No description available.

Innate antiviral defence depends on virus recognition and cytokines like interferon (IFN) that upregulate many interferon-stimulated genes (ISGs). Virus recognition normally relies on pattern recognition receptors binding to virus-associated nucleic acid motifs, but virus activity may provide an additional means for the cell to recognize infection. Enveloped viruses fuse with a cell membrane during entry and membrane fusion by virus-like particles or the purified protein p14 are sufficient to upregulate ISGs in the absence of viral nucleic acid. This thesis examines the mechanism by which cells recognize membrane fusion and how this affects downstream signalling and upregulation of ISGs. We found that membrane perturbation by enveloped virus particles or p14 triggered cytosolic Ca2+ oscillations important for antiviral defence. Surprisingly, Ca2+ signalling seemed to act upstream of nucleic acid sensing pathways during enveloped virus infection. In the absence of viral nucleic acid, p14 triggered a Ca2+-dependent antiviral response to dsRNA. It is still unclear how p14 might trigger recognition of endogenous dsRNA. We found that enveloped virus particles trigger IRF3-mediated upregulation of interferon as well as direct IFN-independent upregulation of ISGs. Furthermore, while some viruses like HCMV trigger widespread IRF3 activation, other viruses like SeV upregulate IRF3 and IFN in a minority of infected cells. This disparate response to infection can lead to different biological outcomes when measured at the population level. Our work highlights the complexity of the response to enveloped virus particles, despite the absence of replication. Further work is necessary to understand how membrane perturbation is recognized and how this interfaces with nucleic acid sensing. While nucleic acid sensing is sufficient to upregulate antiviral ISGs, other signals like membrane perturbation may provide important contextual cues during infection. This will be important to understand moving forward as virus-like particles are used more and more for research and clinical applications. / Thesis / Doctor of Philosophy (PhD) / Cells rely on pattern recognition receptors for innate antiviral defence. While the study of pattern recognition has focused on virus-associated nucleic acid motifs, disruptions of the cellular environment during virus infection could similarly warn the cell. Membrane perturbation during enveloped virus entry is associated with upregulation of antiviral interferon-stimulated genes. This thesis examines the mechanism of membrane perturbation sensing and different antiviral signalling pathways activated by non-replicating enveloped virus particles. We found evidence that membrane perturbation triggers cytosolic Ca2+ signalling which may act as a co-stimulatory signal for recognition of incoming viral nucleic acid. We initially thought enveloped virus particles were recognized through a common pathway, but have since learned that recognition is more complex. Further work is necessary to understand how membrane perturbation and nucleic acid sensing interface during enveloped virus infection and what role this plays in antiviral defence.

Circulating monocytes have the capacity to mature into either macrophages or dendritic cells in tissue, both of which play important roles in the induction and effector phase of immune response. In TB, the macrophages are the central player in the host-bacteria interaction as the main mycobacterial reservoir. In HIV disease, monocyte lineage cells are one of the two main cell types (along with CD4+ T-cells) in sustaining intracellular HIV infection. Monocytes are heterogeneous population with three functionally distinct subsets namely classical, intermediate and non-classical monocytes. The three subsets exist in a continuum, and have a certain plasticity or flexibility to develop into multiple roles depending on the local and tissue environment. In the current study we sought to evaluate the frequencies of these three subsets in participants with TB, HIV and TB/HIV co-infection. While previous studies had shown that the intermediate and non-classical monocyte subsets were elevated relative to classical monocytes, very little had been done in these disease groups regarding more comprehensive characterization of these subsets. In particular, we wished to quantitate the expression of multiple sets of cell surface and intracellular molecules of high relevance using multi-parameter flow cytometry from a functional point of view. In publication I, we evaluated Toll-like receptors (TLRs) expression in each of the study cohorts. TLRs are vital pattern recognition receptors by monocyte lineage cells and signal the induction of crucial functions. We focused on three such TLRs (TLR2, TLR4 and TLR9) which have been shown to be involved in many monocyte lineage cell interactions with mycobacterial and HIV infections. We observed enhanced expression of TLR2 and TLR4, but not TLR9 in TB and HIV. TLR4 was particularly high in patients with TB, but also in HIV. We observed comparable increase of TLR4 irrespective of monocyte subset. However, TLR2 expression exhibited a different pattern. Levels among the most prominent classical monocyte subsets were identical in all four cohorts, healthy controls (HC), HIV, TB, and TB/HIV co-infection. In contrast, TLR2 expression was significantly elevated in both participants with HIV and TB, but not with participants with TB/HIV co-infection in the intermediate monocyte subset. We also observed correlations between TLRs and plasma cytokines that were disease and TLR specific. In publication II, we observed elevated chemokine receptors (CRs) expression which above healthy controls and exhibit a pattern of disease preference. Thus, CCR2 and CX3CR1 were the highest in participants with TB, followed by HIV and TB/HIV co-infection, whereas CCR4 and CCR5 were highest in participants with HIV, and less elevated in TB. CCR2 and CX3CR1 are critical for migration of monocytes to sites of TB infection, as determined by murine models. CCR4 and especially CCR5 have been implicated in migration of cells to distant organs but more as co-receptors for HIV infection. Thus, the observed pattern of CRs expression in these monocytes in different disease states would predict greater availability of these cells or their receptors for interaction with either TB or HIV organisms. From the perspective of the pathogen this would lead to enhanced “substrate”, whereas from the perspective of the host, this could lead to greater immune potential. As a final point, we also observed that the pattern of disease association of CRs was independent of the monocyte subset. In publication III, we explored the expression of Programmed cell death-ligand 1 (PDL1) on the three monocyte subsets. Like many of the other molecules we have addressed in this thesis, PDL1 expression was enhanced in participants with HIV, TB, and TB/HIV co-infection. Among participants with HIV, PDL1 was correlated with HIV-1 viral load. The enhanced expression was apparent in all three subsets, but it was particularly prominent in the intermediate monocyte subset. Moreover, PDL1 expression was the highest in participants with TB/HIV co-infection. The implications behind these observations is that the subset thought to have the greatest potential for T cell antigen presentation had the highest levels of the T cell down-regulatory PDL1 molecule in the cohort of patients particularly participants with TB/HIV co-infection. Participants with TB/HIV co-infection have the greatest potential to be immuno-compromised and as a result the very need for enhanced not depressed APCs function. In addition, we also observed the PDL1 levels were correlated with multiple plasma, mostly pro-inflammatory,cytokines. We analyzed cytokine mRNA levels of total monocytes to address the source of the cytokines but mRNA levels did correlate with neither plasma cytokine nor PDL1 levels. Considering all the phenotype analysis in each of the three studies together we could see two patterns emerging. In one scenario,surface molecules expression patterns were disease specific but independent of monocyte subset expression. In other words, whatever the underlying mechanism(s) involved in their regulation, those mechanisms apparently acted similarly in all three subsets. In another scenario, expression of surface molecules showed disease specific patterns, but molecules were particularly enhanced in the intermediate monocyte subsets. These findings imply that there exist mechanisms to modulate surface phenotypes and functions that are unique to a given subset. In conclusion, we have comprehensively defined the density of multiple molecules expressed by different subsets of monocytes and explored their differences in participants with TB, HIV and TB/HIV co-infection, as well as their correlations with microbial indices and plasma cytokines. Many molecules levels were elevated to some extent in all disease cohorts, but we observed patterns of expression which were particularly elevated in TB (CCR2, CX3CR1, and TLR2), those in HIV (CCR4, CCR5) and those on both (TLR4, PDL1). Molecule-disease associations were either independent of monocyte subset, or most readily revealed in a single monocyte subset. TB/HIV co-infection did not follow a consistent pattern in association with monocytes markers, in some cases more resembling TB, in others HIV, in others neither. Finally, to proof one possible mechanism of association between disease and monocyte phenotype, we explored correlations between monocyte markers and plasma cytokines. We observed significant positive and negative associations, frequently unique to a single monocyte subset or disease cohort, such as TB/HIV co-infected cohort. Collectively, the results imply that there are likely multiple mechanisms involved at many levels regulating the phenotype and function of monocytes, and these differ in different disease states.:Abbreviations ............................................................................................................ 3 Abstract ..................................................................................................................... 4 1. Introduction ........................................................................................................... 6 1.1 Epidemiology of Tuberculosis and Human Immunodeficiency virus ................... 6 1.2 The immunological response to TB and HIV ....................................................... 7 1.2.1 Innate immunity of TB ...................................................................................... 7 1.2.2 Innate immunity of HIV .................................................................................... 11 1.2.3 Immune checkpoint regulation in TB and HIV.................................................. 13 1.3 The role of monocytes in TB, HIV and TB/HIV ................................................... 14 1.3.1 Monocytes ....................................................................................................... 14 1.3.2 Abnormalities of monocytes in TB ................................................................... 15 1.3.3 Abnormalities of monocytes in HIV ................................................................. 16 1.3.4 Abnormalities on monocytes in TB/HIV co-infection ....................................... 17 1.4 The rationale for the thesis ................................................................................ 17 2. Objectives ............................................................................................................ 19 3. Publications .......................................................................................................... 20 4. Summary .............................................................................................................. 65 References ............................................................................................................... 69 Annex I: Author contribution ..................................................................................... 76 Annex II: Declaration of independent writing of the work ......................................... 77 Annex III: Curriculum Vitea ....................................................................................... 78 Annex V: Acknowledgment........................................................................................ 82 Annex VI ................................................................................................................... 84

Doctor of Philosophy / Department of Biology / Lynn E. Hancock / Enterococci are gram-positive bacteria typically found as commensals in the gastro-intestinal tracts of most mammals. Enterococci, most notably Enterococcus faecalis and Enterococcus faecium, have become problematic causative agents of several nosocomially acquired infections including urinary tract infections, bacteremia, surgical sight infections, and endocarditis. These bacteria must first overcome the innate immune response in order to establish infection. Many bacteria produce capsular polysaccharides that contribute to pathogenesis by helping the microbe evade the host innate immune response. The capsular polysaccharide produced by E. faecalis has been shown to play a role in pathogenesis; however the mechanisms of innate immune avoidance were unknown. Moreover, the number of capsule serotypes produced by E. faecalis and the genetic differences that contribute to capsule serospecificity were in doubt. In the current study it is made clear that only two capsule serotypes are produced by E. faecalis and that both capsule serotypes contribute to evasion of the host innate immune system. This work shows two mechanisms by which the capsule of E. faecalis contributes to immune evasion. First, the presence of capsule inhibited complement mediated phagocytosis through limiting the detection of opsonic complement protein C3 on the surface of the bacteria. Secondly, the presence of capsule altered cytokine signaling of macrophages by shielding bacterial components from detection. Many pathogenic strains of E. faecalis also produce an extracellular protease known as gelatinase (GelE). This work also shows a novel mechanism involving GelE in innate immune evasion through the degradation of the anaphylatoxin C5a. Degradation of C5a by GelE resulted in decreased neutrophil recruitment in vitro. A rabbit model of endocarditis was employed to assess the effect of GelE production on disease development and progression. Rabbits infected with GelE producing strains had increased bacterial burdens in the heart compared to rabbits infected with strains that were GelE negative. Reduced phagocyte infiltration at primary and secondary infection sites was also observed in rabbits infected with GelE producing strains compared to GelE negative strains. The work presented here demonstrates that both the capsular polysaccharide and GelE play roles in E. faecalis evasion of innate immune responses. Moreover, these pathogenic determinants would be suitable targets for developing alternative therapeutics used to treat E. faecalis infections.

Secreted surface proteins are an innate immune defense component employed by animals to inhibit invading microbes. Surface proteins have not been documented in plants, even though the aerial leaf surface, or phylloplane, is a major site of pathogen ingress. We have discovered novel proteins, termed phylloplanins, which accumulate on leaf surfaces of Nicotiana tabacum, and we have isolated the gene Phylloplanin that is unique in gene databases. Natural and E. coli-expressed phylloplanins inhibit spore germination and limit leaf infection by the oomycete pathogen Peronospora tabacina. We investigated the site of phylloplanin biosynthesis using biochemical techniques. These techniques included radiolabeling of detached trichome glands, radiolabeling of epidermal peels, analysis of leaf water washes of various Nicotiana plants, and examination of guttation fluid, leaf vein contents, and extracellular fluid. From these experiments, we tentatively conclude that phylloplanins are produced by hydathodes, or an unknown surface secreting system, but not by glandular secreting trichomes. Future experiments with the phylloplanin promoter, whose elucidation is described herein, and its fusion to a reporter gene (GUS or GFP), will undoubtedly provide further insight into the location of phylloplanin biosynthesis and deposition. We suggest that the hydrophobic nature of phylloplanins aids in their dispersal over the leaf surface. Phylloplanins constitute a first-point-of-contact, rapid response, innate immune deterrent to pathogen establishment on N. tabacum leaf surfaces, and are the first studied representatives of a novel protein class in the plant kingdom. Further study of leaf surface proteins is justified to understand further their roles in plant defense, and to investigate their potential in agricultural biotechnology. Additionally, we describe miscellaneous observations we have made during the course of this research. Low molecular mass proteins (as yet uncharacterized) are washed from leaf surfaces of sunflower, soybean, and other plants. Pathogenesis-related (PR-)-5a, a known antifungal protein, was found to be present on the leaf surfaces of healthy plants, although its function there remains unknown. A phylloplanin homologue from Arabidopsis appears to be antibacterial. Further study of this protein is warranted. We note that proteins can also be recovered from N. tabacum root surfaces, or the rhizoplane, but we have not further characterized these proteins. In summary, novel surface-accumulated proteins, termed phylloplanins, and the gene encoding these have been discovered in N. tabacum. An antifungal function for phylloplanins is reported, and evidence was found for a unique mechanism of surface deposition.

Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing through highly regulated pathways that involve the phagocytosis of bacteria and the generation of reactive oxygen species by the NADPH oxidase complex. The Rac small GTPases have prominent roles in the regulation of neutrophil signaling pathways but the research strategies used to analyze their functions in live cells have been limited, since neutrophils are terminally differentiated and difficult to manipulate genetically. In this thesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils that allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely new way, in real time. Using this technique, I show that a bacterial protein known to inhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP stimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac isoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate free-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible for ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the activation of cofilin and Arp2/3. Finally, these observations led to the analysis of the mechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge determines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis iii based on their different aminoacid residues in the polybasic domain. This mechanism depends on lipid metabolism and the accumulation of negatively charged lipids at cellular membranes. During chemotaxis, neutrophils have a polarized accumulation of negatively charged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the lipid metabolism that occurs at the phagosome membrane decreases its negativity and selectively recruits Rac2. All together, this thesis describes the study of primary neutrophil functions from a new angle and adds some valuable information to the comprehension of effective neutrophil activation based on the analysis of Rac isoforms.

Arboviruses are transmitted between vertebrate hosts by arthropod vectors, such as mosquitoes or ticks. In vertebrates arboviruses cause cytopathic effects and disease, however, arbovirus infection of arthropods usually results in persistence. Control of arboviral infection is mediated by the arthropod’s immune system. Pathways such as RNAi, JAK/STAT, Toll and IMD have previously been implicated in controlling arbovirus infections. In contrast, the antiviral role of other pathways in mosquitoes, such as melanisation, is unknown. Using high through output 454 sequencing the transcriptome of U4.4 cells infected with the model arbovirus Semliki Forest virus (SFV)(Togaviridae, Alphavirus) was generated. This experiment revealed intriguing patterns of differential transcript abundance that suggest a broad impact of SFV infection in U4.4 cells, such as in metabolism, cell structure and nucleic acid processing. SFV infection induces differential expression of genes in pathways such as apoptosis, stress response and cell cycle. Most interestingly, this study indicated that melanisation might have an antiviral role in mosquitoes. In arthropods, melanisation is a process involved in wound healing and antimicrobial defences. Phenoloxidase (PO), a key enzyme involved in melanisation, is cytotoxic and therefore kept in its inactive form, prophenoloxidase (PPO), until activation is triggered. The PPO activation process is tightly regulated by serine protease inhibitors (serpins) which inhibit the proteolytic activation reaction. In this thesis I demonstrate that the supernatant of cultured Aedes albopictus-derived U4.4 cells contains a functional proPO-activating system, which is activated by infection with bacteria and virions of SFV. Activation of this pathway reduces the spread and infectivity of SFV in vitro and in vivo. In order to further characterise the PO cascade and its antiviral role the serpins in Ae. albopictus were also investigated. Using the transcriptome sequencing and bioinformatics we identified and classified 11 serpins. We silenced each of the serpins and monitored PPO levels and antiviral activity showing that homologues to drosophila’s serpin- 27a plays a role in melanisation against SFV in vitro. Collectively, these results characterise the mosquito PO cascade as a novel immune defence against arbovirus infection in mosquitoes.

The use of antibiotic growth promoters has been limited all around the world because of the concerns about antibiotic resistant bacteria and the presence of antibiotic residues in poultry products. Yeast-derived products are rich sources of ß1,3-1,6-glucan, mannan polysaccharides, and nucleotides and are considered as possible antibiotic alternatives due to their potential intestinal health benefits, growth promotion, and immune system stimulation. The objectives of the current research were: (1) to the evaluate effect of yeast products derived from yeast Saccharomyces cerevisiae on growth performance, gut histomorphology, and innate immune response of broiler chickens; (2) to investigate the effect of yeast products, including distillers dried grains with solubles (DDGS), on innate and antibody-mediated immune response following immunization with different antigens; and (3) to examine the effect of yeast-derived products and DDGS on growth performance, incidence of necrotic enteritis (NE), and local innate immunity in broiler chickens challenged with Clostridium perfringens. Overall, supplementation of diets with yeast products did not affect growth performance of broilers. However, the diets containing yeast cell walls (YCW) and nucleotides increased the villus height in the jejunum and enhanced the number of goblet cells in the ileum. Inclusion of diets with yeast products did not activate the innate immune response of birds under non-pathogen challenge conditions. However, the diet containing YCW activated Th2 cell-mediated immune response in birds immunized with sheep red blood cells and bovine serum albumin. Furthermore, supplementation of diets with YCW and DDGS in birds challenged with Escherichia coli lipopolysaccharide, activated the systemic innate immune response. Regarding antibody-mediated immune response, when compared to the control, serum antibody titer and specific antibody response against different antigens were not affected by dietary treatments. In the C. perfringens challenge study, growth performance, NE lesions and C. perfringens counts in the intestine were not affected by yeast-derived products. However, diets containing YCW and nucleotides stimulated the local innate immune response of birds by upregulation of cytokines and receptors involved in innate immunity. Such findings suggest that the immune-adjuvant like properties of YCW and nucleotides activate the innate immunity of broiler chickens following immunization or challenge with different antigens. / October 2015

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Michael Kanost / Insects secrete antimicrobial peptides as part of the innate immune response. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. Fractions containing activity against S. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome databases. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. Diapausin-1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to diapausin-1, each with 6 conserved Cys residues. Diapausin-1 was produced as a recombinant protein in Escherichia coli. Purified recombinant diapausin-1 was active against S. cerevisiae, with IC₅₀ of 12 μM, but had no detectable activity against bacteria. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. In addition, diapausin-1 mRNA levels increased in midgut and fat body at the wandering larval stage prior to pupation, suggesting developmental regulation of the gene. Our results indicate that synthesis of diapausin-1 is part of an antifungal innate immune response to infection in M. sexta. Biophysical analysis showed that diapausin-1 binds to the β-1,3 glucan component of the S. cerevisiae cell wall. A second insect peptide investigated in this project was M.sexta stress-response peptide 1(SRP1), an immune-related peptide upregulated under different stress conditions including immune-challenge. Preliminary results for NMR structure determination are presented. Most of the amino acid residue spin systems were assigned, and we determined the connectivities of many amino residues as a first step to solve the NMR structure. The circular dichroism spectrum of SRP1 indicates that the peptide lacks alpha-helical structure and may contain beta strands and turns.

Os insetos são capazes de sobreviver em diversos ecossistemas do planeta e, mesmo estando constantemente expostos à ameaça de infecção microbiana, permanecem livres de infecções na maior parte do tempo. Essa capacidade de sobrevivência aliada à larga distribuição dos insetos em regiões totalmente diferentes tem estimulado a pesquisa de novos agentes terapêuticos nesta classe devido à descoberta de diversos componentes de mecanismos inespecíficos de combate à infecção, sendo possível sua aplicação no controle de diversas doenças. Todavia, apesar de um grande número de moléculas de defesa ter sido identificado a partir de vários insetos, pouca informação sobre suas aplicações está disponível. Desta forma, o presente trabalho elucida o perfil transcriptômico geral e dos genes de defesa do tegumento de Podalia orsilochus durante sua fase larval. Como consequência, os transcritos e os dados obtidos permitirão o auxílio em pesquisas posteriores, seja para comparação, citação, conhecimento biológico e das respostas de defesa ou das relações de filogenia do animal. / The insects are able to survive in diverse ecosystems on earth, and even being constantly exposed to the threat of microbial infections, remain free of infection for most of the time. This survivability combined with the wide distribution of insects in totally different regions has stimulated the search for new therapeutic agents in this class due to the discovery of several components of nonspecific mechanisms to fight infection, and possible implementation in the control of various diseases. However, despite a large number of defense molecules have been identified from various insects, little information is available on their applications. Thus, this paper elucidates the general transcriptomic profile and integument of defense gene Podalia orsilochus during their larval stage. As a result, the transcripts and the data obtained will aid in further research, to compare, reference, biological knowledge and defense or animal phylogeny relationships.