms of mechanical, optical and reliability performance as well as define a set of design rules to support creation of robust and efficient light modules, also using a set of new, commercially available elastomeric, polymer materials. The developments are illustrated with demonstration devices

NASA Thesaurus. Volume 2: Access vocabulary

The NASA Thesaurus -- Volume 2, Access Vocabulary -- contains an alphabetical listing of all Thesaurus terms (postable and nonpostable) and permutations of all multiword and pseudo-multiword terms. Also included are Other Words (non-Thesaurus terms) consisting of abbreviations, chemical symbols, etc. The permutations and Other Words provide 'access' to the appropriate postable entries in the Thesaurus

Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts

© 2018 American Chemical Society. Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo typesñYsmall molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications and challenges to implementation. Throughout, we highlight specific mechanisms of membrane disruption and suggest areas in need of further experimentation. We hope the concepts discussed in our review inspire scientists and engineers with further ideas to improve intracellular delivery

R-Pyocin Regulation, Release, and Susceptibility in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a major determinant of declining lung function in individuals with cystic fibrosis (CF). P. aeruginosa possesses many intrinsic antibiotic resistance mechanisms and isolates from chronic CF lung infections develop increasing resistance to multiple antibiotics over time. Chronic infection with P. aeruginosa remains one of the main causes of mortality and morbidity in CF patients, thus new therapeutic interventions are necessary. R-type pyocins are narrow spectrum, phage tail-like bacteriocins, specifically produced by P. aeruginosa to kill other strains of P. aeruginosa. Due to their specific anti-pseudomonal activity and similarity to bacteriophage, R-pyocins have potential as additional therapeutics for P. aeruginosa, either in isolation, in combination with antibiotics, or as an alternative to phage therapy. There are five subtypes of R-pyocin (types R1-R5), and it is thought that each P. aeruginosa strain uniquely produces only one of these, suggesting a degree of strain-specificity. P. aeruginosa from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. It is known P. aeruginosa populations in CF chronic lung infection become phenotypically and genotypically diverse over time, however, little is known of the efficacy of R-pyocins against heterogeneous populations. Even less is known regarding the timing and regulation of R-pyocins in CF lung infections, or if P. aeruginosa utilizes R-pyocin production during infection for competition or otherwise – which may influence pressure towards R-pyocin resistance. In this work, I evaluated R-pyocin type and susceptibility among P. aeruginosa isolates sourced from CF infections and found that (i) R1-pyocins are the most prevalent R-type among respiratory infection and CF strains; (ii) a large proportion of P. aeruginosa strains lack R-pyocin genes entirely; (iii) isolates from P. aeruginosa populations collected from the same patient at a single time point have the same R-pyocin type; (iv) there is heterogeneity in susceptibility to R-pyocins within P. aeruginosa populations and (v) susceptibility is likely driven by diversity of LPS phenotypes within clinical populations. These findings suggest that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage, which is important for consideration of antimicrobials as therapeutics. To investigate the prevalence of R2-pyocin susceptible strains in CF, I then utilized 110 isolates of P. aeruginosa collected from five individuals with CF to test for R2-pyocin susceptibility and identify LPS phenotypes. From our collection we i) estimated that approximately 83% of sputum samples contain heterogenous P. aeruginosa populations without R2-pyocin resistant isolates and all sputum samples contained susceptible isolates; ii) we found that there is no correlation between R2-pyocin susceptibility and LPS phenotypes, and iii) we estimate that approximately 76% of isolates sampled from sputum lack O-specific antigen, 42% lack common antigen, and 27% exhibit altered LPS cores. This finding highlights that perhaps LPS packing density may play a more influential role in mediating R-pyocin susceptibility in infection. Finding the majority of our sampled P. aeruginosa populations to be R2-pyocin susceptible further supports the potential of these narrow-spectrum antimicrobials despite facing heterogenous susceptibility among diverse populations. In order to evaluate how R-pyocins may influence strain competition and growth in CF lung infection, I assessed R-pyocin activity in an infection-relevant environment (Synthetic Cystic Fibrosis Sputum Medium; SCFM2) and found that (i) R-pyocins genes are transcribed more in the CF nutrient environment than in rich laboratory medium and (ii) in a structured, CF-like environment, R-pyocin induction is costly to producing strains in competition rather than beneficial. Our work suggests that R-pyocins may not be essential in CF lung infection and can be costly to producing cells in the presence of stress response-inducing stimuli, such as those commonly found in infection. In this thesis I have studied R-pyocin susceptibility, regulation and release utilizing a biobank of whole populations of P. aeruginosa collected from 11 individuals with CF, as well as the CF infection model (SCFM) to understand the mechanisms of R-pyocin activity in an infection-relevant context and the role R-pyocins play in shaping P. aeruginosa populations during infection. The findings of this work have illuminated the impact of P. aeruginosa heterogeneity on R-pyocin susceptibility, furthered our understanding of R-pyocins as potential therapeutics, and built upon our knowledge of bacteriocin-mediated interactions.Ph.D

Genetic analysis of developmental traits associated with enhanced winter survival in autumn-seeded rye (Secale cereale L.).

The abstract of this item is unavailable due to an embargo