Macrophage – cryptococcus interactions during cryptococcosis

The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic effector cells. Cryptococcus can survive and proliferate within macrophages, and is also capable of escaping into the intracellular environment via a non-lytic mechanism (‘expulsion’) and can be transferred directly from one cell to another (lateral transfer). In the first part of this thesis, I demonstrate that enhanced Th2, but not Th1, cytokine levels lead to increased intracellular cryptococcal proliferation but lower levels of cryptococcal expulsion. In the second part, I describe the generation and characterisation of GFP-expressing derivates of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Furthermore, I have developed a method to effectively and rapidly investigate macrophage parasitism by flow cytometry that preserves the accuracy of current approaches but offers a four-fold improvement in speed. The final part dissects the regulation and induction of mitochondrial tubularisation in hypervirulent C. gattii strains and describes the first steps towards a comparative mitochondrial genome sequencing approach to identify the underlying molecular mechanisms

Macrophage – cryptococcus interactions during cryptococcosis

The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic effector cells. Cryptococcus can survive and proliferate within macrophages, and is also capable of escaping into the intracellular environment via a non-lytic mechanism (‘expulsion’) and can be transferred directly from one cell to another (lateral transfer). In the first part of this thesis, I demonstrate that enhanced Th2, but not Th1, cytokine levels lead to increased intracellular cryptococcal proliferation but lower levels of cryptococcal expulsion. In the second part, I describe the generation and characterisation of GFP-expressing derivates of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Furthermore, I have developed a method to effectively and rapidly investigate macrophage parasitism by flow cytometry that preserves the accuracy of current approaches but offers a four-fold improvement in speed. The final part dissects the regulation and induction of mitochondrial tubularisation in hypervirulent C. gattii strains and describes the first steps towards a comparative mitochondrial genome sequencing approach to identify the underlying molecular mechanisms.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Effect of the capsular material of cryptococcus neoformans on the interplay between Mmicroglial cells and Neutrophils.

Doctoral Degree. University of KwaZulu-Natal, Durban.Cryptococcal meningitis is an important opportunistic infection in immunocompromised patients. It has been well established that a distinguishing feature of this form of meningitis is a relatively low neutrophil count in the cerebrospinal fluid (CSF) compared to bacterial meningitis. There has been speculation and research undertaken previously to understand this phenomenon, however, little information is available in human studies. Furthermore, there is insufficient information on expression and function of Toll-like receptors (TLR) in the human central nervous system (CNS). The work presented here investigated the effect of the capsular material of a series of clinical isolates of Cryptococcus neoformans on neutrophil recruitment at the site of infection and determined whether downregulation occurs at the level of TLR expression. This was done in a multiple component study. Clinical information was collected from patients with cryptococcal meningitis and baseline blood and CSF investigations were performed, which included the quantification of neutrophils in CSF and blood specimens. The size of the Cryptococcus capsule was measured in each isolate and shed capsular material was quantified in individual CSF specimens. The extent of neutrophil chemotaxis inhibition by individual strains of C. neoformans was determined by using a Transwell migration assay. Toll-like receptor (TLR)2 and TLR4 gene expression induced by individual C. neoformans isolates in human microglial cells was quantified. The possible associations among these experiments were subsequently evaluated. As anticipated, a paucity of neutrophils in the CSF was observed. The cryptococcal capsule was larger in isolates of patients with lower CSF neutrophil counts. In addition, patients with lower CSF neutrophil counts shed more capsular material in the CSF. Chemotaxis inhibition occurred in close to 70% of tested isolates. The concentration of shed capsular material in this group was higher compared to the group with no chemotaxis inhibition. Patients presenting with fever had higher CSF neutrophil counts as well as elevated intracranial pressures. The majority of isolates expressed downregulation for TLR2 and TLR4 in microglial cells exposed to C. neoformans. CSF neutrophil counts were lower in this group. These findings imply that the capsular components of C. neoformans downregulated recruitment of neutrophils into the CSF. Downregulation of neutrophil recruitment was observed at the level of TLR expression

Interactions between Streptococcus pyogenes and the Human Immune System

Streptococcus pyogenes is a common human pathogen causing mild infections such as tonsillitis and pharyngitis, but is also the cause of life-threatening conditions. To be able to infect the human host, S. pyogenes express different virulence factors. The surface-expressed M protein is a major virulence factor of S. pyogenes, mediating resistance to killing by human neutrophils. Results presented in this thesis show that in the presence of plasma from non-immune individuals, S. pyogenes avoids killing by human neutrophils by M protein-mediated inhibition of adhesion to the neutrophils. By comparing a wild type strain and a strain deficient in M protein-expression, the interactions between S. pyogenes and human neutrophils were studied. The interactions between S. pyogenes and human neutrophils in the presence of non-immune plasma are mediated mainly by complement components deposited on the bacterial surface and CD11b/CD18 expressed on the neutrophil surface. This interaction leads to the effective ingestion and killing of the bacteria. The activation of CD11b/CD18 leads to the activation of tyrosine kinases and the subsequent activation of the small Rho GTPase Cdc42. In blood from immune humans, S. pyogenes are opsonized and killed. In this thesis, we show that killing is mediated by Abs directed against the N-terminal part of the M protein and that these Abs activate the complement system. In the presence of serum from immune individuals, the killing of S. pyogenes is mediated through activation of the complement system by M protein-specific Abs and activation of CD11b/CD18 and Cdc42 and subsequently phagocytosis. Almost all strains of S. pyogenes bind fibrinogen (Fg) and the Fg-binding is associated with members of the M protein family. We mapped the binding of Fg to the B repeats of the M1 and M5 proteins and demonstrated that the Fg-binding is important for these bacteria to resist killing when incubated in human blood. The conditions under which the interactions between S. pyogenes and human neutrophils were investigated in the studies described above resemble the situation in bacteremia (a low multiplicity of infection, MOI, i.e. bacteria: neutrophil ratio). However, at the initial stages of infection, occurring at skin or mucosal surfaces, the bacteria to neutrophil ratio is much higher. Using two different model systems to study the interactions of S. pyogenes and phagocytes at a higher MOI, we found that this interaction is mediated by fibronectin deposited at the bacterial surface and the integrins ?5?1 and ?v?3 expressed on the phagocytes

Exploiting complement for targeted therapies against Pseudomonas aeruginosa infection and breast cancer

Complement is the primary defense mechanism against pathogens and in antitumor immunity. Complement activating Multimeric compleXes (CoMiX), a novel class of antibody-based therapeutics, activate the complement at the surface of pathogens or target cells. In this work, we have evaluated their potential in targeting and eliminating both Pseudomonas aeruginosa (P. aeruginosa) and HER2-positive breast cancer. These multimeric complexes are composed of three distinct functional components: a targeting moiety, an oligomerization domain, and an effector component. In the context of P. aeruginosa infections, CoMiX were designed to target the Psl-exopolysaccharide of the bacterium. The effector component of CoMiX in this case was either an Fc region, which activated the classical complement pathway and immune cells, or Factor H-related protein 1 (FHR1), which competed with the complement inhibitor Factor H. In vitro studies demonstrated CoMiX's ability to bind to P. aeruginosa isolates, activate complement, and directly kill the bacteria. Importantly, CoMiX was shown to reduce dramatically bacterial load and mortality in a mouse model of acute P. aeruginosa lung infection. For HER2-positive breast cancer, CoMiX were engineered to target HER2-expressing tumor cells. The effector component in this case was either FHR4, which activated the alternative complement pathway, or a triple Fc dimer, which activated the classical complement pathway and promoted NK cell activation and phagocytosis by macrophages. In vitro studies demonstrated CoMiX's ability to activate complement, induce complement-dependent cytotoxicity, and stimulate immune cell responses. In vivo studies in a xenograft model of human breast cancer showed that CoMiX could effectively inhibit tumor growth and overcome the resistance to trastuzumab. Overall, these studies highlight the versatility and therapeutic potential of CoMiX in targeting and eliminating both bacterial pathogens and cancer cells. Subsequent research and clinical development are essential to comprehensively assess their potential benefits in patients.AER

Interactions of antibody with the capsular polypeptide of Bacillus anthracis

Bacillus anthracis is the etiologic agent of anthrax. The bacterium is surrounded by a capsule entirely assembled from gamma (g)-linked D-glutamic acid (gDPGA). The polymer is produced immediately following spore germination and is a considerable obstacle to the immune system. Previous studies have indicated that monoclonal antibodies (mAbs) reactive with the bacterial capsule are effective reagents for antimicrobial therapy and immunodiagnosis; both applications have tremendous potential in the field of bio-defense. gDPGA is a unique antigen; some of its characteristics include high molecular weight, resistance to degradation, negative charge, and repeating subunits. The work presented in this dissertation was carried out to more fully understand how mAbs interact with gDPGA. We determined that binding may occur in a stereo-selective manner, despite the repetitious character of the polymer. Transfer of a murine IgG3 anti-gDPGA variable region to human IgG1-4 constant domains resulted in severe binding alterations. Affinity maturation of a gDPGA-reactive mAb F26G3 facilitated the assessment of subclass and binding attributes as independent variables. These studies highlighted the importance of capsular reactivity to protection. Finally, the function of murine IgG3-specific post-translational modifications were examined. Deglycosylation altered the fundamental capsular reactivity of murine IgG3. Together, the results indicate that mAb:gDPGA interactions are surprisingly complex. Murine IgG3 anti-gDPGA mAbs retain characteristics not observed in any other murine or human antibody; suggesting a purposeful mechanism to manage encapsulated pathogens

Towards Immuno-profiling of Complex Biological Fluids in Patients Recovering from Major Surgery

The conventional biochemical diagnosis of disease using isolated blood biomarkers must be revisited by clinicians, replacing it with a multi-biomarker, personalised profile of human health. A label-free nanoparticle array technology, Liscar, has been developed. It is capable of performing rapid, multi-biomarker assays from complex biological samples which, if employed to assay the Complement cascade of the innate immune system, has the potential for a novel systemic profile of patient health. An assay for IgG has been developed on the Liscar platform with a detection limit of 380 ± 100 ng/mL IgG in model sera. Furthermore, addition of a chaotropic agent to the complex sample is shown to improve the accuracy of the IgG assay. Competitive binding between nonspecific interfering proteins and specific target analytes (IgG) at the sensor surface is studied, and a quantitative mathematical model is developed to analyse the data, yielding evidence for the active displacement of albumin by IgG-antigen binding. With a sensitive, accurate multi-biomarker detection platform, a systemic profile of patient health may be possible by examination of the Complement cascade. The Complement system can be activated by a variety of immunological challenges, causing large numbers of activation biomarkers to be produced quickly. Assays for three activation markers, C3d, TCC and Bb are developed, with detection limits of 0.864 ACS Units, 2.32 ng/ml and 54.7 ng/ml respectively. Complement activation was tested in a prospective cohort study of 45 patients undergoing major abdominal surgery. Patient recovery was monitored from admission to ~60 hours postoperatively by Complement activation and consumption using C3d, TCC, Bb, C3 and C4 as biomarkers. A response profile was obtained for the entire cohort for C3 and C4 assays by normalising with respect to individual analyte levels on admission, against which individual responses are compared. 22% of patients in the study suffered postoperative complications, and 73% showed Complement activation by increased levels of C3d, as expected from the initial trauma of surgery. Expansion of the trial is needed to establish clinical significance and utility, especially in relation to the presymptomatic diagnosis of disease

Responses of the innate immune system to the human pathogen Streptococcus pyogenes

Streptococcus pyogenes, auch bekannt als Gruppe A Streptokokkus (GAS), ist ein bedeutendes humanes Pathogen, das für eine Reihe von Krankheiten verantwortlich gemacht wird. Diese Krankheiten können relativ harmlos sein, wie zum Beispiel Mandelentzündung, aber auch extrem gefährlich für den Patienten, wie zum Beispiel invasive Infektionen der Haut und der darunterliegenden Gewebe, die zu einer nekrotisierenden Fasziitis, Sepsis oder zu einem streptokokkalen toxischen Schock-Syndrom führen können. Bakterielle Pathogene werden von Zellen des angeborenen Immunsystems durch spezielle Rezeptoren, die auf die Erkennung von bestimmten bakteriellen Mustern von Bakterien spezialisiert sind, erkannt. Die Toll-like Rezeptoren (TLRs) sind die wohl am besten charakterisierte Familie unter diesen Rezeptoren und jedes Pathogen wird von mindestens einem dieser Rezeptoren erkannt. Jedoch sind die Moleküle, die für die Aktivierung einer Immunantwort gegen S. pyogenes verantwortlich sind, noch nicht bekannt. Wir konnten zeigen, dass S. pyogenes zur Aktivierung einer inflammatorischen Antwort in aus dem Knochenmark von Mäusen gewonnenen Makrophagen führt, die durch die Produktion von Zytokinen wie TNF-alpha und IL-6 gekennzeichnet ist. Diese Aktivierung ist im Fall von S. pyogenes abhängig von dem Adapter-Molekül MyD88, jedoch unabhängig von den wichtigsten bakteriellen Rezeptoren TLR2, TLR4 und TLR9. Zusätzlich konnten wir zeigen, dass die Infektion von Zellen des angeborenen Immunsystems mit S. pyogenes zu einer Produktion von Typ I Interferonen (IFNs) und einer anschließenden Aktivierung des Jak/Stat Signaltransduktionsweges führt. Experimente mit Makrophagen, denen bestimmte Komponenten des IFN- Signaltransduktionsweges fehlen (IRF3, IRF1, STAT1, IFNAR1) und mittels siRNA-vermittelter Inaktivierung einer anderen Komponente dieses Signalwegs (TBK1) führten zu dem Schluss, dass die IFN-Produktion unterhalb eines noch nicht bekannten Rezeptors abhängig von IRF3, TBK1 und IFNAR1 aber unabhängig von bekannten TLRs geschieht, obwohl eine partielle Abhängigkeit von MyD88 beobachtet wurde. Ferner deuteten mit Enzymen verdaute streptokokkale Extrakte darauf hin, dass streptokokkale DNA für die Induktion der IFN- Produktion wichtig ist. Allerdings sind auch in diesem Fall die Moleküle die für diese Induktion noch nicht bekannt. Zusammengefasst wurde in dieser Studie gezeigt, dass Streptococcus pyogenes in der Lage ist, gleichzeitig mehrere Signaltransduktionswege im Wirt zu aktivieren. Dies könnte zu der bekannten Eigenschaft des Bakteriums beitragen, viele schwere inflammatorische Krankheiten hervorzurufen.Streptococcus pyogenes, also known as group A streptococcus (GAS), is an important human pathogen responsible for a variety of diseases ranging from mild (e.g. tonsillitis) to more severe infections (e.g. invasive infections of skin and soft tissues that can develop into necrotizing fasciitis, sepsis or lethal toxic shock syndrome). Bacterial pathogens are recognized by cells of the innate immune system through pattern recognition receptors (PRRs). Among them, the family of Toll-like receptors (TLRs) is the best characterized class of PRRs and virtually all pathogenic bacteria are recognized by at least one of them. However, the molecule(s) responsible for the activation of an immune response against S. pyogenes are not known yet. We were able to demonstrate that activation of an inflammatory response in murine bone-marrow derived macrophages (BMDMs) against S. pyogenes, involving the production of cytokines such as TNF-alpha and IL-6, depends on the adaptor molecule MyD88, but not on the prototype bacterial receptors TLR 2, 4 and 9. Moreover, we could show that infection of innate immune cells with S. pyogenes causes the production of type I interferons (IFNs) and subsequent activation of the Jak/Stat signaling pathway. Experiments using BMDMs from mice deficient in components of the interferon pathway (IRF3, IRF1, STAT1, IFNAR1) and siRNA-mediated knock down of another important gene (TBK1) from this pathway revealed that IFN-production occurs downstream of an unknown PRR via IRF3, TBK1 and IFNAR1, but without the involvement of any described TLRs, although a partial dependence on MyD88 was observed. Furthermore, digested streptococcal extracts suggest that streptococcal DNA is required for the induction of IFN-beta production. However, the receptor molecule which promotes the induction of the interferon response stays elusive. An experimental mouse model of skin infection using IFNAR1-deficient mice emphasized the important role of type I IFNs during the host immune response against GAS. Taken together, our work demonstrates that Streptococcus pyogenes has the ability to induce multiple signalling pathways within the host, which may contribute the known high capability to cause severe inflammatory diseases