Campylobacter jejuni is an enteric bacterium that causes human gastroenteritis worldwide. Some C. jejuni strains exhibiting human ganglioside-like lipooligosaccharide (LOS) structures, such as GM1 ganglioside, can induce an autoimmune neuropathy of the peripheral nervous system known as the Guillain-Barré syndrome (GBS). This GBS-inducible determinant is encoded by a gene cluster, which shows a high degree of variation among C. jejuni strains. The experiments presented in this thesis were conducted to give a better insight into the LOS synthesis genes in relation to the pathophysiology of C. jejuni. Firstly, a C. jejuni strain without GM1-like molecules was shown to be able to take up large DNA fragments, including LOS synthesis genes, from a strain expressing GM1-like molecules and consequently be transformed into a number of potential GBS-inducible transformants, which exhibited a high degree of genetic and phenotypic diversity. The ability of C. jejuni to take up and integrate foreign DNA explains the genome plasticity observed in this pathogen. Secondly, while attempting to analyse transcription of the LOS gene cluster, neither published methods nor any commercially available kits for RNA isolation could produce DNA-free RNA from C. jejuni. Combinations of these methods were trialled and only the combination of RNAzolB, TURBO DNase treatment, and acid phenol extraction was able to produce DNA-free RNA. The RNA isolated from most C. jejuni strains showed different RNA patterns to that of other bacteria. In addition the RNA from C. jejuni seemed closely associated with DNA compaired to RNA from other organisms. This might be caused by species-specific DNA conformation or chromatin structure. Thirdly, bidirectional transcription was observed in the LOS gene cluster. Both DNA strands were transcribed but transcription of the non-coding strands was at a lower rate, and both sense and antisense transcripts of each LOS gene tested were responsive to acid stress. This unusual transcription might have a potential effect on the expression of the GBS-inducing determinant. Finally, one of the LOS genes, the htrB gene, was further analysed. It was shown that expression of the htrB gene affects morphology, viability, growth ability, and sensitivity to stress environments. These results showed that the LOS molecule of C. jejuni is involved in many processes and is an important molecule for survival.

Campylobacter jejuni is one of the leading bacterial causes of human gastroenteritis world wide and has been linked to several severe complications including autoimmune syndromes which can result in paralysis. Despite being the subject of much study, C. jejuni remains a major public health burden in both developing and developed nations. There is currently no vaccine available for protection against this pathogen and the mechanisms important for C. jejuni pathogenesis are not fully defined. This study has employed a range of experimental approaches to investigate the molecular mechanisms involved in C. jejuni pathogenesis. Lipooligosaccharides (LOSs) are surface structures and known virulence factors of C. jejuni which are involved in serum resistance, resistance to phagocytic killing, endotoxicity and adhesion. Mutagenesis studies targeting the putative LOS biosynthesis genes wlaRF, wlaTA, wlaTB, wlaTC and waaV were performed in order to characterise the proteins encoded by each of these six genes and assess their potential role in C. jejuni pathogenesis in vitro. The gene product of wlaTA was found to be essential for C. jejuni survival and therefore a knock out mutant could not be generated. Phenotypic characterisation of four knock-out mutants confirmed that each gene contributed to the construction of the LOS molecule as all four mutants produced a truncated LOS moiety and altered their immunoreactivity. Further analysis determined that the production of complete LOSs was important for C. jejuni to invade and adhere to both human and chicken cells in vitro. This study identified a link between the inactivation of two LOS biosynthesis genes and the loss of motility, another important virulence factor. A major source of human C. jejuni infection is contact with contaminated poultry. However, C. jejuni exists as a commensal in chickens. It is currently not known why C. jejuni is pathogenic to humans and not to chickens and the differences between these two hosts represent pathogenic and non-pathogenic environments respectively. These environmental differences were exploited in this study. The four conditions investigated were temperature, blood, bile and host cells in vitro. Five different C. jejuni strains (NCTC11168, 81116, HB93-13, a recent human enteritis isolate and a recent chicken isolate) were subjected to modelled

Repetitive tetramers of the DNA sequence 5'-CAAT-3' are present in several loci associated with lipooligosaccharide (LOS) phase variation in Haemophilus influenzae type b (Hib). In an attempt to identify H. somnus phase-variable LOS genes, the presence of CAAT repeats within the H. somnus 738 genome was confirmed using a (CAAT)7 probe. A 3.9 kb EcoRI fragment that reacted with the probe was cloned and sequenced. Sequence analysis confirmed the presence of 31 CAAT repeats downstream of two potential start codons, and indicated that small or large proteins would be encoded depending on the number of CAAT repeats. The larger gene products showed 46% amino acid homology to Lex2b from Hib, which influences LOS phase variation in that species. In H. somnus, this gene was named lob1 (lipooligosaccharide biosynthesis gene). Sequence analysis showed that randomly selected colonies most frequently contained 33 CAAT repeats in lob1, corresponding to a 294 amino acid product. Colonies selected for negative reactivity to mAb 5F5 were significantly more likely to have different numbers of CAAT repeats in lob1 than randomly selected colonies. The presence of lob1 in trans altered the LOS profile of a non-phase variable strain of H. somnus, and caused increased levels of reactivity to polyclonal antisera made to purified LOS from strain 738. Based on the ability of this gene to alter the LOS profile of a non-phase varying strain and the correlation of changes in CAAT repeats with mAb 5F5 reactivity, lob1 appears to be involved in LOS biosynthesis and phase variation. / Master of Science

<p>This thesis focuses on host and pathogen specific interactions during invasive disease. We have investigated the role and impact of different virulence factors of <i>Neisseria gonorrhoeae, N. meningitidis</i> and <i>Streptococcus pyogenes</i> on host epithelial cells and <i>in vivo</i>. </p><p><i>N. gonorrhoeae</i> cause the sexually transmitted disease gonorrhoea and <i>N. meningitidis</i> is the most common cause of bacterial meningitis and may be leathal to the host within hours of infection. The neisserial type IV pili were shown to have an important impact on host cells for the induction of pro-inflammatory and other cellular defence transcriptional responses. Furthermore, <i>N. meningitidis</i> generally induced an earlier response compared to <i>N. gonorrhoeae</i>, probably as a result of the meningococcal capsule. The role of <i>N. meningitidis</i> serogroup B lipooliogsaccharide was investigated during invasive disease. Bacterial invasion of host cells and blood survival as well as virulence in vivo was dependent on the integrity of the LOS structure. </p><p><i>S. pyogenes</i> may cause a variety of diseases ranging from uncomplicated diseases such as 'strep-throat' to more severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. <i>S. pyogenes</i> ScpC protease degrade interleukin 8 during necrotizing fasciitis. We investigated the role of ScpC in systemic disease and observed enhanced virulence by bacteria unable to degrade IL-8. Following an intravenous infection of mice pro-inflammatory cytokines and complement activation was induced by the ScpC negative mutant compared to the wild-type and correlated with higher bacteremia. These data indicate that the precense of the ScpC protease has an important impact on the host for the outcome of streptococcal sepsis. Another phagocytic escape mechanism of <i>S. pyogenes</i> is their ability to coat themselves with host proteins. We observed that released complement control protein, CD46, bound to the streptococcal cell surface. CD46 has been shown to interact with the streptococcal M protein and have now been found to bind to the surface of the bacteria in a growth phase dependent manner. We observed a more aggressive disease development in CD46 transgenic mice after an intravenous infection with an M6 serotype, resulting in higher mortality of CD46 transgenic mice compared with control mice. These data indicate that CD46 may confer a protection to the streptococci during early stage of systemic infection and contributes to the understanding of immune evsion of <i>S. pyogenes</i>.</p>

This thesis focuses on host and pathogen specific interactions during invasive disease. We have investigated the role and impact of different virulence factors of Neisseria gonorrhoeae, N. meningitidis and Streptococcus pyogenes on host epithelial cells and in vivo. N. gonorrhoeae cause the sexually transmitted disease gonorrhoea and N. meningitidis is the most common cause of bacterial meningitis and may be leathal to the host within hours of infection. The neisserial type IV pili were shown to have an important impact on host cells for the induction of pro-inflammatory and other cellular defence transcriptional responses. Furthermore, N. meningitidis generally induced an earlier response compared to N. gonorrhoeae, probably as a result of the meningococcal capsule. The role of N. meningitidis serogroup B lipooliogsaccharide was investigated during invasive disease. Bacterial invasion of host cells and blood survival as well as virulence in vivo was dependent on the integrity of the LOS structure. S. pyogenes may cause a variety of diseases ranging from uncomplicated diseases such as 'strep-throat' to more severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. S. pyogenes ScpC protease degrade interleukin 8 during necrotizing fasciitis. We investigated the role of ScpC in systemic disease and observed enhanced virulence by bacteria unable to degrade IL-8. Following an intravenous infection of mice pro-inflammatory cytokines and complement activation was induced by the ScpC negative mutant compared to the wild-type and correlated with higher bacteremia. These data indicate that the precense of the ScpC protease has an important impact on the host for the outcome of streptococcal sepsis. Another phagocytic escape mechanism of S. pyogenes is their ability to coat themselves with host proteins. We observed that released complement control protein, CD46, bound to the streptococcal cell surface. CD46 has been shown to interact with the streptococcal M protein and have now been found to bind to the surface of the bacteria in a growth phase dependent manner. We observed a more aggressive disease development in CD46 transgenic mice after an intravenous infection with an M6 serotype, resulting in higher mortality of CD46 transgenic mice compared with control mice. These data indicate that CD46 may confer a protection to the streptococci during early stage of systemic infection and contributes to the understanding of immune evsion of S. pyogenes.

Histophilus somni virulence factors include expression and antigenic variation of lipooligosaccharide (LOS). Phosphorylcholine (ChoP) is often expressed on H. somni LOS and also undergoes antigenic variation. In this study, five genes that play a role in expression and antigenic variation of ChoP, lic1ABCD and glpQ, were identified in the genome sequence of H. somni through sequence homology with Haemophilus influenzae genes. The open reading frame (ORF) of lic1A contained a variable number of tandem repeats of the tetranucleotide unit 5'-AACC-3'. Slipped strand mispairing in the repeat region during replication leads to shifting the downstream reading frame in and out of frame with the start codon, thus controlling phase variation of lic1A expression. Removal of the repeats from lic1A, cloning the gene in E. coli, and performing a functional assay on the product indicated that lic1A encodes a choline kinase and that the repeats were not required for expression of a functional gene product. Variation in the number of repeats in lic1A correlated with the antigenic variation of ChoP expression in strain 124P, but not in strain 738. This result supported previous findings that antigenic variation of ChoP expression in strain 738 is controlled through extension/truncation of the LOS outer core. Therefore, these results indicated that the lic1ABCD and glpQ genes control expression and antigenic variation of ChoP on the LOS of H. somni and that there are two possible mechanisms for ChoP antigenic variation. The role of H. somni expression of ChoP in colonization of the host respiratory tract was also examined. Experimental infection in the natural host showed that the population of H. somni that expresses ChoP was enriched in the bacteria that colonized the respiratory tract. In addition, bacteria expressing ChoP were able to aggregate bovine platelets through binding to the platelet activating factor receptor (PAF-R), which is also present on epithelial and endothelial cells. These results indicated that ChoP may play a role in the process of colonization and subsequent systemic invasion of host tissues, which may occur through binding of ChoP to PAF-R. Bacteria that did not express ChoP were more prevalent in systemic sites, indicating that ChoP expression may be disadvantageous for the organism during systemic dissemination. / Ph. D.

Haemophilus parasuis est un pathogène porcin causant la maladie de Glässer caractérisée par de la polysérosite fibrineuse, polyarthrite, méningite et septicémie. La pathogenèse de l’infection et les facteurs de virulence sont encore mal connus. Le site de colonisation de Haemophilus parasuis dans le tractus respiratoire supérieur est controversé. Pour accéder à la circulation sanguine, H. parasuis doit envahir la muqueuse. H. parasuis adhère à des cellules épithéliales porcines de trachée (NPTr). Pour accéder au système nerveux central et causer la méningite, H. parasuis doit traverser la barrière hémato-méningée. H. parasuis adhère à et envahit des cellules endothéliales porcines de microvaisseaux cérébraux (PBMEC) provenant de la BBB. Le but de cette étude était d’étudier certaines interactions entre H. parasuis et son lipooligosccharide (LOS), et des cellules endothéliales et épithéliales porcines. Les résultats démontrent que l’adhésion de H. parasuis Nagasaki aux NPTr et aux PBMEC est en partie médiée par son LOS. H. parasuis induit l’apoptose des NPTr et des PBMEC, mais le LOS ne semble pas impliqué. H. parasuis, et à un niveau moindre son LOS, stimulent la sécrétion d’interleukine- (IL) 6 et d’IL-8. Différentes souches de H. parasuis sérotypes 4 et 5 (sérotypes les plus prévalents en Amérique du Nord) stimulent également les NPTr et PBMEC à produire IL-6 et IL-8. Les résultats suggèrent que le LOS de H. parasuis joue un certain rôle dans la pathogenèse de l’infection, mais d’autres composantes bactériennes sont également impliquées. / Haemophilus parasuis is a swine pathogen that causes Glässer’s disease characterized by fibrinous polyserositis, polyarthritis, meningitis and septicemia. The pathogenesis of the infection and virulence factors are not well known. Whether the upper respiratory tract is the site of colonization of H. parasuis is still a controversial issue. H. parasuis must invade the mucosa to gain access to the bloodstream. H. parasuis is able to adhere to newborn pig trachea cells (NPTr). H. parasuis must then cross the blood-brain barrier to gain access to the central nervous system in cases of meningitis. H. parasuis is able to adhere to and invade porcine brain microvascular endothelial cells (PBMEC). The aim of this work was to study the interactions between H. parasuis, its lipooligosccharide (LOS), and porcine endothelial and epithelial cells. Results showed that adhesion of H. parasuis Nagasaki to NPTr and PBMEC was partially mediated by its LOS. H. parasuis induced NPTr and PBMEC apoptosis, although purified LOS does not seem to be involved. H. parasuis, and to a lesser extent its LOS, stimulated the release of interleukin- (IL) 6 and IL-8. Field strains of H. parasuis serotypes 4 and 5 (the most prevalent serotypes in North America) also induced the production of IL-6 and IL-8. Results suggest that H. parasuis LOS plays a role in the pathogenesis of the infection, but other bacterial components are also involved.

Haemophilus parasuis est un pathogène porcin causant la maladie de Glässer caractérisée par de la polysérosite fibrineuse, polyarthrite, méningite et septicémie. La pathogenèse de l’infection et les facteurs de virulence sont encore mal connus. Le site de colonisation de Haemophilus parasuis dans le tractus respiratoire supérieur est controversé. Pour accéder à la circulation sanguine, H. parasuis doit envahir la muqueuse. H. parasuis adhère à des cellules épithéliales porcines de trachée (NPTr). Pour accéder au système nerveux central et causer la méningite, H. parasuis doit traverser la barrière hémato-méningée. H. parasuis adhère à et envahit des cellules endothéliales porcines de microvaisseaux cérébraux (PBMEC) provenant de la BBB. Le but de cette étude était d’étudier certaines interactions entre H. parasuis et son lipooligosccharide (LOS), et des cellules endothéliales et épithéliales porcines. Les résultats démontrent que l’adhésion de H. parasuis Nagasaki aux NPTr et aux PBMEC est en partie médiée par son LOS. H. parasuis induit l’apoptose des NPTr et des PBMEC, mais le LOS ne semble pas impliqué. H. parasuis, et à un niveau moindre son LOS, stimulent la sécrétion d’interleukine- (IL) 6 et d’IL-8. Différentes souches de H. parasuis sérotypes 4 et 5 (sérotypes les plus prévalents en Amérique du Nord) stimulent également les NPTr et PBMEC à produire IL-6 et IL-8. Les résultats suggèrent que le LOS de H. parasuis joue un certain rôle dans la pathogenèse de l’infection, mais d’autres composantes bactériennes sont également impliquées. / Haemophilus parasuis is a swine pathogen that causes Glässer’s disease characterized by fibrinous polyserositis, polyarthritis, meningitis and septicemia. The pathogenesis of the infection and virulence factors are not well known. Whether the upper respiratory tract is the site of colonization of H. parasuis is still a controversial issue. H. parasuis must invade the mucosa to gain access to the bloodstream. H. parasuis is able to adhere to newborn pig trachea cells (NPTr). H. parasuis must then cross the blood-brain barrier to gain access to the central nervous system in cases of meningitis. H. parasuis is able to adhere to and invade porcine brain microvascular endothelial cells (PBMEC). The aim of this work was to study the interactions between H. parasuis, its lipooligosccharide (LOS), and porcine endothelial and epithelial cells. Results showed that adhesion of H. parasuis Nagasaki to NPTr and PBMEC was partially mediated by its LOS. H. parasuis induced NPTr and PBMEC apoptosis, although purified LOS does not seem to be involved. H. parasuis, and to a lesser extent its LOS, stimulated the release of interleukin- (IL) 6 and IL-8. Field strains of H. parasuis serotypes 4 and 5 (the most prevalent serotypes in North America) also induced the production of IL-6 and IL-8. Results suggest that H. parasuis LOS plays a role in the pathogenesis of the infection, but other bacterial components are also involved.

Targeted delivery of drugs directly to the lung epithelium is a promising, though challenging, strategy for the treatment of diseases that affect the lung tissues, such as infections caused by cell-penetrating pathogens, cystic fibrosis, and cancer. With appropriate surface functionality, such as through the attachment of ligands that recognize receptors on cellular surfaces, particulate carriers show improved efficiency in penetrating cells in vitro. A useful class of ligands is produced by many natural human pathogens that infect the respiratory tract. A variety of phylogenetically distinct respiratory bacterial pathogens, such as Haemophilus influenzae, invade host cells in the upper airways by binding of the platelet-activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms. By expressing host carbohydrate structures, including phosphorylcholine (ChoP), as a terminal structure on the LOS, the bacteria exhibit molecular mimicry of the host and are able to evade the host immune system. The effectiveness of LOS to induce cellular uptake of the bacteria is dependent on the specific glycoform, with higher ChoP content inducing more bacterial adherance into the lung epithelial. These ligands naturally expressed on bacterial cell surfaces can be isolated and utilized as targeting ligands for delivery vehicles. The studies described in this thesis focus on the development of particulate drug carriers coated with LOS bacterial ligands to enhance the targeting and binding of the carriers to the lung epithelium. Three NTHi clinical isolates were screened to select the strain with the highest ChoP level, and NTHi 3198, an isolate from a patient with chronic obstructive pulmonary disease (COPD), was selected due to its high ChoP activity. LOS from NTHi 3198 was isolated from the bacterial cell membrane, and its activity verified using dot immunoblot and ELISA techniques. Particles (0.2 and 1 µm) composed of polystyrene or poly(lactic-co-glycolic acid) were passively coated with 0.005-50 µg/mL of the isolated LOS 3198 with or without gelatin, coated with gelatin alone, or left uncoated. The LOS coating on the particles was verified using either XPS or ELISA. The association of particles with human bronchial epithelial cells was investigated using two cell culture models, 16HBE14o- and Calu-3, as a function of particle concentration and incubation time. The expression of PAFR on both cells types was confirmed, though the expression of PAFR on 16HBE14o- cells was significantly greater than on Calu-3 cells. Enhancement of 0.2 µm particle-cell association was achieved through coating of the particles with LOS. However, no significant difference in particle-cell association was observed for the 1 µm particles based on particle coating. Control particles of 0.2 µm size, those coated with gelatin (with or without LOS) or uncoated, exhibited low cell binding with a maximum of about 10-18% of cells associated with particles. The ability of the LOS ligand to enhance particle-cell association was coating concentration dependent, with a low coating concentration of LOS having little effect on association, but a concentration 1000-fold higher causing a doubling of the percentage of cells associated with particles at 24 hours. This enhancement was attributed to increased cellular binding of the 0.2 µm particles to the cell surface by confocal microscopy, and was further increased by activating the PAFR prior to incubation with particles. These results suggest the potential application of LOS as a targeting ligand for lung epithelial cells, especially under conditions where PAFR has been activated, such as occurs in lungs infected with Haemophilus influenzae. A significant reduction in particle-cell association was observed when particles were incubated with Calu-3 cells due to the presence of mucus on the cellular surface. This suggests that further optimization of the drug carrier system is needed to efficiently overcome the mucosal fluids.

Haemophilus parainfluenzae (Hp) and H. influenzae (Hi) are closely related members of the Pasteurellaceae family and are common commensal bacteria of the human nasopharynx. Whilst Hi is frequently implicated in meningitis, otitis media and respiratory tract infections, reports of pathogenic behaviour by Hp are very rare. Lipopolysaccharide (LPS) is a key component of the Gram negative cell wall, and its structure influences the ability of Haemophilus to interact with the host and evade immune clearance. A better understanding of the differences in LPS structure between Hi and Hp could help to ascertain which parts of the molecule are important for commensal and pathogenic behaviour. Hi LPS comprises lipid A, a conserved oligosaccharide inner core, and an oligosaccharide outer core that differs between strains. The latter is partly phase variable by the slipped strand mispairing during replication of DNA repeat tracts within several LPS biosynthesis genes. Very little was known about LPS in Hp so we investigated its biosynthesis and structure in a panel of 20 Hp carriage isolates. Using PCR, DNA sequencing and Southern analysis we demonstrated that Hp possesses homologues of the Hi lipid A and inner core LPS synthesis genes and a few of the genes for outer core synthesis; however, homologues of the Hi phase variable outer core genes were largely absent and did not contain repeat tracts. The results of immunoblotting and collaborative structural analysis were consistent with this data. Phosphocholine, a phase variable Hi LPS epitope that has been implicated in otitis media, was found to be absent in Hp LPS due to the lack of four genes required for its biosynthesis and incorporation. The introduction of these genes into Hp led to the phase variable addition of phosphocholine to the LPS, indicating that there is no fundamental reason why Hp could not use a similar mechanism of variation to Hi if it was advantageous to do so. SDS-PAGE data suggested the presence of O-antigens (repeated chains of sugars) in many of the Hp strains, an unusual feature for Haemophilus, and all of the strains were found to contain a potential O-antigen synthesis locus. Each locus encodes homologues of several glycosyltransferases in addition to either the Wzy polymerase- or ABC transporter-dependent mechanisms of O-antigen synthesis and transport. Comparisons of wild type and isogenic mutant strains showed that the O-antigen enhances resistance to complement-mediated killing and appears to affect adhesion to epithelial cells in vitro. Hp is a successful commensal organism but lacks the flexibility of adapting its LPS using repeat-mediated phase variation, potentially limiting its range of host niches.