Innate immune evasion of microbes

The environment is dominated by bacteria and every day our body has to defend itself against microbes. Since our immune system is very effective only the minority of microbes is able to enter and survive within the body. The complement system is the first line of innate immunity. It is able to recognize and distinguish between the host s own cells and non-host, such as microbes, via activation of three pathways: classical, lectin, and alternative pathway. All the pathways lead to C3b deposition onto target cell surfaces and eventually elimination of the target. To avoid destruction of host cells complement has to be strictly regulated and the main regulator of the alternative pathway is the elongated protein composed of twenty domains, factor H. Several microbes acquire host factor H via domains 5-7 and 19-20 onto their surfaces to protect themselves against complement attack. There is an increasing number of microbes with resistance against several antimicrobial agents. Therefore a detailed knowledge of host-pathogen interactions on structural and functional levels is important for the development of novel mechanisms to fight microbial infections. In this work we found that Bordetella pertussis and the closely related B. parapertussis utilized host factor H to protect themselves against complement attack. Furthermore, we revealed that pathogens representing Gram-positive and Gram-negative bacteria, and a yeast, bound factor H via a conserved binding site on the domain 20. By utilizing the specific binding site the microbial proteins enhanced the interaction between C3b and factor H. This resulted in a more efficient inactivation of C3b and subsequently enhanced evasion of complement mediated damage. While the number of pathogens reported to bind factor H is increasing, it is known that some pathogens such as Staphylococcus aureus do not bind factor H but instead bind C3b via secreted molecules, i.e. extracellular complement binding protein (Ecb). We showed that factor H deposition on the surface of S. aureus could be induced by the formation of tripartite complexes between Ecb, C3b, and factor H. The results revealed that S. aureus is able to utilize host factor H in three ways - to promote the tripartite complex formation so that the C3 convertase cannot be formed, to prevent complement receptor 1 -binding leading to impaired opsonophagocytosis, and to prevent degradation of C3b to iC3b thereby preventing recognition of S. aureus by phagocytic receptors. The results presented in this thesis provide detailed understanding on host- or pathogen interactions on structural and functional basis that contribute to the development of new vaccines and antimicrobial compounds.Miljön domineras av bakterier och varje dag måste kroppen vår försvara sig mot mikrober. Eftersom immunförsvaret vårt är mycket effektivt vill endast en minoritet av mikroberna komma in och överleva i kroppen. Medfödd immunitet är kroppens första försvarslinje där komplementsystemet utgör en viktig del. Komplement kan känna igen och skilja mellan kroppens egna celler och celler som inte hör hemma i kroppen, såsom mikrober, genom aktivering av tre reaktionsvägar: klassisk, lektin, och den alternativa vägen. Alla vägarna leder till C3b avsättning på målcellens yta och slutligen eliminering av cellen. För att undvika eliminering av kroppens egna celler måste komplement regleras strikt och den huvudsakliga regulatorn av den alternativa vägen är et protein som heter faktor H. Faktor H består av tjugo domäner. Flera mikrober skyddar sig mot komplementattack ved att binda faktor H via domänerna 5-7 och 19-20 på sina ytor. Det finns ett ökande antal mikrober med resistens mot flera antimikrobiella medel. Därför är det viktig med detaljerad kunskap om värd-patogen interaktioner för att kunna utveckla nya mekanismer som kan bekämpa mikrobiella infektioner. I detta arbete fann vi att Bordetella pertussis och den närbesläktade B. parapertussis skyddade sig mot komplementattack ved att förvärva faktor H på deras ytor. Vidare upptäckte vi att olika patogener (Gram-positiva och Gram negativa bakterier och en jäst), binder faktor H via et specifikt bindningsställe på domänen 20. Genom att utnyttja det specifika bindningsstället förbättrar de mikrobiella proteinerna interaktionen mellan C3b och FH. Detta resulterade i en verkningsfull inaktivering av C3b och komplement. Medan antalet patogener som rapporterats binda faktor H ökar, är det känt att vissa patogener, såsom Staphylococcus aureus, inte binder faktor H utan istället binder C3b via utsöndrade molekyler, till expempel extracellular complement binding protein (Ecb). Vi visade att avsättning av faktor H på ytan av S. aureus kunde induceras genom bildande av et komplex mellan Ecb, C3b och faktor H. Resultaten visade att S. aureus kan utnyttja faktor H på tre olika sätt: 1) främja bildande av komplexet (Ecb, C3b, faktor H) så att enzymet som klyver C3 inte kan bildas, för att förhindra komplementreceptor 1 -bindning vilket leder till försämrad upptag genom fagocytos, och för att förhindra nedbrytningen av C3b till iC3b därigenom förhindra igenkännande av S. aureus av receptorer på fagocyter. Sammanfattningsvis har denna avhandling gett en förbättrat förståelse om interaktioner mellan värd och patogen på strukturella och funktionella nivå som möjligtvis kan användas för att utveckla nya vaccin och antimikrobiella medel

Staphylococcal protein Ecb impairs complement receptor-1 mediated recognition of opsonized bacteria

Staphyloccus aureus is a major human pathogen leading frequently to sepsis and soft tissue infections with abscesses. Multiple virulence factors including several immune modulating molecules contribute to its survival in the host. When S. aureus invades the human body, one of the first line defenses is the complement system, which opsonizes the bacteria with C3b and attract neutrophils by release of chemotactic peptides. Neutrophils express Complement receptor-1 [CR1, CD35) that interacts with the C3b-opsonized particles and thereby plays an important role in pathogen recognition by phagocytic cells. In this study we observed that a fraction of S. aureus culture supernatant prevented binding of C3b to neutrophils. This fraction consisted of S. aureus leukocidins and Efb. The C-terminus of Efb is known to bind C3b and shares significant sequence homology to the extracellular complement binding protein [Ecb). Here we show that S. aureus Ecb displays various mechanisms to block bacterial recognition by neutrophils. The presence of Ecb blocked direct interaction between soluble CR1 and C3b and reduced the cofactor activity of CR1 in proteolytic inactivation of C3b. Furthermore, Ecb could dose-dependently prevent recognition of C3b by cell-bound CR1 that lead to impaired phagocytosis of NHS-opsonized S. aureus. Phagocytosis was furthermore reduced in the presence of soluble CR1 [sCR1). These data indicate that the staphylococcal protein Ecb prevents recognition of C3b opsonized bacteria by neutrophil CR1 leading to impaired killing by phagocytosis and thereby contribute to immune evasion of S. aureus.Peer reviewe

Developing a Multivariate Prediction Model for Sleep Apnea from Polysomnography Data

Recent years have seen a rapid expansion of consumer sleep monitoring devices, using a wide variety of cheap and accessible sensor technology. However, these products tend to be seriously lacking in terms of validation, clinical or at all, and have very limited medicinal usefulness. On the other side, the gold standard of sleep medicine is polysomnography, a highly comprehensive and resource intensive method that sacrifices any and all practicality for its high accuracy. The essential motivation behind this thesis is a desire to unite these two worlds through extensive validation and multivariate data modelling techniques. This thesis focuses on building a predictive model for sleep apnea, a sleep disorder characterized by cessation of or periods of shallow breathing during sleep. The work consists of accessing and exporting anonymized polysomnography recordings done at St. Olav's hospital between 2012 and 2015, as well as reconfiguring the data to a more well-structured format. A selection of preprocessing and data reduction techniques are described and implemented, and multivariate analysis and modelling is performed on several different configurations of the data. Using a subset of 10 sensors from the polysomnography data and Partial Least Squares Regression, a model with good predictive ability for a characteristic of sleep apnea is achieved, that also performs well under validation. However, more data would have to be included before any proper conclusions can be drawn. We therefore end the work by proposing an outline for an extensive research project involving both polysomnography scored by trained experts and a selection of commercial wearable physiological monitors, which might eventually lead to a commercial at-home sleep monitor with a validated clinical ability to recognize respiratory disturbances in its users

A pilot study of impulse radio ultra wideband radar technology as a new tool for sleep assessment

Study Objectives To validate Impulse radio ultra wideband pulse-doppler radar technology against polysomnography (PSG) for sleep assessment. Methods In all, 12 participants were recruited and their overnight sleep was assessed both by a Novelda XeThru radar and PSG. Two subjects had two nightly recordings, whereas 10 had one recording. Epoch by epoch (30 seconds) comparisons from bedtime to rise time were conducted. Concordance was estimated in terms of the mean difference between the radar and the PSG estimates regarding sleep onset latency, wake time after sleep onset and total sleep time. In addition, accuracy, sensitivity, specificity and Cohen kappa were calculated. Results The mean difference (minutes) between the radar and the PSG registrations was −5.7 minutes (standard deviation [SD] = 22.1 minutes) for sleep onset latency, 6.4 minutes (SD = 32.5 minutes) for wake after sleep onset, and 1.5 minutes (SD = 24.6 minutes) for total sleep time. The mean values obtained for accuracy, sensitivity, specificity and Cohen kappa were 0.931, 0.961, 0.695 and 0.670, respectively. Conclusion Impulse radio ultra wideband radar technology is a promising tool in terms of affordable and practical objective sleep assessment. Further technical development and more validation studies are needed in order to conclude about the utility potential of this device

A pilot study of impulse radio ultra wideband radar technology as a new tool for sleep assessment

Study Objectives: To validate Impulse radio ultra wideband pulse-doppler radar technology against polysomnography (PSG) for sleep assessment. Methods: In all, 12 participants were recruited and their overnight sleep was assessed both by a Novelda XeThru radar and PSG. Two subjects had two nightly recordings, whereas 10 had one recording. Epoch by epoch (30 seconds) comparisons from bedtime to rise time were conducted. Concordance was estimated in terms of the mean difference between the radar and the PSG estimates regarding sleep onset latency, wake time after sleep onset and total sleep time. In addition, accuracy, sensitivity, specificity and Cohen kappa were calculated. Results: The mean difference (minutes) between the radar and the PSG registrations was −5.7 minutes (standard deviation [SD] = 22.1 minutes) for sleep onset latency, 6.4 minutes (SD = 32.5 minutes) for wake after sleep onset, and 1.5 minutes (SD = 24.6 minutes) for total sleep time. The mean values obtained for accuracy, sensitivity, specificity and Cohen kappa were 0.931, 0.961, 0.695 and 0.670, respectively. Conclusion: Impulse radio ultra wideband radar technology is a promising tool in terms of affordable and practical objective sleep assessment. Further technical development and more validation studies are needed in order to conclude about the utility potential of this devic

Staphylococcal protein Ecb impairs complement receptor-1 mediated recognition of opsonized bacteria

Staphyloccus aureus is a major human pathogen leading frequently to sepsis and soft tissue infections with abscesses. Multiple virulence factors including several immune modulating molecules contribute to its survival in the host. When S. aureus invades the human body, one of the first line defenses is the complement system, which opsonizes the bacteria with C3b and attract neutrophils by release of chemotactic peptides. Neutrophils express Complement receptor-1 [CR1, CD35) that interacts with the C3b-opsonized particles and thereby plays an important role in pathogen recognition by phagocytic cells. In this study we observed that a fraction of S. aureus culture supernatant prevented binding of C3b to neutrophils. This fraction consisted of S. aureus leukocidins and Efb. The C-terminus of Efb is known to bind C3b and shares significant sequence homology to the extracellular complement binding protein [Ecb). Here we show that S. aureus Ecb displays various mechanisms to block bacterial recognition by neutrophils. The presence of Ecb blocked direct interaction between soluble CR1 and C3b and reduced the cofactor activity of CR1 in proteolytic inactivation of C3b. Furthermore, Ecb could dose-dependently prevent recognition of C3b by cell-bound CR1 that lead to impaired phagocytosis of NHS-opsonized S. aureus. Phagocytosis was furthermore reduced in the presence of soluble CR1 [sCR1). These data indicate that the staphylococcal protein Ecb prevents recognition of C3b opsonized bacteria by neutrophil CR1 leading to impaired killing by phagocytosis and thereby contribute to immune evasion of S. aureus

Effect of Ecb on binding of C3b to human red blood cells.

<p><i>A</i>, RBC were incubated with C3b with or without Ecb. After washings, C3b bound to RBC was detected with flow cytometry using an antibody recognizing the C3c fragment (FI, fluorescence intensity). The assay was performed three times with duplicates and mean SD values are shown. <i>B</i> is a representative histogram of RBC incubated with C3b in the presence of Ecb (0–1.5 and 0–1.8, respectively). Student’s two-tailed <i>t</i>-test was used to determine the statistical significances (* <i>p</i><0.05; ** <i>p</i><0.01).</p