Microbiology & Immunology - Theses
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ItemEstablishing two systems to study MAIT cell responses during bacterial infection( 2019)Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells, which are abundant in humans and also present in many mammals, including mice [1]. MAIT cells express semi-invariant T cell receptors (TCR) are activated by recognizing microbial vitamin B2 (riboflavin) metabolites presented on non-classical major histocompatibility complex (MHC) class I related protein 1 (MR1) [1-6]. Upon activation, MAIT cells can either directly kill infected cells or secrete IFN, IL-17, TNF and other functional molecules that enhance the anti-microbial activity of other immune cells [7, 8]. These characteristics suggest an important role played by MAIT cells in anti-bacterial immunity in mammals. Bacterial infections are major problems in clinical settings. While MAIT cells have shown antibacterial properties, clinically relevant in vivo animal models have not been extensively developed for further exploration of the mechanisms of MAIT cell protection. Using three clinically common bacteria; Klebsiella pneumoniae, Staphylococcus aureus, and Escherichia coli, this study established bacterial peritonitis models in mice for MAIT cell research. The three bacteria were all able to produce antigens that stimulate MAIT cells in vitro. For mouse infection models, the optimal dose of K. pneumoniae for inducing productive but nonlethal infection was not established in this study. S. aureus and E. coli peritonitis incurred mild and robust MAIT cell responses in vivo, respectively. Further experiments with E. coli peritonitis showed that MAIT cells accumulated in the intraperitoneal cavity, the spleen and liver, which contributed to bacteria control. The finding suggests possible MAIT cell-based treatments in clinical conditions. While in vitro studies have shown that MAIT cells can be stimulated by various types of cells upon infection, there have been few investigations into how MAIT cells are activated in vivo. In the second project presented in this thesis, the contributions of antigen presenting cells (APCs) in initiating activation in MAIT cells was studied with Francisella tularensis infection. We found that APCs were burdened with bacteria in the early phase of infection. With an ex vivo cellular co-culture assay was specifically optimized for assessing MAIT cell activation induced by the F. tularensis burdened APCs, we found that alveolar macrophages contributed to the best activation of MAIT cells in an MR1 dependent manner. Other cell subsets: monocytes, neutrophils and dendritic cells were also capable of inducing a mild activation. In summary, the present study helped optimize two novel systems for MAIT cell research. With the ex vivo cellular co-culture system, this thesis contributes to understanding how APCs activate MAIT cells in vivo upon bacterial infection. With the in vivo peritonitis models, this study introduces clinically relevant bacteria to the current MAIT cell research in mice, and ultimately to inform and complement clinical research. It is hoped that the insights gained from this study could be of assistance to current research methods and to achieve a better understanding of MAIT cell responses during bacterial infection.
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ItemEvolution of an emerging pathogen: atypical enteropathogenic Escherichia coli( 2016)Atypical enteropathogenic Escherichia coli (aEPEC) is an important emerging pathogen that is causally associated with diarrhoea in children in both industrialised and developing nations. The current definition for aEPEC is the presence of the locus of enterocyte effacement (LEE) pathogenicity island and the absence of genes for bundle forming pili (bfpA) and Shiga toxin (stx). Classical approaches to categorise clinical isolates of aEPEC have shown that they are heterogeneous for sequence type, lipopolysaccharide (O-) and flagella (H-) surface antigens, antimicrobial resistance profiles and virulence genes. Associations of aEPEC with diarrhoea are inconsistent, suggesting that the current definition of aEPEC lacks the ability to distinguish between virulent and less virulent isolates. For this thesis, I used comparative genomic analysis to explore the evolution and population structure of aEPEC. A total of 196 putative aEPEC isolates were collected during the Global Enteric Multicenter Study (GEMS) from seven countries in South Asia and Sub-Saharan Africa. These isolates were subjected to whole genome sequencing and 185 were con rmed in silico as aEPEC. The core aEPEC genomes were rst compared with publicly available genomes with phylogenomic analysis revealing ten globally distributed clonal lineages of aEPEC. Investigation into the evolution of the LEE showed that aEPEC clones were associated with different LEE subtypes. Analysis of genetic variation within the LEE revealed three major lineages, comprised of a total of 30 different subtypes. The three LEE lineages were shown to have different preferred tRNA insertion sites on the chromosome and to be associated with different complements of known non-LEE-encoded effector genes. The genes encoding the type III secretion system (T3SS) were found to limited in their evolution whilst genes encoding immunogenic proteins have accumulated extensive genetic diversity suggesting that they are subject to diversifying selection. This genetic variation was utilised in the development of a LEE typing scheme, the utility of which was demonstrated using isolates from public health investigations. The diversity of O- and H-antigens was explored in the aEPEC collection, and a method as developed to facilitate in silico serotyping from short read data. This approach was validated by serological phenotyping of 197 EPEC isolates, before being used to characterise other clinically relevant isolates. Antimicrobial resistance of GEMS aEPEC isolates was characterised phenotypically and genetically, revealing high levels of resistance across geographical sites and clonal lineages. Regional differences between the GEMS sites, that may be due to differences in local drug use, was determined to be a key driver for the acquisition and retention of gene content conferring antimicrobial resistance. Antimicrobial resistance could be reliably predicted from genome data for the majority of drug classes. In summary, this study examined the evolution of the emerging pathogen, aEPEC and provides a framework for future studies. The methods and schemes developed in this study may be used for characterising aEPEC isolates, and other E. coli isolates in general.
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ItemCharacterisation of the type III effector NleE/OspZ from enteropathogenic Escherichia coli and Shigella flexneri( 2016)The gastrointestinal pathogens enteropathogenic E. coli (EPEC) and Shigella species utilise a type III secretion system (T3SS) to deliver bacterial effectors into host cell. A wide range of eukaryotic cell processes, including cytoskeletal organisation, host inflammatory responses, cell survival, apoptosis and phagocytosis, are modulated by T3 effectors during EPEC and Shigella infection. For example, T3 effectors from EPEC are required for the formation of attaching and effacing (A/E) lesions that are characterized as the effacement of microvilli and the actin pedestal formation on infected epithelial cells. Many EPEC effectors have homologues in other Gram-negative bacterial pathogens such as Shigella, Salmonella and Pseudomonas. The T3 effector NleE from EPEC plays a key role in the inhibition of NF-κB activation. NleE inactivates the ubiquitin-chain binding activity of the host proteins TAB2 and TAB3 by modifying the NZF domain of these host proteins through S-adenosyl methionine (SAM) dependent cysteine methylation activity. Here we found that the homologue of NleE from Shigella flexneri serotype 6, termed OspZ, was also able to bind TAB3 and decrease IL-8 transcription by inhibiting the NF-κB signalling pathway. Furthermore, OspZ also exhibited methyltransferase activity upon TAB3 to induce methylation in the presence of SAM. Previously, we identified the C-terminal motif 209IDSYMK241 of NleE as essential for the inhibition of NF-κB activation. Using yeast two hybrid protein interaction studies, we found that NleE6A, where 209IDSYMK241 was replaced with six alanine residues, retained the ability to bind TAB3. In contrast, the conserved region between amino acids 34 to 52 of NleE/OspZ, especially the motif 49GITR52, was critical for TAB3 binding. Furthermore, TAB2 and a recently identified NZF domain containing substrate, ZRANB3, were also associated with NleE/OspZ through the 49GITR52 motif. NleE mutants lacking 49GITR52 had no inhibitory effect on NF-κB-dependent luciferase activity and during infection, wild-type NleE but not NleE49AAAA52 restored the ability of an EPEC nleE mutant to inhibit IL-8 production. Similar results were observed during the infection with Shigella flexneri 6 where IL-8 expression levels were significantly higher in HT-29 cells infected with a Shigella flexneri 6 ospZ mutant strain compared to wild type Shigella flexneri 6. In contrast to NleE and OspZ, NleE49AAAA52 and OspZ49AAAA52 showed no ability to methylate TAB2 and TAB3. In addition, ectopic expression of an N-terminal fragment of NleE (NleE34-52) in HeLa cells blocked the ability of wild-type NleE to suppress IL-8 secretion during EPEC infection. NF-κB signalling is the one of the most important host immune defences against pathogenic bacterial infection, thereby the NF-κB pathway is frequently targeted for inactivation by bacterial virulence factors. Bacterial effectors often subvert host cell processes by targeting specific host proteins through posttranslational modifications such as phosphorylation, methylation and ubiquitination. This study characterized the substrate recognition mechanism of the T3 effectors NleE/OspZ which block NF-κB signalling during EPEC E2348/69 and Shigella flexneri 6 infection. In summary, we have identified a unique substrate-binding site in NleE and OspZ, which is required for the ability of NleE/OspZ to inhibit the host inflammatory response.
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ItemControlled production of tryptophan by genetically-manipulated strains of Escherichia coli( 1992)The tryptophan productivity of the genetically-manipulated strain JP4153 was increased 2.5-fold by introducing pMU78, a medium copy-number plasmid carrying a feedback-resistant trp operon. JP4153(pMU78) produced 23.5 g/l of tryptophan at a rate of 0.7 g/l/h when grown at 37 degrees C in a defined glucose and ammonium salts medium in a bench-scale fermentor. During prolonged cultivation in the presence of antibiotic, the recombinant strain generated faster-growing, production-defective variants, which harboure mutated derivatives of pMU78. Insertion sequences were responsible for the two predominant types of mutation. The plasmid element ISI02 mediated deletions extending into the promoter-proximal region of the plasmid-borne trp operon. ISI0-Right, a chromosomal element, inserted into the promoter/trpE region of the plasmid. Three methods were employed to increase the structural stability of JP4153(pMU78) during the course of the production process. First, the growth of seed cultures was carried out at 30 degrees C, the permissive temperature for the trpS378 mutation carried by the host strain. Second, the seed culture medium was modified by the addition of yeast extract, which appeared to reduce the selective disadvantage conferred by the plasmid. Third, ISI02was deleted from pMU78 to create pMU88. (For complete abstract open document)
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ItemType II secretion and biofilm formation in enteropathogenic Escherichia coli( 2013)Gastroenteritis is a leading cause of infantile morbidity and mortality in the developing world. As such, research into the infectious agents that cause diarrhoeal disease is imperative to better understand their pathogenesis and identify novel preventatives and therapeutics. Enteropathogenic Escherichia coli (EPEC) is a common cause of gastroenteritis in children under the age of two years. Clinical manifestations of EPEC infection include dehydration, failure to thrive and in extreme cases, death. EPEC infection is characterised by the production of attaching and effacing lesions throughout the intestine; which cause destruction of the brush border microvilli and lead to absorption defects. The production of these lesions requires a type III secretion system (T3SS), a common secretion system of Gram-negative bacteria and the major virulence determinant of EPEC. Apart from the T3SS, EPEC are thought to possess several additional secretion systems, including a putative type II secretion system (T2SS). The T2SS is important for virulence in other bacterial strains, including the closely related enterotoxigenic E. coli (ETEC), however its contribution to EPEC pathogenesis is unknown. The aim of this work was to investigate the functionality, biological roles and substrates of the T2SS of EPEC. The T2SS was found to be functional and capable of secreting one major substrate, renamed SslE. SslE is a high molecular weight lipoprotein with no known biological role, although bioinformatic analysis suggested that it may be involved in carbohydrate degradation. After identifying SslE, we attempted to determine the function of this secretion pathway, finding biological roles for T2SS and its substrate in biofilm formation and virulence. However, as biofilm formation in EPEC is poorly understood, these two phenotypes could not be conclusively linked. This left two open questions surrounding the T2SS, one regarding the role of biofilm formation in EPEC and the other concerning the existence of additional substrates. With regard to this second aim, two additional minor substrates for this pathway were identified using a comparative proteomics approach. These proteins, YmgD and TraT, have not been previously described to be secreted, and have never been linked to biofilm formation. Whether these proteins are involved in biofilm formation or virulence in a similar manner to SslE, or are involved in novel functions is yet to be determined. To approach the question regarding biofilm formation and virulence, the phenomenon of biofilm formation was investigated in EPEC using a transposon mutagenesis approach. Upon screening a transposon library for biofilm mutants, we found that genes important for EPEC biofilm formation were broadly similar to those involved in biofilm formation by E. coli K-12, and largely dissimilar from those in other pathogenic E. coli, such as enterohaemorrhagic E. coli (EHEC). Specifically, genes identified from the screen included those previously known to be associated with biofilm formation, such as those encoding fimbrial, flagellar and LPS biosynthesis, as well as some novel genes. Understanding these novel mutations will facilitate studies to determine if biofilm formation is a virulence determinant of EPEC. To summarise, the T2SS of EPEC is functional and capable of secreting at least three natural substrates: SslE, TraT and YmgD. The secretion system itself and its major substrate, SslE, are required for both biofilm formation and virulence, although the contribution of biofilms to virulence is unknown. EPEC biofilm formation resembles that by E. coli K-12, although novel genes are required that may be used in the future to explore the links between biofilm formation and virulence. This information adds to our knowledge of EPEC as a pathogen and could potentially be used to design novel therapeutics and preventatives for EPEC infection, lessening the global impact of this important pathogen.