Dissertations / Theses on the topic 'Nontypeable Haemophilus influenzae'

Thesis (M.S.)--University of Missouri-Columbia, 2004.
"December, 2004." The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

Haemophilus influenzae is an opportunistic pathogen that forms part of the human nasopharyngeal microbiota. This microorganism is classified into encapsulated and nonencapsulated or nontypeable (NTHi) isolates, depending on the presence of a polysaccharide capsule. Although H. influenzae is a common respiratory commensal, it is also able to cause several infections, especially in patients with comorbidities. The most common respiratory infections in which H. influenzae can be identified as the main etiological agent are exacerbations in patients with Chronic Obstructive Pulmonary Disease (COPD), community-acquired pneumonia (CAP), cystic fibrosis, and otitis media. In addition, this pathogen is also a common cause of invasive infections such as bacteraemia and meningitis. Before the introduction of the conjugate vaccine, H. influenzae serotype b (Hib) was the main cause of meningitis in children under five years. However, effective childhood vaccination has caused a dramatic reduction in Hib and allowed the expansion of NTHi, which is becoming more relevant in both respiratory and invasive infections. In this thesis, we studied three different aspects of the epidemiology of NTHi since the introduction of the vaccine. Our study focused on molecular genotyping, antimicrobial resistance and adhesion and biofilm formation of NTHi isolates from healthy children and from adult patients with CAP, COPD and invasive diseases. In the first part of this thesis, we set out to characterize the NTHi populations that are involved in adult infections in Bellvitge hospital. Furthermore, as humans are the only reservoir of NTHi, we aimed to identify the oropharyngeal carriage rate in healthy children attending day care centres in Oviedo. The aim of the second part of this thesis was to determine the antimicrobial susceptibility profile of clinical NTHi isolates, placing emphasis on the molecular characterization of B-lactam and fluoroquinolone resistance, the main antimicrobials used in the treatment of NTHi infections. The last part of this thesis focused on adhesion and biofilm formation. Biofilm is one of the mechanisms that microorganisms have developed in order to protect themselves and survive in hostile environments. Once the biofilm structure is formed, it is difficult to eliminate and, as a consequence, biofilm-associated infections commonly show recurrent symptoms. Although biofilm formation by NTHi remains controversial, biofilm-like structures have been observed in middle-ear mucosa in experimental chinchilla models of otitis media. Taken together, all the studies discussed in this thesis can improve our understanding of the clinical epidemiology of NTHi populations since the introduction of vaccination and of the mechanism of biofilm formation in clinical isolates of this microorganism.
Haemophilus influenzae és un patogen oportunista que forma part de la microbiota nasofaríngia humana. Aquest microorganisme es classifica en soques capsulades i no capsulades o no tipables (HiNT) depenent de la presència d’una càpsula polisacarídica. Tot i que H. influenzae és un comensal respiratori comú, posseeix la capacitat de causar diferents infeccions, especialment en pacients amb malalties de base. Les infeccions respiratòries més freqüents causades per H. influenzae són les exacerbacions agudes en pacients amb Malaltia Pulmonar Obstructiva Crònica (MPOC), pneumònia adquirida en la comunitat (PAC), exacerbacions en pacients amb fibrosis quística i otitis mitjana. A més, aquest patogen és també una causa freqüent de malalties invasives com bacterièmia i meningitis. Abans de la introducció de la vacuna conjugada, H. influenzae serotipus b (Hib) fou la causa principal de meningitis en nens/es menors de cinc anys d’edat. No obstant, l'efectiva vacunació ha causat un dramàtic descens del Hib permetent l’expansió dels HiNT, que s’estan convertint en un patogen més rellevant tant en infeccions respiratòries com en infeccions invasives. Els objectius plantejats en aquesta tesis, foren l’estudi de tres aspectes de la epidemiologia dels HiNT en l'etapa posterior a la introducció de la vacuna en Barcelona: la genotipificació molecular, la resistència antibiòtica i la formació de biofilm en soques d’HiNT aïllades de nens/es sans i de pacients adults amb PAC, MPOC i malalties invasives. L’objectiu de la primera part d’aquesta tesis fou caracteritzar les poblacions d’HiNT involucrades en les infeccions en pacients adults de l’hospital de Bellvitge així com la determinació de la freqüència de colonització orofaríngia d’HiNT en nens/es sans que van a llars d’infants en Oviedo. L’objectiu de la segona part de la tesis fou determinar els perfils de susceptibilitat antibiòtica dels aïllats clínics d’HiNT, emfatitzant en la caracterització molecular de la resistencia a B-lactàmics i fluoroquinolones, ja que són els antibiòtics més utilitzats en el tractament de les infeccions per HiNT. L’última part de la tesis està enfocada a l’estudi de l’adhesió i la formació de biofilm. El biofilm és un dels mecanismes que els microorganismes han desenvolupat per a la protecció i supervivència en ambients hostils. Una vegada l’estructura del biofilm està formada és molt difícil d’eliminar i, com a conseqüència, les infeccions associades a biofilm presenten símptomes recurrents. Tot i que la formació de biofilm per HiNT roman controvertida, estructures tipus biofilm s’han observat en la mucosa de l’oïda mitjana en models experimentals d’otitis mitjana en xinxilla. En conjunt, tots els estudis discutits en aquesta tesis contribueixen a ampliar el coneixement de la epidemiologia clínica, la resistència antibiòtica i la formació de biofilm de les poblacions d’HiNT en un període posterior a la introducció de la vacuna.

Nontypeable Haemophilus influenzas (NTHi) is recognised as a significant human pathogen causing mild to severe respiratory tract infections. At present, no vaccine is available for prevention of infection caused by this pathogen. Several outer membrane proteins (OMPs) of NTHi and its lipooligosaccharide have been investigated as possible vaccine antigens against NTHi infections. Previous investigations in our laboratory have shown that OMP26 from an NTHi 289 strain was able to significantly enhance pulmonary clearance of NTHi in a rat model in which animals were immunised via intestinal Peyer's patches and then boosted intratracheally (Kyd and Cripps, 1998; El- Adhami et al., 1999). In recent studies, the OMP26, when used as a parenteral immunogen, was also highly effective at inducing immune responses that led to significantly enhanced clearance of the chinchilla nasopharynx (Kyd et al., 2003). These studies indicate significant potential of the OMP26 as a candidate vaccine antigen and warrant further investigations for development of a vaccine against NTHi. This thesis focussed on the immunological and structural characterisation of the NTHi vaccine candidate, OMP26. Peptides of OMP26 were used as tools to localise the immunologically important regions of the OMP26. Two different E. coli expression systems, the GST gene fusion and the 6xHis tagged systems, were employed to construct the OMP26 peptides. It was found in this study that, despite efforts to optimise the system, the GST-fusion protein system failed to produce consistent results for the purification and storage of the OMP26 peptides. In contrast, the 6xHis tagged system exhibited more reliable outcomes in the production of the recombinant OMP26 peptides and the stability of the stored purified peptides. As such, the purified OMP26 peptides from the 6xHis tagged system were chosen to map major regions of immunological significance for the OMP26 protein. The regions of the OMP26 which are involved in the induction of the acquired immune responses have been identified in the present study. Based on the antigen specific lymphocyte proliferation assay, the dominant T cell epitopes for OMP26 were located between amino acid residues 95 and 197 (T3+T4 region). These identified T cell epitopes exhibited the capability of efficient T cell activation, suggesting that the epitopes within the T3+T4 region potentially had the highest affinity for binding to the MHC molecules than did any other OMP26 region. Using two different assay systems, ELISA and BIA, the predominant B cell epitopes of OMP26 were located between amino acid residues 45 and 145 (T2+T3 region). This region was also found to be immunodominant across all animal species tested, and with all immunisation regimens used. Flow cytometry analysis also revealed that these particular epitopes were expressed on the surface of NTHi cells. By integration of the data obtained from these current experimental studies and the computational analysis of the OMP26 sequence, two hypothetical models of the OMP26 were also proposed in this study. The significant outcomes obtained in this thesis provide a better understanding of the specificity of the host immune responses to the OMP26 protein These findings provide great benefit not only for the development of a future NTHi vaccine but for the development of the peptide-based immunodiagnostic reagents as well. These diagnostic reagents will be valuable, in particular, for the evaluation of efficacy of an NTHi vaccine in humans that may include OMP26 or specific conformational structures. Future studies are still required to further define the minimum epitope length required for the B and T cell responses identified in this study. The significance of these responses in immune protection against NTHi infection also requires further investigations. Human immune responses also need to be determined, but this can only be achieved following clinical trial studies.

Pulmonary infection caused by nontypeable Haemophilus influenzae (NTHi) is a significant cause of morbidity and mortality in both industrialised and developing countries. Previous work from this group resulted in the development of a respiratory model in rodents which has precipitated studies into the pathogenesis of infection by NTHi and investigation of the humoral and cellular mechanisms by which the bacteria are cleared from the lung. Comparison of mucosally immunised with non-immunised animals has demonstrated that not only are bacteria cleared more rapidly from the lungs, but there is a more rapid response and resolution of inflammatory factors in the mucosally immunised animals following challenge with NTHi. This inflammatory response is partially regulated by the ability of the mucosally immunised animals to rapidly produce, then control the production of tumour necrosis factor (TNF)-a. The TNF-a is produced by both macrophages and type I pneumocytes in the alveoli and also by the endothelial cells lining the blood vessels in the lungs. Immunocytochemical studies have identified cellular subsets accumulating in the lung at various time points following infection. Marked differences in cellular infiltration into the lung tissue were noted between immunised and non-immunised animals after challenge with NTHi. Immunised animals demonstrated an early influx of macrophages, CD8+ T cells and Y8+ T cells, followed by enhanced expression of the MHC-II marker, cellular infiltration by polymorphonuclear leukocytes and finally an increased number of both B cells and CD4+ T cells. In contrast, non-immunised animals did not demonstrate any proliferation nor extravasation of lymphocytes or increased expression of MHC-II before total bacterial clearance had occurred. Polymorphonuclear leukocyte infiltration occurred in the non-immunised animals, however at a later time than that seen in immunised animals. Challenging rodents to establish persistent infection highlighted the inappropriately aggressive white blood cell response to an initial challenge when bacteria may be masked by other substances, followed by the inability to amplify the polymorphonuclear leukocyte response on repeated challenge with NTHi. This hyporesponsiveness in the macrophage population, shown by lack of detectable TNF-a production, concomitant with low numbers of NTHi resulted in a continuously high number of macrophages in the alveoli and the possibility of increased damage to the lung tissue. The requirement for cell surface TNF-a and CD8+ T cells to enhance the clearance of NTHi from the lungs further strengthens previous in vitro and in vivo findings of the possible significance of cellular invasion as a mechanism of pathogenicity for NTHi. This thesis has contributed to the understanding of both the immune response to and the pathogenicity mechanisms of pulmonary infection with NTHi. Kinetic studies identifying cellular responses and cytokine levels have emphasised the ability of mucosal immunisation to increase the rate of immune response and resolution of inflammation to NTHi infection in the lung. Observations demonstrating a requirement for macrophages and CD8+ T cells in mechanisms associated with enhancing NTHi clearance from the lung will lead to further investigations.

One of the major challenges of vaccine development is the conservation of immunogenicity and protective efficacy through the stages of design, production, formulation and delivery. The critical issue is that how and in what form an antigen is taken up by antigen presenting cells for proteolytic processing and presentation to the immune system bound to MHC can have dramatic effects on the activation of Th cells to drive clonal responses and induction of immunological memory. Nontypeable Haemophilus influenzae (NTHi) is a pathogenic commensal of the human respiratory tract that causes diseases with enormous socioeoconomic burdens. There is no licensed vaccine, although the potential for vaccination with outer membrane components to reduce the incidence of disease caused by NTHi has recently been demonstrated in clinical trials. The issue of immunomodulation was explored in this thesis in the context of the further evaluation of a leading NTHi vaccine candidate, the outer membrane protein OMP26. The efficacy of recombinant OMP26 (rOMP26) against NTHi challenge has been previously demonstrated in mice, rats and chinchillas. In rats, efficacy was shown to be restricted to the precursor form (containing the signal peptide) and not the mature form of rOMP26. The immunodulatory effects of changes to the rOMP26 structure were further investigated in this thesis. A range of structural variants of rOMP26 were constructed in view of reducing extraneous plasmid-derived sequence from the antigen and to introduce a unique cysteine residue as a potential conjugate site for multivalent vaccine development (Chapter 2). It was demonstrated that minor structural changes to rOMP26 such as the addition, deletion, modification or relative positioning of a single amino acid or bulky group, designed to increase the efficiency of production or introduce (cysteine) conjugation sites, altered the expression of the protein in E. coli and the immunogenicity in Balb/C mice. Furthermore, in contradiction to the published report (El-Adhami et al. 1999) and a new study in rats (Chapter 3), there was no positive effect of the signal peptide in mice, with precursor and mature forms of rOMP26 equally immunogenic (Chapter 2). Following confirmation of the need to retain the signal peptide for the immunogenicity of rOMP26 in rats, a precursor form (rOMP26VTAL) in which the conserved n-region of the signal peptide was deleted, and shown to reduce the efficiency of the cleavage of the signal peptide by signal peptidase during protein overexpression in E. coli (Chapter 3). Not only did this deletion result in an increase the yield and stability of the purified precursor protein, but rOMP26VTAL was highly immunogenic and enhanced the clearance of NTHi from the lungs of challenged rats. The potential for signal peptides to be exploited as an immune-enhancing moiety in a proteinaceous vaccine is discussed. Following the development of rOMP26VTAL as a production optimised variant of rOMP26, the next step was to test the feasibility of rOMP26VTAL as a component of a multivalent vaccine (Chapter 4). Two chimeras were constructed with LB1(f)2,1,3, a trivalent synthetic B-cell epitope from the extracellular loop 3 region of the P5 fimbrin protein of NTHi, positioned at the N- or C-terminus of rOMP26VTAL. The solubility of rOMP26VTAL was affected by the fusion, with both chimera constructs expressed only in the insoluble fraction, thus requiring a denaturing protocol for purification. Although rLB1(f)2,1,3-OMP26VTAL was expressed and purified as a more stable protein and in greater yield than rOMP26VTAL-LB1(f)2,1,3, the relative positioning of the fusion was important and rOMP26VTAL-LB1(f)2,1,3 was significantly more immunogenic in rats than rLB1(f)2,1,3-OMP26VTAL. In addition, rOMP26VTALLB1( f)2,1,3, but not rLB1(f)2,1,3-OMP26VTAL induced a significant degree of bacterial clearance following pulmonary challenge with NTHi, in levels comparable to the highly efficacious rOMP26VTAL construct. In the third part of the thesis, bacterial ghosts were evaluated as a novel mucosal delivery technology for rOMP26VTAL and rOMP26VTAL-LB1(f)2,1,3, (Chapter 5). To mimic the natural presentation of OMP26 and P5 fimbrin antigens on the cell surface of NTHi, an OmpA� sandwich fusion surface display system was developed for the outer membrane expression of the OMP26 constructs in E. coli ghosts. Following gut immunisation, but not intranasal immunisation even when co-administered with the cholera toxin�derived adjuvant CTA1-DD, bacterial ghosts were successful at presenting OMP26VTAL and rOMP26VTAL-LB1(f)2,1,3 to the immune system for the induction of enhanced clearance of NTHi in the rat pulmonary challenge model. Although this study was the first to demonstrate enhanced bacterial clearance induced by heterologous antigens expressed in the outer membrane of bacterial ghosts, future studies with ghosts will require optimisation of the expression levels of the OmpA� fusion proteins possibly to avoid cross-reactive responses related to high doses of ghosts in the inoculum. This thesis presents data that both supports the further evaluation of rOMP26 constructs for clinical trials, and has demonstrated the significant effects of structural changes, method of production and delivery system can have on the immunogenicity of a candidate vaccine. Such knowledge will contribute to and provide some new approaches for enhancing the efficiency of vaccine development against a range of diseases including those caused by NTHi. Major Outcomes: 1. Demonstration that the immunogenicity of rOMP26 antigen constructs is affected by structural modifications and their positioning within the construct, and by the delivery system. 2. Development of rOMP26VTAL, an rOMP26 construct with the KNIAK sequence deletion of the signal peptide n-region. This protein retains the immunogenicity and protective efficacy of rOMP26, but is produced with reduced cleavage of the signal peptide, resulting in higher yields and a stable protein. Lacks extraneous plasmidderived multiple cloning site sequence, and is produced in high yield as a stable protein. 3. Construction of a NTHi rOMP26VTAL-LB1(f)2,1,3 chimera antigen that induced enhanced clearance of NTHi in an acute pulmonary challenge model in rats. 4. Development of an OmpA� surface display system for the expression of rOMP26 antigen constructs in the outer membrane of E. coli/bacterial ghosts 5. Bacterial ghosts were successful as delivery vehicles for rOMP26 candidate vaccine constructs when delivered in the gut.

Heterogeneity in immunodominant outer membrane proteins has been proposed as a significant factor in the failure of an NTHi infection to induce immune protection against subsequent infections. This study has examined the vaccine potential of three outer membrane proteins in an attempt to identify conserved regions that could be targeted by an immune response after vaccination. The three proteins investigated were: TbpB, P5 and P48 (HI0164). The optimal route of immunisation in clearing a bolus inoculum of NTHi to the lung in the rat has been shown to be a combination of gut sensitisation with a respiratory boost and this regime was used in the present study. A panel of NTHi isolates was assessed to determine the frequency with which strains were able to bind transferrin and thus be targeted by a TbpBspecific immune response. A high proportion of strains was able to bind transferrin with similar frequencies in isolates associated with infection and those from normal throat swabs. A protocol was developed to purify nonlipidated recombinant TbpB from NTHi using a glutathione-Stransferase (GST)-rTbpB fusion protein and Glutathione-Sepharose affinity chromatography. Mucosal-directed immunisation with rTbpB significantly enhanced clearance of an NTHi challenge to the lung, however, whilst rTbpB-specific antibodies were cross-reactive on Western immunoblots, the cross-reactivity was variable in both transferrin binding inhibition assays and bactericidal activity. This suggested that the rTbpB-specific humoral response would be variable in the recognition of heterologous NTHi isolates. The secondary structure of P5 has been controversial with several reports suggesting that P5 was a fimbrin protein composed of coiled coils. In this present study the interstrain variation in P5 amongst isolates from diverse anatomical sites, as well as computer prediction methods and spectrophotometric analysis, generated a model of P5 based on the homologous E. coli protein, OmpA. This model suggested a B-barrel conformation with no evidence of coiled coils. Synthetic peptides corresponding to conserved regions of P5 that were thought to be surface exposed, as well as a region (H3) with some homology to a protective epitope in the P. aeruginosa protein, OprF, were then combined with a "promiscuous" T cell epitope from the measles virus F protein (MVF) and used for immunisation studies. Whilst variable protection was seen with the peptides, the MVF/H3 peptide was the most efficacious of the antigens assessed in this study in enhancing clearance of NTHi. This occurred in the absence of detectable peptide- or PS-specific antibody leading to the suggestion that cell mediated responses may have played an important role in enhancing clearance in this model. The highly conserved nature of the region in P5 represented by the H3 peptide suggests that further study should be focused on this peptide as a potential NTHi vaccine candidate. The last antigen, P48, is homologous to a A. pleuropneumoniae antigen, AopA, which has been proposed to have potential as a vaccine component against pleuropneumonia in pigs. Sequence analysis of the gene encoding P48 from several isolates showed that this protein was well conserved. Recombinant P48 was purified from a GST-rP48 fusion protein and used for immunisation, which also conferred significant protection. However, immunisation with rP48 was not as efficacious as immunisation with the MVF/H3 peptide. Whilst immunisation with rP48 induced high antibody titres, no bactericidal activity could be detected indicating that bactericidal antibody had not contributed to the observed clearance. In addition, the rP48- specific serum IgG was predominantly of the IgG2a isotype suggesting that Thl cell mediated responses had been induced by immunisation with rP48.

An animal model of respiratory infection was used to determine the effect of various factors, thought to influence the ability of the host to clear bacteria, on the host?s innate response to an NTHi lung infection. Mucosal immunisation with NTHi has previously been shown to enhance the clearance of NTHi from the lung in an animal model of infection through the increased recruitment of phagocytes. Comparisons of cytokine and chemokine kinetic profiles were made in order to determine differences between innate and acquired immune response and the way in which mucosal immunisation controls the innate immune response to NTHi. Increased production of proinflammatory cytokines and chemokines in the early stages of NTHi lung infection enhanced the ability to clear bacteria from the rat lung in the immune animals through the increased recruitment of phagocytes to the site. Mucosal immunisation was found to alter the cytokine and chemokine mRNA profiles of CD4+ and CD8+ cells, with increased levels of MCP-1 protein being detected in both types of immune cells. An antecedent viral infection has been shown to increase the chance of developing a respiratory bacterial infection. The NTHi model of respiratory infection was used to characterise the effect that a viral infection had on the host response to the host?s innate response to a bacterial infection and the ability to clear the bacteria. The host?s ability to clear NTHi from the rat lung was enhanced by an antecedent viral infection through alterations to the innate immune response and the cytokine and chemokine kinetic profiles. The use of a mutant strain of NTHi deficient in a component of Lipooligosaccharide (LOS), Phosphorylcholine (ChoP), was utilised as a tool to characterise the innate immune response to LOS. Animals challenged with the LOS mutant strain had a reduced inflammatory response to NTHi through the decreased production of pro-inflammatory cytokines and chemokines and the reduced recruitment of phagocytes to the site of infection. This thesis has contributed valuable information to enable a better understanding of the host?s innate immune response to respiratory infection. This study has identified the role of cytokines and chemokines in the innate response to a respiratory bacterial infection and the enhanced ability of the host to clear NTHi from the lung.

COPD is a chronic inflammatory disease of the airways where many patients have recurrent lower airway bacterial infection, most commonly nontypeable Haemophilus influenzae (NTHi). Corticosteroids are commonly used anti-inflammatory drugs in COPD with limited clinical benefit. Previous studies focused on corticosteroid responsiveness in COPD did not consider the role of airway NTHi infection. Alveolar macrophages are the main inflammatory cells in COPD pathogenesis, a shift in their phenotype was highlighted in COPD patients. Some bacteria can modify alveolar macrophage phenotype to persist in the lower airways. I have optimised a clinically relevant in vitro model of NTHi infection; human alveolar macrophages were stimulated with an increasing load of live NTHi clinical isolate (R2846). NTHi provoked time-dependent release of TNF-α, IL-6, CXCL8 and IL-10 from alveolar macrophages, which was correlated with bacterial growth, lysis and phagocytosis in the model. Furthermore, NTHi load was inversely correlated with IL-10 release. These findings suggest that NTHi infection is a dynamic inflammatory process in human alveolar macrophages and pointed to the possible role of IL-10 in the NTHi persistence in the lower airways. NTHi-induced cytokines in alveolar macrophages showed reduced corticosteroid responsiveness, CXCL8 was particularly corticosteroid unresponsive cytokine. NTHi-induced glucocorticoid receptor (GR) phosphorylation at ser 226 residue, which would encourage GR nuclear exportation. This might be one possible mechanism of reduced corticosteroid response in the model. In line with the latter finding, NTHi impaired the corticosteroid-induced GR nuclear localisation, which was partially reversed by p38 MAPK inhibitor (BIRB-796). These results suggest the role of NTHi in corticosteroid unresponsiveness in COPD alveolar macrophages. NTHi-induced cytokine release in alveolar macrophages was mediated by NF-κB, p38 and ERK MAPK pathways. Combination of corticosteroid (dexamethasone) with p38 and ERK MAPK inhibitors (BIRB-796 and AZD6244 respectively) showed a potential synergistic anti-inflammatory effect in the model. Therefore, inhibitors of MAPK pathways might serve as future anti-inflammatory therapies in NTHi-infected COPD patients. NTHi in vitro infection caused upregulation of the pro-inflammatory (TNF-α, CXCL8, CD38) and the anti-inflammatory (IL-10) markers’ mRNA levels in COPD alveolar macrophages. Meanwhile, NTHi downregulated the antigen-presentation molecule (HLA-DR) and the scavenger receptors (CD14, CD36, CD163 and CD206) mRNA levels in COPD alveolar macrophages. Moreover, sputum macrophages from NTHi-infected stable COPD patients showed lower mRNA levels of CD36 and HLA-DR. These findings suggest that the NTHi modification of alveolar macrophage functions, especially antigen presentation and efferocytosis, might be a possible mechanism of NTHi chronic infection and COPD disease progression.

Bien qu’il existe un vaccin contre haemophilus influenzae (hi) type b, aucun n’est disponible contre la forme non encapsulee (nthi) responsable d'infections du systeme respiratoire telle que l'otite de l'oreille moyenne. Cette infection entraine des sequelles auditives dans 20% des cas et reste la premiere cause de visite pediatrique dans les pays developpes. L’enjeu de ce travail a ete de developper un systeme permettant l'analyse de l'expression genomique durant les phases de l'infection. Et ce dans le but d’acquerir une meilleure comprehension de la pathogenese de nthi et d’identifier les antigenes de surface transcrits in vivo, representant des cibles potentielles pour un vaccin. Les sequences des genes de hi ont ete analysees pour determiner les domaines conserves, et repertories dans une banque de donnees: "the hi genomic master key". Celle-ci permet la construction d’amorces et sondes pour l’etude de ces genes dans des souches de sequences inconnues. Les protocoles necessaires a la manipulation, l’isolation et la purification d’arn ont ete mis en place et adaptes aux conditions experimentales. Une nouvelle methode d’acquisition d’echantillons ex vivo dans le chinchilla a ete developpee afin d’augmenter la quantite de bacteries disponibles pour l’analyse microarray. L’affinement des connaissances des mecanismes infectieux de nthi, par cette nouvelle methode, a des retombees cliniques pour les traitements antibiotiques et le developpement de vaccins. La plateforme microarray pour l’etude du transcriptome in vivo a ete etablie et a permis pour la premiere fois l’etude in vivo du transcriptome au cours des phases de colonisation et d’infection d’un pathogene
While a successful capsular vaccine has been developed for encapsulated serotype b haemophilus influenzae (hi), no vaccine exists for unencapsulated hi responsible for respiratory tract infections. These include otitis media that can result in sequelae in 20% of cases and remains a primary reason for pediatrician visits in developed countries. The goal of this study was to develop a system to analyze hi genes expression genome-wide during the different stages of disease. Successful comprehensive analysis at this level would provide critical insight into the pathogenesis of nthi during the course of otitis media as well as in vivo expression of particular surface antigens and thus potential candidate vaccine targets associated with infection. An initial screen of all publicly available nthi genes was carried out and conserved sequences then referenced in a database: called the hi genomic master key database. This database allows for subsequent screening of vaccine target candidates in any nthi isolate of unknown genomic sequence. All rna procedures and protocols needed for gene expression analyses were developed and adapted to the specific experimental conditions of this study. Additionally, the novel multiple consecutive lavage sampling methodology was developed to increase the amount of ex vivo bacterial mrna available for microarray analysis. This sampling method also provided insights into nthi induced otitis media with resulting clinical implications. Finally, microarray ex vivo gene expression analyses were used here for the first time to study nthi in vivo transcriptional profile genome-wide during the course of nasopharyngeal colonization and middle ear infection

Otitis media (OM) is one of the most common bacterial diseases of childhood and include both acute and chronic forms of disease (1). Whereas acute OM (AOM) is a disease with rapid onset and is short in duration, chronic forms of OM can persist for weeks to months. The most severe form, chronic suppurative OM (CSOM) occurs upon OM with tympanic membrane rupture and is characterized by purulent drainage from the middle ear space that persists for at least 2 weeks or longer. There are 709 million cases of acute OM and 65 million to 330 million episodes of chronic suppurative OM (CSOM) occurring each year, worldwide (2, 3). Complications of CSOM result in the deaths of at least 50,000 children under 5 years of age in resource-poor regions across the world (4, 5) and morbidity associated with OM is significant for all children worldwide. Hearing loss due to OM is associated with developmental delays in behavior, language acquisition and education, all factors with profound influence that last well into adulthood (6-8). Significantly, prevention of the first incidence of OM curb extensive antibiotic prescription and surgical intervention in young children and is projected to reduce subsequent episodes, limiting the disease-related sequellae (9). Nontypeable Haemophilus influenzae (NTHI) is a primary causative agent of OM, in addition to multiple upper and lower respiratory tract diseases (10). In children, NTHI is the predominant bacterium implicated in chronic OM, recurrent OM and OM for which treatment has failed (11-14). Moreover, whereas Streptococcus pneumoniae was the primary pathogen in OM prior to the use of pneumococcal conjugate vaccines, NTHI now also predominates during acute OM (15-17). NTHI, a typically benign commensal inhabitant of the human nasopharynx, possesses numerous determinants that facilitate its persistence, including outer membrane adhesive proteins and lipooligosaccharide (18). Perturbation of innate and/or physical immune defenses can result in disease distally, for example within the middle ear during OM, sinuses in rhinosinusitis, lungs in chronic cough and during episodes of exacerbations of chronic obstructive pulmonary disease (COPD) and cystic fibrosis (19). Moreover, the ability of this bacterium to rapidly form a biofilm, a community of bacteria that is frequently polymicrobial and is highly recalcitrant to the action of antibiotics and resistant to clearance by host immune effectors (20) promotes disease chronicity. This compilation of my research, presented for consideration herein, describes the pre-clinical development of vaccines against NTHI-induced OM with a focus on two critical adhesive proteins expressed by NTHI, outer membrane protein P5 (OMP P5) and the Type IV pilus (Tfp), selected from my research performed over the past 22 years. Adhesin OMP P5 binds to mucin, intercellular adhesin molecule- 1 (ICAM1) and carcinoembryonic antigen-related adhesion molecule-1 (CEACAM1) (21-25), and I identified that NTHI Tfp engages ICAM1 (26). Furthermore, NTHI Tfp performs multiple biological functions which include competence, twitching motility and biofilm formation (27-31). I show that antibodies directed against the majority protein subunit of NTHI Tfp, PilA, induces active dispersal of NTHI from established biofilms, an outcome that is dependent on and coordinated with expression of the quorum signaling molecule, LuxS (29). Toward development of NTHI OMP P5 and Tfp-directed vaccine immunogens, I performed epitope mapping studies that revealed minimal immunodominant and immunoprotective domains within each native protein (32-34). These data guided the subsequent design of a novel chimeric immunogen, as a multi-component vaccine may be necessary to provide maximal protection against this heterogeneous bacterial species (35). I validated pre-clinical efficacy of each immunogen in chinchilla models of NTHI-induced experimental OM (29, 34-36). Although immunisation via intramuscular or subcutaneous injection is wellestablished and proven to be one of the most effective strategies for disease prevention, non-invasive delivery methods have the potential to promote greater compliance, reduce costs associated with production and administration and extend the reach of vaccines to underserved regions (37). To explore this premise, I considered transcutaneous immunisation (TCI). My first efforts involved rubbing vaccine formulations on to the pinnae (or outer ears) of chinchillas (38, 39), a strategy that was later refined to utilize a small circular bandaid applied to the postauricular region behind the ear as a delivery device and administration route (40, 41). I examined the influence of adjuvantation, skin hydration and homing of antigenpresenting cell to local lymphoid tissues as essential factors toward induction of protective immunity. I characterised the durability of immunity induced by skin vaccination and validated TCI as a highly effective strategy to induce protective immune responses in experimental models of NTHI-induced OM (38-41). Improving vaccination methodologies for young children, particularly those in resource-poor regions of the world, was a key consideration in TCI conceptualization and refinement. Collectively, these data demonstrate the simplicity of bandaid immunisation which, combined with highly effective antigens, that target two critical NTHI adhesive proteins admixed with a potent adjuvant, has tremendous potential to enable greater reach of vaccines against NTHI and diseases of the respiratory tract, including OM.
Thesis (Professional Doctorate)
Doctor of Philosophy by Publication (PhD)
School of Medicine
Griffith Health
Full Text

Moraxella bovis, Moraxella catarrhalis and Nontypeable Haemophilus influenzae (NTHi) are Gram-negative, oxidase positive, pathogenic microorganisms. M. bovis causes Infectious Bovine Keratoconjunctivitis, known (IBK) as ‘pink-eye’ in cattle worldwide. The disease is of economic importance as it leads to substantial economic loss in the cattle and dairy industries. There are antibiotic treatments available to treat M. bovis associated infections, but they tend to be ineffective at controlling disease outbreaks. Importantly, the current series of antibiotics used to treat IBK have shown occurrence of resistance due to beta-lactamase enzyme produced by the bacteria. There does exist a pilin-based vaccine for IBK which has been approved for use in Australia, although it is problematic because it is not protective against all strains of M. bovis. M. catarrhalis and NTHi are human respiratory tract opportunistic pathogens responsible for otitis media in children and exacerbate chronic obstructive pulmonary disease in adults. Similar to M. bovis, both bacteria have been shown to produce β-lactamase, which has led to the emergence of antibiotic resistance. There is no licenced vaccine for M. catarrhalis or NTHi infections. In the past two-decades studies on M. catarrhalis lipooligosaccharide (LOS) have suggested that this cell surface glycolipid could potentially be incorporated into vaccines. This is based on immunogenicity in a mouse model and role in adherence and invasion of host epithelia and serum resistance. It is also conserved among strains. M. bovis lipooligosaccharide (LOS) is not well studied. Structural analysis of wild-type M. bovis strain Epp63 oligosaccharide (OS from LOS) have identified the core OS as containing eleven sugar residues, including Kdo (the number of Kdo residues is still unknown) with a branched structure. Interestingly, this core OS has an unusual terminal open chain (1S)-GalaNAc residue and lacks heptose residues in its inner core. Recent studies have also elucidated the structure of the cell surface glycans in other strains (M. bovis Mb25 capsule and M. bovis Epp63 LOS), but whether the unusual structural features are present in other strains of M. bovis, was not known. This study elucidated the OS structure and identified the presence of capsular polysaccharide in M. bovis strains Mb25 and L183/2. NMR spectroscopy of the OS from M. bovis Mb25 and L183/2 showed that the structural characteristics of Epp63 and Mb25 strains are shared and that L183/2 OS lacks the terminal (1S)-GalaNAc residue. Strain L183/2 has the same capsular polysaccharide as strain Mb25, namely unsulfated chondroitin, whereas strain Epp63 does not express a capsule. The biological activity of M. bovis Epp63 LOS and the role LOS play in causing disease is not known, therefore a series of biological assays were investigated using OS mutants with varying glycan length. It was identified that LOS truncation affected M. bovis Epp63 susceptibility towards the antibiotics novobiocin and chloramphenicol, but not to nalidixic acid, polymyxin B, rifampin, Tween 20, Triton X-100 or vancomycin. Highly truncated Epp63 OS mutant was found to be susceptible to the bactericidal activity of bovine serum. The growth rate of wildtype L183/2 was significantly slower than that of wild-type Mb25 or Epp63, however, the Epp63 OS mutants showed reduced growth rates compared to the wild-type. Limulus amebocyte lysate (LAL) assay revealed that L183/2 (1.3 x104 EU/mL) and Mb25 (8.9 x 103 EU/mL) had significantly higher toxicity than Epp63 (3.8 x 103 EU/mL; p<0.0001 when compared to L183/2 and Mb25) or Epp63 mutant OS strains (p<0.0001 when compared to L183/2 and Mb25). LOS truncation (from mutant Epp63 strains) affected bacterial interaction with mammalian cells, with increasing truncation inversely correlating with bacterial adherence to Chang conjunctival and HeLa cells. Adherence assays revealed that the Epp63 strain had lower adherence levels than Mb25 or L183/2 strains to Chang or HeLa cells, which decreased with OS truncation. L183/2 was found to exhibit higher levels of adherence than Mb25 or Epp63 to the cell lines. These observations collectively indicate that the OS moiety of the LOS is a significant component in facilitating membrane integrity to preserve normal cell growth and colonisation, maintaining complement resistance of the bacteria to bovine serum and susceptibility towards hydrophobic agents/antibiotics. Interestingly, it also suggests that the OS may possibly play a role in toxicity, at least as measured by LAL assay (lipid A structure of the mutants needs to be elucidated in-order to conclusively indicate OS role). Unlike M. bovis, there is already substantial information known about M. catarrhalis LOS. This information has been exploited here towards vaccine studies utilising M. catarrhalis LOS. Firstly, we investigated the biological role of M. catarrhalis LOS to confirm its suitability as a vaccine antigen. The outer membrane glycan from M. catarrhalis 2951 and 3292 wild-type and 2951lgt1/4Δ mutant strains was isolated, O-deacylated and conjugated to an outer membrane protein (rOMP26VTAL) from NTHi via an adipic dihydrazide linker; to produce the vaccine candidates referred to as 2951dLOS-rOMP26VTAL, 3292dLOS-rOMP26VTAL and 2951lgt1/4ΔdLOS-rOMP26VTAL, respectively. This allowed us to design a single vaccine that would be effective against both M. catarrhalis and H. influenzae. Three subcutaneous immunizations using 2951dLOS-rOMP26VTAL and 3292dLOS-rOMP26VTAL induced an antibody response to their respective serotype antigens and to rOMP26VTAL and NTHi. Confirming the efficacy of the conjugate vaccine, three subcutaneous immunizations using 2951lgt1/4ΔdLOS-rOMP26VTAL elicited an antigen-specific IgG response to all antigens tested (antigens from serotypes A, B, rOMP26VTAL and NTHi). Antisera from serotype A and B conjugates induced bactericidal activity against their respective serotype-specific strains, with no cross-reactivity observed. Antisera from 2951lgt1/4ΔdLOS-rOMP26VTAL immunisation showed protective efficacy against serotype A and B strains. These results indicate that 2951lgt1/4ΔdLOS-rOMP26VTAL is a promising vaccine candidate, but requires further investigation in a challenge model. Overall, the studies within this thesis show that M. bovis OS has a role in bacterial attachment, sensitivity towards bactericidal activity of bovine serum and endotoxin activity of tested strains. This thesis also shows that M. catarrhalis LOS is an important bacterial antigen that can be utilised for vaccine development. Additionally, OMP26, being highly-conserved across NTHi strains and immunogenic makes it the ideal carrier protein for respiratory related vaccine development. Combining the LOS from M. catarrhalis and the rOMP26VTAL from NTHi paves the path for the development of a single vaccine that may prevent respiratory infections caused by these two predominant pathogens.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Nontypeable Haemophilus influenzae (NTHi), Streptococcus pneumoniae and Moraxella catarrhalis are common bacterial agents of otitis media which is a major cause of morbidity in young children. Mucosal immune responses are an integral part of the immune defense against middle ear infection and it is known that certain populations, including Australian Aboriginal children, are highly susceptible to disease. The current study focussed on the development of the mucosal immunity to the three bacterial pathogens in Aboriginal and non-Aboriginal children from birth to two years of age, living in the Kalgoorlie-Boulder region of Western Australia. Salivary and breast milk IgA levels were measured by the enzyme Linked immunosorbent assay. The measured IgA levels, combined with socio-economic, demographic and bacteriological data were analyzed statistically to determine the influential factors on the mucosal IgA response in these children over time. This study found that each antigen-specific IgA examined followed a distinct ontogeny pattern and IgA responses differed significantly according to age, indigenous status and feeding type. Indoors smoke exposure, maternal smoking, and sibling day care attendance had some impact on salivary IgA levels in the children. However, household crowding and the presence of older siblings had the most significant impact on salivary IgA levels for children of different age groups. These two factors were correlated to increased nasophayrngeal colonization by H. influenzae, S. pneumoniae and M. catarrhalis and colonization status was also found to influence salivary IgA levels in the children. No correlation between maternal breast milk IgA levels and child salivary IgA levels was observed. The results suggest that the degree of exposure to environmental factors rather than immunological deficit is responsible for the observed differences in salivary IgA responses between Aboriginal and non-Aboriginal children and modifying these factors could lead to a reduction in the burden of otitis media experienced by the children. Further studies correlating specific salivary IgA levels to diseases such as otitis media will reveal the role of specific salivary IgA responses in the prevention of infection by respiratory pathogens.

Thesis (Ph.D.)--State University of New York at Buffalo, 2008.
Title from PDF title page (viewed on Dec. 3, 2008) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Murphy, Timothy F. Includes bibliographical references.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that is associated with a range of respiratory infections, including acute exacerbations of chronic obstructive pulmonary disease (COPD), and community acquired pneumonia (CAP). Macrolide antibiotics such as azithromycin are being more frequently used to manage these conditions, including those where NTHi may be involved, despite macrolides having relatively poor antibiotic activity against H. influenzae (azithromycin MICs of wild-type strains typically cover the range of 0.25-4 μg/mL). The efficacy of these antibiotics in managing these conditions is further threatened by the emergence of strains of NTHi exhibiting high-level macrolide resistance. A range of different mechanisms of macrolide resistance are recognised broadly within bacteria. Resistance has been attributed to the presence of mutations in structural components of the ribosomal binding site of macrolides. Such mutations can occur in the 23S rRNA gene, as well as in the L4 and L22 structural proteins. Increased expression of inherent macrolide efflux mechanisms such as acrAB resulting from mutations in regulatory regions of these efflux pumps have also been recognised as a potential cause of macrolide resistance. Finally, resistance has also been attributed to the acquisition of macrolide resistance genes (AMRGs), which are readily disseminated among species on mobile genetic elements. There are a large number of different AMRGs and associated proteins, and the mechanisms by which they produce resistance vary. For example, the erm genes encode enzymes which modify the ribosomal binding site, the mef genes encode alternate efflux systems, and an additional group of genes encode enzymes which directly deactivate the macrolide. High-level macrolide resistance in NTHi is uncommon and has historically been attributed to chromosomal mutations in ribosomal structural elements or regulatory regions controlling acrAB. In contrast, AMRGs have not been widely associated with NTHi and a number of studies have failed to detect these genes in this particular species. There has been a single study in which these genes have been reported to be highly prevalent in NTHi. In that study, among a collection of 106 NTHi isolates from children with cystic fibrosis enrolled in a placebo-controlled azithromycin trial, all isolates had at least one AMRG, with many of the isolates carrying two or three of these genes. However, the phenotypic effect of these genes was not consistent; only 27 of the AMRG-carrying isolates exhibited phenotypic resistance to macrolides. While the findings of that study identify the emergence and potential spread of AMRGs in NTHi, it raises a number of questions regarding the prevalence of these genes within NTHi isolates more broadly and the role they play in generating a resistant phenotype. In Chapter 4 of this thesis, a collection of 186 NTHi respiratory isolates of variable azithromycin resistance phenotype (azithromycin MIC range: 0.09 to >256 μg/mL; MIC50: 1.5 μg/mL; MIC90: 3 μg/mL) derived from both cystic fibrosis and non-cystic fibrosis patients was established and analysed for; 1) the presence of macrolide resistance-associated L4, L22 and 23S rRNA mutations, and 2) the presence of the AMRGs erm(A), erm(B), erm(C), erm(F), mef(A) and mef(E). For the detection of the AMRGs, two methods were used; 1) a novel PCR using locked nucleic acid dual-labelled hydrolysis probes, and 2) the original primer set used by the authors of the previous study. While L22 and 23S rRNA alterations were detected in 2 isolates with high-level macrolide resistance (azithromycin MIC ≥ 256 μg/mL), none of the isolates were found to carry any of the AMRGs using the novel PCR detection method. When using the primers described in the original study, mef(A) and erm(A) were detected in a number of isolates. Subsequent analysis of these amplicons. revealed them to be false positive results, raising questions as to the possibility of false positive results in the original study. Over 100 different AMRGs have been recognised, and the development and increasing availability of whole genome sequencing (WGS) techniques now allows for efficient and thorough analysis of sequences for the detection of antibiotic resistance mechanisms. In Chapter 5 of this thesis, WGS was utilised to further investigate the presence of a broad selection of other AMRGs (n=72) in NTHi, using the SPANDx pipeline. WGSs of two isolates of NTHi exhibiting high-level macrolide resistance obtained from the study of Chapter 4, as well as an additional 89 publically available WGSs of NTHi isolates of variable resistance phenotype, were examined in the study. None of the specified AMRGs were detected among this collection of WGSs. In addition, the WGSs of the 2 isolates from Chapter 4 with high-level macrolide resistance were interrogated for any AMRGs using the Comprehensive Antibiotic Resistance Database (CARD), and none were detected. Both isolates underwent further WGS analysis to confirm the L22 and 23S findings in Chapter 4; one isolate carried R88P in L22 and C2611G in 23S rRNA and the other isolate carried A2058G in 23S rRNA, all previously associated with decreased macrolide susceptibility in H. influenzae. Alterations in regulatory regions of acrAB were also detected in both isolates. Finally, transformation studies using donor genomic DNA from these 2 isolates were performed on H. influenzae Rd KW20. While none of the transformants that were generated exhibited as high an azithromycin MIC as the donors, a number of different regions of donor origin were detected in various transformants. The role of these regions in generating resistance in individual transformants was not clear but, with respect to the lower MICs exhibited by these transformants, the findings suggested a multifactorial aetiology for the high-level macrolide resistance seen in the donor isolates. In the Roberts et al. study, the effect of the AMRGs appeared to be inconsistent, with some isolates not exhibiting increased MICs (compared to a typical wild-type strain). The effect of these AMRGs on macrolide susceptibility in H. influenzae remains to be established. As a result, the aim of Chapter 6 was to transfer select AMRGs (erm(A), erm(B), erm(C), mef(A) and mef(E)) to H. influenzae Rd KW20 and examine the phenotypic effect of expression of these genes. Initially, attempts were made to conjugatively transfer these AMRGs from select Gram positive donors to H. influenzae Rd KW20. These attempts were unsuccessful. As a result, the AMRGs were cloned into the shuttle vector pLS88 and H. influenzae Rd KW20 was transformed with these constructed plasmids by electroporation. Clones were generated with a range of approaches, including with and without the native regulatory regions of the AMRG inserts, and in the former, tested for expression with and without the presence of an inducing agent. High-level expression of erm(A), erm(B) and erm(C) was demonstrated in at least some of the various conditions and resulted in increased macrolide resistance in these transformants. In contrast, expression of mef(A) and mef(E) did not have an effect on macrolide resistance. In the Roberts et al. study, conjugative transfer of mef(A) to H. influenzae Rd KW20 resulted in a moderate increase in azithromycin and erythromycin MICs; our findings therefore suggest that mef only increases MICs in combination with other macrolide resistance mechanisms such as msr(D) (found downstream of mef and not covered by our cloned inserts) or underlying chromosomal alterations. The above AMRGs are commonly encountered among human pathogens which share a respiratory niche with NTHi, including Staphylococcus aureus and various respiratory Streptococcus spp.. A number of other AMRGs, such as erm(42), msr(E) and mph(E), are typically encountered among animal commensals and pathogens such as Pasteurella spp. and Mannheimia spp.. H. influenzae is closely related to these pathogens and previous studies have demonstrated that they are able to exchange resistance determinants, including beta-lactamases. Therefore, the work of Chapter 7 explored the potential for conjugative transfer of erm(42), msr(E) and mph(E) (carried on the mobile multiresistance integrative and conjugative element ICEPmu1) and associated macrolide resistance from a bovine Pasteurella multocida isolate to H. influenzae Rd KW20. Transconjugants generated in this study were found to carry a truncated form of ICEPmu1 that lacked msr(E) and mph(E) but carried erm(42); transconjugants were found to exhibit increased erythromycin and clindamycin resistance. This truncated ICEPmu1 was successfully transferred from primary transconjugants to secondary H. influenzae Rd KW20 recipients, indicating that transfer functions were retained during conjugation. The acquisition of ICEPmu1 did not appear to have an impact on the fitness of H. influenzae Rd KW20 and the ICE was found to be stable in the absence of antibiotic selective pressure. In summary, the major findings of this thesis are that AMRGs are not widespread in NTHi and that the high prevalence of a selected set of these genes described in one recent study is probably unique to the circumstances of that study. Although complex regulatory regions in many AMRGs mean that expression and associated resistance may be dependent on the specific genetic context, we have shown that under favourable conditions, erm(A), erm(B) and erm(C) can produce macrolide resistance in H. influenzae in their own right, but this is unlikely for the mef genes. Although we were unable to demonstrate conjugative transfer of common AMRGs from respiratory Gram positive organisms to H. influenzae, we were able to demonstrate conjugative transfer of an AMRG encoding multi-resistance replicon from a closely related organism of animal origin. This replicon produced macrolide resistance, was stably maintained without a significant fitness cost and was capable of ongoing conjugative transfer within H. influenzae. This thesis concludes that there is little evidence for the imminent and widespread emergence of AMRG associated macrolide resistance in NTHi; however, the increasing use of macrolides in both animals and humans and the prevalence of AMRGs in other organisms suggest that it is prudent to undertake ongoing periodic surveillance for these genes in NTHi.

Nontypeable Haemophilus influenzae (NTHi) is a major opportunistic pathogen that causes a variety of infections in the respiratory tract, including community-acquired pneumonia, acute exacerbations of chronic obstructive pulmonary disease and otitis media. Collectively, these infections and subse-quent complications impose a significant global burden of disease. The impact of NTHi-associated disease is further amplified by the rapidly expanding spectrum and prevalence of antibiotic resistance, and the lack of an effective vaccination strategy. Consequently, novel preventative or therapeutic approaches that do not rely on antibiotic susceptibility or stable vaccine targets are becoming more attractive. One such approach may involve exploiting the bacterium’s nutritional dependency for host-derived iron-containing haem, the acquisition of which is a major determinant of NTHi survival and pathogenesis within the respiratory tract. Strategies that interfere with the acquisition of this essential nutrient may therefore have a significant impact on the ability of NTHi to cause disease. The limited availability and nutritional demand for haem-iron promotes a highly competitive environ-ment between NTHi and other upper respiratory tract inhabitants. We therefore hypothesised that an upper respiratory commensal capable of outcompeting NTHi for haem-iron may have utility as a probiotic therapy by generating an environment inhospitable for NTHi growth. Recently, nasopharyn-geal isolates of the closely related commensal Haemophilus haemolyticus (Hh), were discovered with the capacity to inhibit NTHi growth by secretion of a novel bacteriocin-like substance (herein referred to as haemophilin; Hpl). Hpl was found to possess structural characteristics consistent with that of a haem-binding protein and thus, we proposed a model by which Hpl inhibits NTHi growth by restricting access to haem. In Chapter 3, this hypothesis was tested by generating and comparing the NTHi-in-hibitory capacity of an hpl knockout to the wild-type strain under varying concentrations of haem or recombinant Hpl. These experiments indicated that Hpl was only inhibitory under haem-limited con-ditions. The loss of NTHi-inhibitory activity in media recovered from the knockout strain, and the di-rect correlation between NTHi-inhibitory activity and hpl expression across different Hh strains, con-firmed that Hpl was the primary mediator of NTHi growth inhibition by Hh. In addition to the loss of NTHi-inhibitory capacity, the knockout also displayed poor growth on haem supplemented media compared to the wild-type strain, indicating a defect in haem utilisation and a role for Hpl in Hh haem acquisition/utilisation. Growth of NTHi and Hh in media supplemented with either Hpl or the equiva-lent concentration of free haem demonstrated that Hpl-bound haem is available to Hh as a nutritional source of haem, but not to NTHi. To further characterise the biological role and genetic determinants of Hpl production, whole genome sequences of Hh isolates containing the hpl open reading frame (n=24) were generated. Among all isolates and publicly available genomes, the hpl gene occurred in a conserved gene cassette containing genes for a putative secretion protein and a putative Hpl/haem receptor. Together with supporting proteomic investigations, these findings provided evidence that Hpl has a role in haem acquisition by Hh, and that NTHi-inhibitory activity occurs through haem star-vation. We therefore proposed the possibility that competition from Hpl-producing Hh (Hh-Hpl\(^+\)) could antagonise NTHi colonisation in the respiratory tract. The feasibility of this approach was tested in chapter 4 by direct in vitro competition assays between NTHi and Hh strains with varying capacities to produce Hpl. Subsequent changes in NTHi growth rate and fitness, in conjunction with hpl expression analysis, were employed to assess the NTHi-inhibitory capacity of Hh strains. The growth rate of NTHi was significantly impaired during co-culture with Hh strains containing hpl (Hh-hpl\(^+\)), but not with strains lacking the hpl open reading frame (Hh-hpl-), including the knockout strain generated in Chapter 3. During an extended co-culture assay, the com-petitive advantage of Hh-hpl\(^+\) strains was evident within two generations, culminating in a total loss of NTHi fitness over subsequent generations. Hh strains capable of high levels of hpl expression, were able to compete with NTHi more effectively, providing a strong link between the NTHi-inhibitory phe-notype, hpl expression and favourable outcomes during competitive growth with NTHi in vitro. These findings demonstrate that Hh-Hpld to quantitatively compare the oropharyngeal carriage load of NTHi and Hh populations with the Hh-hpl+ or Hh-hpl\(^+\) strains possess characteristics desirable in a probiotic, warranting further investigation into the utility of these strains in preventing NTHi interactions with host cells. In chapter 5, the capacity of Hh-Hpl\(^+\) strains to disrupt NTHi association with airway epithelial cells was tested to determine their probiotic utility against the requisite nasopharyngeal colonisation stage of NTHi infection. Cell culture models of nasopharyngeal (D562) and lung (A549) epithelia were pre-treated with Hh strains with different levels of hpl expression prior to NTHi challenge. NTHi attach-ment and invasion was significantly reduced in cell monolayers pre-treated with Hpl or Hh strains with high levels of hpl expression. Among all Hh-Hpl\(^+\) strains the production of Hpl was found to be stimulated in response to NTHi challenge and nasopharyngeal cell exposure. Pre-treatment with Hh-Hpl\(^+\) strains was more effective than the purified Hpl protein alone, such that Hh-Hpl\(^+\) cell numbers 10-100-fold lower than that of the NTHi challenge load, were sufficient to significantly inhibit NTHihost-cell interactions. These data suggest that conditions in the nasopharyngeal niche might support high levels of expression of hpl in Hh-Hpl\(^+\) strains with associated protection against NTHi adhesion and attachment. Based on the in vitro findings presented in Chapters 3-5, we hypothesised that natural pharyngeal carriage of Hh strains with the Hh-hpl\(^+\) genotype would be associated with a lower prevalence and/or density of NTHi colonisation in healthy individuals. Chapter 6 describes an in vivo human study involving the collection of oropharyngeal swabs from 257 healthy adults in Australia between 2018 and 2019. Real-time PCR was used to quantitatively compare the oropharyngeal carriage load of NTHi and Hh populations with the Hh-hpl\(^+\) or Hh-hpl\(^-\) genotype. The likelihood of acquiring/maintaining NTHi colonisation status over a two- to six-month period was also assessed in individuals that carried either Hh-hpld to quantitatively compare the oropharyngeal carriage load of NTHi and Hh populations with the Hh-hpl\(^+\) or Hh-hpl\(^-\) (n = 25) or Hh-hpl\(^+\) (n = 25). The carriage of Hh-hpl\(^+\) was associated with a significantly lower proportionate density and prevalence of concurrent NTHi carriage. Additionally, colonisation with high densities of Hh-hpl\(^+\) correlated with lower NTHi carriage loads and a lower likelihood of acquiring/maintaining NTHi colonisation status between visits. These findings suggest a potential pro-tective role of Hh-hpl\(^+\) strains against NTHi pharyngeal colonisation in vivo. In summary, the work presented in this thesis provides in vitro and in vivo evidence which supports the therapeutic potential of Hh-Hpl\(^+\) against NTHi by inhibiting growth and host-cell interactions re-quired for pathogenesis. These findings encourage translational studies of a probiotic which can be applied to the upper respiratory tract as a strategy to prevent NTHi infections which is not compro-mised by the limitations associated with standard antibiotic or vaccination strategies.

Bacterial colonization of the upper respiratory tract is the first step in the pathogenesis of nontypeable Haemophilus influenzae (NTHi) disease. Examination of the determinants of NTHi colonization process has been hampered by the lack of an appropriate animal model. To address this, we have developed a model of NTHi colonization in adult rhesus macaques that involves intranasal inoculation of 1x105 CFU and results in persistent colonization of the upper respiratory tract for at least three weeks with no signs of disease, mimicking asymptomatic colonization of humans. Using this model, we assessed the contributions to colonization of the HMW1 and HMW2 adhesive proteins. In competition experiments, the parent strain expressing both HMW1 and HMW2 was able to efficiently out-compete an isogenic mutant strain expressing neither HMW1 nor HMW2. In experiments involving inoculation of single isogenic derivatives of NTHi strain 12, the strains expressing HMW1 or HMW2 or both were able to colonize efficiently, while the strain expressing neither HMW1 nor HMW2 colonized inefficiently. Furthermore, colonization resulted in antibody production against HMW1 and HMW2 in one-third of the animals, demonstrating that colonization can be an immunizing event. In conclusion, we have established that NTHi is capable of colonizing the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response. The HMW1 and HMW2 adhesive proteins play a major role in the process of colonization.

After establishing that the HMW1 and HMW2 proteins are colonization factors we further investigated the determinants of HMW1 function. HMW1 is encoded in the same genetic locus as two other proteins, HMW1B and HMW1C, with which HMW1 must interact in order to be functional. Interaction with HMW1C in the cytoplasm results in the glycosylation of HMW1. By employing homologues of HMW1C that glycosylate HMW1 in slightly different patterns we show that the pattern of modification is critical to HMW1 function. Structural analysis showed a change in protein structure when the pattern of HMW1 modification differed. We also identified two specific sites which must be glycosylated for HMW1 to function properly. These point mutations did not have a significant effect on protein structure, suggesting that glycosylation at those specific sites is instead necessary for interaction of HMW1 with its receptor. HMW1B is an outer membrane pore through which HMW1 is transported to reach the bacterial cell surface. We observed that HMW1 isolated from the cytoplasm has a different structure than HMW1 isolated from the bacterial cell surface. By forcing HMW1 to be secreted in a non-HMW1B dependent manner, we show that secretion alone is not sufficient for HMW1 to obtain a functional structure. This leads us to hypothesize that there is something specific in the interaction between HMW1 and HMW1B that aids in proper HMW1 folding.

The NTHi HMW1C glycosyltransferase mediates unconventional N-linked glycosylation of HMW1. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. To determine if this mechanism of N-linked glycosylation is employed by species other than NTHi, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. LC-MS/MS analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function.

Dissertation

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic human pathogen responsible for diseases such as exacerbations of chronic bronchitis, community acquired pneumonia, otitis media and occasionally conjunctivitis. H. haemolyticus is closely related to NTHi and shares the same respiratory niche as a commensal, but is not an opportunistic respiratory pathogen. Both NTHi and H. haemolyticus can acquire resistance to β-lactam antibiotics via mutations to the ftsI gene and associated amino acid substitutions in penicillin binding protein 3 (PBP3) and the prevalence of such resistant strains is increasing worldwide. The factors associated with pathogenicity of NTHi are complicated, but can include mucociliary interactions, attachment to respiratory mucosa, evasion of mucosal immunity, and invasion of respiratory epithelial cells. There is significant in vivo and in vitro evidence that NTHi can invade and persist within host epithelial cells leading to the hypothesis that this allows the organism to avoid the normal immune response and establish a persistent reservoir for infection. Despite increased understanding of some mechanisms involved with invasion, the relationship between intracellular NTHi and pathogenesis is still unclear and many studies have shown enormous strain-to-strain variation in the in vitro invasive ability of clinical isolates. One of the limitations in understanding the relationship between intracellular NTHi and pathogenesis is the lack of a standardised model for studying invasion, as a very large range of both respiratory and non-respiratory, and primary and immortal cell lines have been used, often without explanation or justification. It is unclear whether an isolate that shows in vitro invasion in one cell type will be similarly invasive in another cell type, and this makes comparisons between studies very difficult. The aims of this thesis were to investigate the effect of respiratory cell types and presence of altered PBP3 on invasion rate of NTHi and Haemophilus haemolyticus. To investigate our aims, we established a large collection of NTHi and H. haemolyticus isolates where the identity had been confirmed previously using a validated PCR algorithm as either NTHi (being positive for hpd#3 or fucK and negative for sodC) or H. haemolyticus (positive for sodC and negative for both hpd#3 and fucK. In this working collection, NTHi isolates were collected from four different sites and clinical conditions such as otitis media, conjunctivitis, lower respiratory tract infection and normal oropharyngeal flora, whereas H. haemolyticus isolates were recovered from the oropharynx of healthy individuals. These isolates were tested for invasion using the gentamicin survival assay with immortalised BEAS-2B (Sigma-Aldrich); isolated from normal human bronchial epithelium of non-cancerous individuals NHBE (Lonza); isolated from epithelial lining of airways above bifurcation of the lungs A549 (Public Health England); epithelial lung carcinoma cells derived from 58 year old Caucasian male and NCI-H292 (ATCC); muco-epidermoid pulmonary carcinoma cells derived from 32 year old female epithelia cell lines. Cell lines were grown and maintained in LHC8 (Gibco), BEGM (Lonza), DMEM growth medium (Sigma-Aldrich) supplemented with 2Mm Glutamine and 10% Foetal Bovine Serum (FBS), and RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FBS respectively at 370C in 5% CO\(_2\). Chapter 3 is a detailed study of the invasive ability of NTHi, possessing normal or PBP3, using four different and widely used respiratory cell types: BEAS-2B, NHBE, A549, and NCI-H292. The focus of this study was to evaluate if there is any difference in invasive ability of NTHi isolates individually, collectively and between isolates with normal and altered PBP3 across each cell type. The results showed that NTHi invasion of respiratory epithelial cells in vitro is both strain dependant and influenced significantly by the type of cell lines used and also confirmed the previous suggestions, provided by Okabe et al. (2010) and Atkins et al. (2014), that isolates with altered PBP3 possess more invasive ability compared to isolates with normal PBP3. Furthermore, the association between altered PBP3 and increased invasion was conserved across each cell line. H.haemolyticus is considered to be a non-pathogenic commensal of the respiratory tract butlittle information is available on its ability to invade epithelial cells in vitro. If in vitro invasion is an indicator of ability for in vivo invasion and is important in the pathogenesis of NTHi infection, then H. haemolyticus would be expected to be comparatively non-invasive. As a result, Chapter 4 examined the invasive ability of H. haemolyticus to the BEAS-2B cell line. The invasion rate of 20 H. haemolyticus isolates were tested with BEAS-2B cell line and then 5/20 isolates were selected to test their invasion rate with other respiratory cell types used previously in this study. The results confirmed that non-invasiveness of H. haemolyticus isolates is consistent with their inability to cause respiratory infections. In conclusion, this thesis has demonstrated the significant variability of invasion results across different epithelial cell lines, highlighting the importance of the choice of cell type in invasion assays as a confounding factor, as the ability of NTHi to invade respiratory epithelial cells in vitro is both strain dependant and influenced significantly by the respiratory cell types. Furthermore, we have shown that the association between altered PBP3 and increased invasion is conserved across all the respiratory epithelial cell types used in this study. Finally, this thesis also revealed the inability of H. haemolyticus isolates to invade respiratory epithelial cell types in vitro, and suggests that this is consistent with their inability to cause opportunistic respiratory tract infections.

Chronic obstructive pulmonary disease (COPD) is a severe and progressive condition characterised by persistent respiratory symptoms and airflow limitation. Around 300 million people in the world have COPD. It has emerged as the third leading cause of mortality, claiming 3.2 million lives worldwide in 2017. An acute exacerbation of COPD, a sudden worsening of respiratory symptoms, is a major cause of morbidity and mortality in COPD patients. Various factors, including biomass smoke exposure and infection with bacteria trigger COPD exacerbations. Nearly half of the world’s population uses biomass fuel for cooking and heating and is therefore at risk of exposure to noxious particles released from the combustion of biomass fuel. Among respiratory bacteria, nontypeable Haemophilus influenzae (NTHi) is a key pathogen implicated in colonisation and damage of airways in COPD patients. In this thesis, I assessed the impact of biomass smoke exposure on inflammation and adherence of NTHi to human bronchial epithelial cells. Due to the lack of a standard and easily accessible procedure for the preparation of biomass smoke, I first devised a simple, cost-effective, and reproducible method for the generation of biomass smoke extracts, in particular, cow dung and wood smoke extracts. Using this method, I generated quantifiable batches of biomass smoke extracts that were utilised for the assessment of cellular responses to different types of biomass smoke. I investigated the effect of biomass smoke extracts on human airway epithelial cells with respect to expression of a known receptor of NTHi, platelet-activating factor receptor (PAFR), and the pro-inflammatory cytokines interleukin 6 (IL-6) and IL-8, using quantitative polymerase chain reaction. In addition, I examined the response of bronchial epithelial cells to adherence of NTHi using immunofluorescence microscopy. I observed an increased inflammatory response in cells exposed to biomass smoke, characterised by induction of significant levels of IL-6 and IL-8 mRNA, in comparison to mock exposed cells. I demonstrated a dose-dependent increase in NTHi adhesion to epithelial cells following exposure to biomass smoke extracts. I further established an association between PAFR expression and the adhesion of NTHi in biomass smoke-exposed cells. Pre-treatment with a known PAFR antagonist, WEB-2086 inhibited biomass smoke-induced adherence of NTHi in airway cells in a dose dependent manner. In addition, pre-treatment of biomass smoke-exposed airway epithelial cells with a novel WEB-2086 analogue, C17 reduced NTHi adhesion in a dose-dependent manner. I next assessed the genomes of 568 NTHi isolates, including 40 newly sequenced clinical isolates collected from patients with different diseases, including COPD. Phylogenetic analysis based on polymorphic sites on the core genome did not provide sufficient resolution to separate COPD strains from other clinical phenotypes, suggesting a similar set of core genes are present in all clinical NTHi isolates. I applied discriminant analysis based on the presence or absence profiles of accessory genes and found a clear distinction between COPD and other disease strains. I then applied a pan genome-wide association study approach to identify the accessory genes associated with COPD. I identified a set of accessory genes that regulate metabolic functions, such as the metabolism of organic acids and oxidation-reduction reactions that regulate cellular respiration to be significantly associated with COPD strains. This result suggests that NTHi associated with COPD may exhibit genetically encoded functional variances to isolates collected from other clinical illnesses. In conclusion, this work advances our understanding of how biomass smoke could contribute to the development and progression of COPD and highlights the potential of PAFR as a therapeutic target for reducing the impact of hazardous biomass smoke exposure on respiratory health. Further, this thesis increases our understanding of gene sets shared by NTHi strains that survive and cause disease in the COPD lung.

碩士
中國醫藥大學
基礎醫學研究所碩士班
102
Nontypeable Haemophilus influenzae [1] is a Gram negative coccobacillus, which belongs to the normal flora of the human respiratory tract. When host immune mechanisms were defective, NTHi could invade into the lower respiratory tract, cause acute exacerbation of bronchial airway disease or chronic colonization. Previous study showed that nanomaterials (e.g., silver, iron, zinc oxide, titanium dioxide) have a variety of applications. Nanoparticles can deliver to the bloodstream or lymph nodes and then transfer to other organs, and easily penetrate the cytoplasmic membrane followed by getting into the nucleus. NTHi is the pathogen which was isolated from lower respiratory tract and secretions from chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD) and chronic bronchitis, and more than 40% to 60% of those patients became acute exacerbations of COPD (AE-COPD). In vitro experiment showed that treatment of RAW264.7 cells with ZnO-nanoparticles (ZnO-NPs) can decrease macrophage clearance of NTHi, and lead to downregulation of NTHi-activated expression of inducible nitric oxide (NO) and translocation of active NF-κB into the nucleus. Additionally, in the study of cigarette smoking extracts [2], we found that treatment of RAW264.7 cells with 10% CSE and exposure to NTHi, the mRNA and protein expression levels of CCL-2, CXCL-2, CXCL-10 were decreased. Our results demonstrate that the initial performance of innate sensing under the exposure of CSE and ZnO-NPs which may affect the clearance of NTHi and therefore lead to disease become more severity.

Biofilm formation has been recognised as an aggregation of bacterial cells surrounded by extracellular polymeric substances (EPS) which are secreted from the cells. Biofilm has a significant role in chronicity of infection and cells are recalcitrant to antibacterial agents when biofilm is formed on abiotic or biotic surfaces. Nontypeable Haemophilus influenzae (NTHi) has been investigated previously in terms of biofilm production ability. This study examined NTHi biofilm formation using a staining assay and relationship to pathogenicity, and an assessment of the spatial distribution of the chemical components by Fourier Transform Infrared (FTIR) spectroscopy. Firstly, a semi-quantitative microtitre plate (MTP) assay for NTHi biofilm formation was developed and validated for in vitro formation of biofilm. This assay was used to evaluate the effect of length of storage time on four fresh NTHi clinical isolates. Isolates were stored at -80Cº for two, four, eight, 12, 24 and 48 weeks before measuring biofilm production. Sixty clinical isolates of NTHi from different body sites were also screened for biofilm formation ability and relationship with the body site. FTIR microspectroscopy was applied to study NTHi biofilm formation and pathogenesis by generating FTIR spectra. FTIR spectroscopy coupled with imaging of the biofilm components were assessed for untreated biofilm, as well as the effect of antibiotic treatment on new biofilm formation and treatment of mature NTHi biofilm. The MTP showed consistent differences between the different NTHi isolates tested in first part of the project with these categorised as high and low biofilm producers. The MTP assay demonstrated that frozen storage of the isolates was not a determinant of biofilm formation. No statistically significant differences in the in vitro biofilm production were found across the clinical NTHi isolates from the nasopharynx (normal flora), ears (otitis media), lung (community acquired pneumonia and bronchiectasis in cystic fibrosis) or the isolates of Haemophilus haemolyticus (oropharynx, normal flora). However, the isolates from eyes (conjunctivitis) demonstrated remarkably consistent low biofilm production compared to the isolates from other body site (P<0.005). In the second part of this project, FTIR spectroscopy was used to analyse the chemical constituents of the different biofilms. Unsupervised multivariate analysis (PCA) of the spectral data showed a chemical distinction between the two categories of biofilm formation (high and low). Analysis of microscopic IR hyperspectral data highlighted the spatial distribution of the different chemical components of the biofilm such as protein and carbohydrate. Analysis of antibiotic treated biofilm by FTIR showed an increase of protein bands in NTHi compared to standard Haemophilus influenzae isolate and untreated biofilm. This project provides detailed information about ability of formation, pathogenesis and chemical composition of biofilms produced by NTHi isolates. Spectral information, with the effect of antibiotic on mature and newly formed NTHi biofilms provides a spatial overview of chemical differences in the bacterial biofilm.

碩士
中國醫藥大學
基礎醫學研究所碩士班
105
From the data released by World Health Organization at 2012, Chronic Obstructive Pulmonary Disease (COPD) causes over three million deaths globally and is climbing up to the third of top ten cause of death at 2012. In many clinical studies, COPD patients usually have symptoms like sputum, long-lasting cough, chronic lung inflammation and air-flow limitation. COPD is an irreversible disease. This condition outdraws the importance of early diagnosis and the proper therapy to COPD. However, COPD is not the top ten cause of death in the low developed countries and undeveloped countries. This condition not only shows the high relation between COPD and the development of a country, but also shows the big health problem in developed country. In addition, the risk factors of COPD could come from air pollution(PM2.5、PM5、PM10、polycyclic aromatic hydrocarbons), cigarette smoke (smoke consumption or second-hand smoke), gene, infection, ageing and so on. In several research studies, cigarette smoke is the major risk factor to trigger COPD. After calculation, over 80 percentage of COPD patient have smoke behavior. Moreover, they are also vulnerable to infection. Nontypeable haemophilus influenzae (NTHi), which is gram-negative coccobacillus bacteria, is the most common pathogen found in sputum from COPD patients. In addition, both cigarette smoke and NTHi infection can lead to lung inflammation, COPD exacerbation and lung cancer. Therefore, we are curious about the immune response and the mechanism of this NTHi infection after cigarette smoke challenge. From the in vitro data, we found bacterial clearance is impaired in cigarette smoke extracts (CSE) treated Raw264.7 cells. The CCL-2, CXCL-2 and CXCL10 mRNA expression were reduced in CSE and NTHi dual treated RAW cells. Moreover, our lab member has previously found that NF-B promoter activity was also attenuated in CSE and NTHi dual treated RAW cells. In order to confirm in vitro results, we set up a whole-body cigarette smoke exposure mouse model. We found cigarette smoke indeed impair lung function within air-flow resistance experiment. The total cells found in BALF were increased in smoke-treated mice or NTHi challenged mice compared to control mice. Yet, this BALF cell increasement was impaired within smoke-treated mice after NTHi challenge.