In the past few years, in vivo recombination technologies have emerged to improve the efficiency and simplicity of genetic engineering in Escherichia coli, Salmonella enterica serovar, and other gram-negative bacteria. Phage λ Red homologous recombination system is used to mediate the accurate replacement of target DNA with PCR-generated ?targeting cassettes? that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Salmonella is far lower than that in Escherichia coli. In this study, I firstly improved the recombineering-based strategy by using linear DNA targeting cassettes that contain long flanking ?arms? of sequence (ca. 1,000 base pairs) homologous to the chromosomal target. This reliable and efficient method enables multiple gene targeting procedures to be performed on a single Salmonella enterica serovar typhi Ty21a (Ty21a) chromosome in a straightforward, sequential manner with high efficiency. Secondly, I applied this improved strategy in construction of Salmonella to be live attenuated oral vaccine and tumor targeting vector. In the first part of this thesis, I describe an improved method in Ty21a. Using this strategy, I inserted three different influenza antigen expression cassettes as well as a green fluorescent protein reporter gene into four different loci on the Ty21a chromosome with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. The immune response of this vaccine was also evaluated by ELISA and ELIspots. In the second part of this thesisi, I use this improved recombineering strategy to engineer bacteria of Salmonella typhimurium (S. typhimurium) as therapeutic agents against solid tumor. In current study, a major challenge for bacterial therapy of cancer is avoiding damage to normal tissues. Consequently the virulence of bacteria must be adequately attenuated for therapeutic use. An alternative approach was developed here. By placing an essential gene under a hypoxia conditioned promoter, S. typhimurium strain SL7207 was engineered to generate strain YB1 that survives only in anaerobic conditions without otherwise affecting its functions. In breast and liver tumor bearing mice models, YB1 grew within tumor, retarding its growth, while being rapidly eliminated from normal tissues. Mice treated with SL7207 were killed by infection within short period time. Inhibition of tumor growth by YB1 was significant and was enhanced by the addition of 5-FU in breast cancer model. The development of an “obligate” anaerobic Salmonella provides a much safer bacterial vector for further development of anti-tumor therapies without compromising the other functions or tumor fitness of the bacterium as attenuation methods normally do. In summary, I have developed an efficient, robust and versatile method in genome-wide Salmonella genetic manipulation. Furthermore, I used this method to construct a recombinant Ty21a antigen-expressing vaccine strain and a tumor targeting YB1 strain. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

食物中毒感染是常見但非常重要的全球性公共健康問題。沙門氏鼠傷寒桿菌乃常被分離出來的細菌性病原體之一。隨著實驗室參考菌株LT2的基因組序列於2001年被發表之後，另外9個沙門氏鼠傷寒菌菌株的基因序列均已陸續進行測序。最近，本實驗室亦對十個本地沙門氏鼠傷寒菌臨床分離菌株的基因序列進行了測序。為了為這些基因組序列提供高品質的註釋，我們把預測的基因組提交到質量控制工具GenePRIMP以識別有潛在錯誤或異常的預測基因。本研究針對血液分離菌株78896和糞便分離菌株1047518的GenePRIMP報告進行人工檢查，並對每個菌株超過270個的基因進行了修訂。此外，本研究亦對上述的10個本地菌株進行了功能註釋。註釋項目包括沙門氏菌致病島（SPIs）、致病因子、tRNA和非編碼小分子RNA、噬菌體和CRISPRs結構等基因組及致病元素。 KEGG通路則提供了進一步的功能註釋。 / 本研究同時對本地的血液和糞便分離菌株，連同國外的臨床分離菌株，進行了廣泛的比對，用以識別全身性沙門氏菌感染的潛在遺傳因素。 本研究進行了以下基因分析：（1）多位點序列分型（MLST）；（2）在小鼠全身性感染中涉及的主調控因子和關鍵元素; 及（3）人類腸胃道感染中涉及的基因。然而，這些分析產生只能對全身性沙門氏菌感染提供有限的見解。然而，透過使用RAST註釋系統，我們於其中三個血液分離菌株中發現了一個的額外的螯鐵蛋白aerobactin鐵採集系統。儘管在體外實驗中，這些血液分離菌株並沒有明顯的生長優勢，但實驗結果表明，在缺乏鐵的培養液中，aerobactin基因的表達水平是比較高的。此外，我們亦於其中四個血液分離菌株中，發現負責細胞色素c熟成(ccm)的基因座均被中斷。這可能改變了這些血液分離菌株中細胞色素c的生物合成途徑。這些鐵採集和同化機制的觀察均為未來全身性沙門氏菌感染的研究提供了可能的發展方向。 / 本研究同時識別了用以分別本地及海外的沙門氏鼠傷寒菌菌株的分子標記，並在鮭魚和生菜的接種實驗中，展現了它們分辨本地及海外菌株的能力。然而，在投入實際應用之前，這些標記尚需要進一步的驗證和測試，以便確定快速檢測方法的有效性。 / Foodborne infection is a common but important public health issue worldwide. Salmonella enterica serovar Typhimurium is frequently isolated from outbreaks as one of the common bacterial causative agents. Following the availability of the genome sequence of the reference lab strain LT2 in 2001, nine genomes of S. Typhimurium had been sequenced since then. Recently, genomes of ten local S. Typhimurium clinical isolates have been assembled in our laboratory. In order to provide high quality annotation of these genome sequences, the predicted gene sets were submitted to the quality control tool GenePRIMP (Gene PRediction IMprovement Pipeline) to identify potentially erroneous and abnormal gene calls. The GenePRIMP reports for the local blood isolate 78896 and stool isolate 1047518 were manually inspected and more than 270 genes were amended individually for each isolate. Functional annotation had also been performed for the 10 local isolates. Genomic and virulent elements including Salmonella Pathogenicity Islands (SPIs), virulence factors, tRNAs and small non-coding RNAs, prophage elements and CRISPRs structures had been annotated. The KEGG pathways provided a further means of functional annotation. / The local blood and stool isolates, together with the sequenced foreign clinical isolates, had also been extensively compared to identify potential genetic determinants of Salmonella systemic infection. (1) Multilocus sequence typing (MLST); (2) Alignment of master regulators and key players of systemic infection in mice; and (3) Analyses of the genes responsible for human gastrointestinal tract infection had been performed. However, these analyses yielded limited insights on systemic infection. Alternatively, using subsystems annotation by RAST, an additional aerobactin siderophore iron acquisition system was shown to be prevalent among three of the blood isolates. Despite no obvious growth advantage was offered to the blood isolates in an in vitro experiment, it was demonstrated that expression of the aerobactin genes was higher in iron-depleted culturing medium. In addition, a disrupted cytochrome c maturation (ccm) locus that may alter the cytochrome c biogenesis pathway was also identified in four of the blood isolates. These observations in iron acquisition and assimilation mechanisms suggest their potential in future direction of Salmonella systemic infection studies. / Molecular markers specific to local and foreign S. Typhimurium isolates were also identified and their utility in differentiating local and foreign isolates was demonstrated in a pilot spiking experiment using raw salmon and lettuce. These markers will require further verification and testing prior to actual application in real-world settings in order to examine the validity of the rapid detection method. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheng, Chi Keung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 124-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract of thesis entitled --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xiii / Abbreviations --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Introduction and Taxonomy --- p.1 / Chapter 1.2 --- Epidemiology of Salmonella Typhimurium infections --- p.2 / Chapter 1.3 --- Pathogenesis of Salmonella Typhimurium infection --- p.4 / Chapter 1.3.1 --- Infection mechanisms --- p.4 / Chapter 1.3.2 --- Salmonella Pathogenicity Islands --- p.6 / Chapter 1.3.3 --- Regulation of virulence --- p.9 / Chapter 1.4 --- Non-typhoid Salmonella (NTS) systemic infection --- p.11 / Chapter 1.4.1 --- Epidemiology of NTS systemic infection --- p.11 / Chapter 1.4.2 --- Salmonella Typhimurium multidrug resistance --- p.12 / Chapter 1.5 --- Salmonella Typhimurium genomics --- p.15 / Chapter 1.5.1 --- Salmonella Typhimurium genome sequencing --- p.15 / Chapter 1.5.2 --- Comparative studies on Salmonella genomes --- p.17 / Chapter 1.6 --- Aims of project --- p.19 / Chapter Chapter 2 --- Curation and detailed annotation of genomes of local Salmonella Typhimurium clinical isolates --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Manual curation of GenePRIMP results --- p.27 / Chapter 2.2.2 --- Salmonella Pathogenicity Islands (SPIs) and virulence factors annotation --- p.29 / Chapter 2.2.3 --- Small RNA and t-RNA annotation --- p.29 / Chapter 2.2.4 --- Phage elements annotation --- p.30 / Chapter 2.2.5 --- CRISPRs annotation --- p.30 / Chapter 2.2.6 --- KEGG annotation --- p.30 / Chapter 2.3 --- Results --- p.32 / Chapter 2.3.1 --- Manual curation of GenePRIMP results --- p.32 / Chapter 2.3.1.1 --- Short genes --- p.35 / Chapter 2.3.1.2 --- Long genes --- p.35 / Chapter 2.3.1.3 --- Unique genes --- p.36 / Chapter 2.3.1.4 --- Overlapped genes --- p.36 / Chapter 2.3.1.5 --- Broken genes --- p.37 / Chapter 2.3.2 --- Salmonella Pathogenicity Islands (SPIs) and virulence factors annotation --- p.37 / Chapter 2.3.2.1 --- Salmonella Pathogenicity Islands (SPIs) annotation --- p.37 / Chapter 2.3.2.2 --- Virulence factors annotation --- p.44 / Chapter 2.3.3 --- Small RNA and t-RNA annotation --- p.44 / Chapter 2.3.4 --- Phage elements annotation --- p.44 / Chapter 2.3.5 --- CRISPRs annotation --- p.50 / Chapter 2.3.6 --- KEGG annotation --- p.51 / Chapter 2.4 --- Discussion --- p.53 / Chapter 2.4.1 --- Manual curation of GenePRIMP results --- p.53 / Chapter 2.4.1.1 --- Gene amendment not required --- p.54 / Chapter 2.4.1.2 --- Genes with boundaries relocated --- p.54 / Chapter 2.4.1.3 --- Genes to be discarded --- p.55 / Chapter 2.4.1.4 --- Gene pairs to be fused --- p.55 / Chapter 2.4.1.5 --- Potential pseudogenes formation --- p.56 / Chapter 2.4.2 --- Salmonella Pathogenicity Islands (SPIs) annotation --- p.57 / Chapter 2.4.3 --- Virulence factors annotation --- p.57 / Chapter 2.4.4 --- Small RNA and t-RNA annotation --- p.58 / Chapter 2.4.5 --- Phage elements annotation --- p.59 / Chapter Chapter 3 --- Identification of genetic determinants of blood invasiveness in local S. Typhimurium clinical isolates --- p.61 / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Materials and Methods --- p.66 / Chapter 3.2.1 --- Multilocus Sequence Typing (MLST) --- p.66 / Chapter 3.2.2 --- Phage elements annotation for foreign isolates --- p.67 / Chapter 3.2.3 --- Alignment of genes inferred to play important roles in NTS systemic --- p.infection67 / Chapter 3.2.4 --- Alignment of genes inferred to involved during infection in the gastrointestinal (GI) tract --- p.68 / Chapter 3.2.5 --- Subsystems assignment using Rapid Annotation using Subsystem Technology (RAST) server --- p.68 / Chapter 3.2.6 --- Growth analysis of local S. Typhimurium clinical isolates in iron-limiting environment --- p.69 / Chapter 3.2.7 --- Reverse transcription and real-time PCR --- p.70 / Chapter 3.2.7.1 --- Primer design and verification --- p.70 / Chapter 3.2.7.2 --- cDNA synthesis and real-time PCR --- p.70 / Chapter 3.3 --- Results --- p.73 / Chapter 3.3.1 --- Multilocus Sequence Typing (MLST) --- p.73 / Chapter 3.3.2 --- Phage elements annotation for foreign isolates --- p.73 / Chapter 3.3.3 --- Alignment of genes inferred to play important roles in NTS systemic infection --- p.74 / Chapter 3.3.4 --- Alignment of genes inferred to involved during infection in the gastrointestinal (GI) tract --- p.79 / Chapter 3.3.4.1 --- Acid tolerance response --- p.79 / Chapter 3.3.4.2 --- Epithelial cells attachment --- p.80 / Chapter 3.3.4.3 --- Epithelial cells invasion --- p.83 / Chapter 3.3.4.4 --- Survival within macrophages --- p.83 / Chapter 3.3.5 --- RAST subsystem analysis --- p.86 / Chapter 3.3.6 --- Growth analysis and aerobactin genes expression --- p.87 / Chapter 3.4 --- Discussion --- p.93 / Chapter Chapter 4 --- Molecular markers identification and testing on selected foodstuff for local S. Typhimurium isolates --- p.97 / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.2 --- Materials and Methods --- p.101 / Chapter 4.2.1 --- Molecular markers identification --- p.101 / Chapter 4.2.2 --- Primer design and verification --- p.101 / Chapter 4.2.3 --- Spiking experiments on selected food samples --- p.103 / Chapter 4.2.4 --- Quantitative TaqMan real-time PCR --- p.103 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- Molecular markers identification --- p.105 / Chapter 4.3.2 --- Spiking experiments and TaqMan real-time PCR --- p.109 / Chapter 4.4 --- Discussion --- p.113 / Chapter 4.4.1 --- Molecular markers identification --- p.113 / Chapter 4.4.2 --- Spiking experiments and TaqMan real-time PCR --- p.114 / Chapter Chapter 5 --- General discussion --- p.116 / Chapter 5.1 --- Manual curation of GenePRIMP results --- p.116 / Chapter 5.2 --- Functional annotation of local S. Typhimurium genomes --- p.118 / Chapter 5.3 --- Systemic infection studies --- p.120 / Chapter 5.4 --- Molecular markers identification and spiking experiments --- p.121 / Chapter 5.5 --- Conclusion and future perspectives --- p.122 / References --- p.124

The genus Salmonella consists of pathogenic gram negative organisms which infect intestines of birds, animals and humans. They are the causative agents of salmonellosis which is characterised by diarrhoea, nausea, fever and abdominal cramps. If not treated in time, salmonellosis can also be fatal. Salmonella genus is divided into two species Salmonella bongori and Salmonella enterica. Salmonella enterica is further divided into six subspecies out of which the subspecies enterica has many of the pathogenic serovars of this species. Salmonella typhimurium is a server in the subspecies enterica of Salmonella enterica species. Transmission of salmonellosis takes place through contaminated food and water. When the organism enters a host, it encounters a range of hostile environments such as acidic pH, lack of oxygen as well as immune response of the host. In order to establish infection, the bacterium needs to survive under stressful conditions and propagate itself. Various proteins are induced in cells under unfavourable conditions that protect them in such situations. One such group of proteins belongs to the Universal Stress Protein (USP) family. Universal Stress Proteins are a set of proteins induced in organisms when it is exposed to a variety of environmental insults including heat shock, nutrient starvation, presence of toxic compounds, etc. Although survival in adverse conditions is mediated by induction of this group of proteins, the precise mechanism of cellular protection has not been elucidated yet. The functional role of a protein is directly related to its three-dimensional structure and hence important insights can be gained regarding the role of these proteins by determining their structures. The structures of two Universal Stress Proteins from S. typhimurium; a single domain protein, YnaF and another tandem USP domain protein, YdaA were determined by X-ray crystallography and biochemical analysis was carried out on them. Guided by structure, plausible roles for both the proteins in stress tolerance of S. typhimurium have been proposed. Additionally, work was also carried out on phosphomannose isomerise from S. typhimurium. Phosphomannose isomerase is a housekeeping enzyme which catalyses the interconversion of mannose-6-phosphate and fructose-6-phosphate. Mannose is important for mannosylation of various lipids and proteins which form an important component of bacterial and fungal cell walls. Presence of a functional phosphomannose isomerise enzyme is important as it helps the organism survive adverse conditions by forming a strong cell wall which shields it from harmful environments. Moreover, phosphomannose isomerase was also found to be essential for virulence of Leishmania mexicana and Cryptococcus neoformans. The structure of phosphomannose isomerase from S. typhimurium was determined in our laboratory in the year 2009. However, in the earlier studies, the catalytically important residues had not been identified and mechanism of isomerisation was not established. Structural analysis, site directed mutagenesis and biochemical assays were used to identify key residues in the active site of StPMI. Identification of these residues might help in deciphering the catalytic mechanism which will eventually be useful to develop inhibitors that arrest the growth of Salmonella as well as other microorganisms. The work reported in this thesis describes the efforts made to enhance our understanding of functional aspects of the two Universal Stress Proteins, YnaF and YdaA and phosphomannose isomerase from S. typhimurium. Chapter 1 begins with a brief introduction to the kinds of unfavourable environments encountered by microorganisms and their strategies of adaptation. This is followed by a review of the literature on Universal Stress Proteins, which are induced in many organisms in response to arrest of or perturbations in the growth rate. Structural, biochemical and evolutionary aspects of members of the family have also been discussed. Subsequently, a brief description of the earlier work carried out on another enzyme important in stress tolerance, phosphomannose isomerase, has been documented. A detailed account of mechanisms of isomerisation carried out by aldose ketose isomerases and identification of important strategies for determination of mechanism of phosphomannose isomerase catalysed reaction have then been provided. The chapter ends with a summary of aims and objectives of the present work. Chapter 2 describes the various experimental techniques and computational methods used during the course of this thesis work. Isolation of plasmids, overexpression and purification of protein, site directed mutagenesis, biochemical assays, crystallisation of proteins, X ray diffraction data collection form a part of the experimental aspect and have been described in detail. Brief descriptions of the programs used and principles behind computational methods used for structure determination (including data processing, phasing, model building and refinement), validation and analysis have also been provided. Chapter 3 includes the structural and functional studies carried out on YdaA, a tandem USP domain protein from S. typhimurium. Expression, purification, crystallisation and structure determination of YdaA in its native and ADP bound forms are described in the chapter. Biochemical assays with radiolabelled ATP showed that YdaA was an ATPase. The crystal structure of YdaA complexed with ATP revealed the presence of ADP (hydrolysis product of ATP) only in the C-terminal domain of the protein. Based on structural analysis and presence of ATP binding motif in the C-terminal domain, it could be hypothesized that ATP hydrolysis activity of the protein is confined to the C-terminal domain of the protein. The N-terminal domain of the protein was found to play another interesting role. A zinc binding site could be identified in the N terminal domain based on structural analysis and elemental X-ray absorption studies done at the synchrotron. Site directed mutagenesis and biochemical experiments suggested that zinc binding in the N-terminal domain was not related to ATPase activity of the C-terminal domain. Additionally, an intermediate of lipid A biosynthesis pathway UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetyl glucosamine was found bound to the N-terminal domain of YdaA. Lipid A is the membrane anchor of polysaccharides in the outer membrane of gram negative organisms and the intermediate occurs at the committed step of the pathway. However, no similarities could be identified between YdaA and members of the relevant biosynthetic pathway. Therefore, YdaA is unlikely to play a catalytic role in the same pathway but can function as a carrier molecule. A plausible link between the N- and C-terminal domains of YdaA could be identified by structural analysis. Many catalytically suitable residues from the N-terminal domain were found to be close to the β-phosphate of ADP bound to the C-terminal domain. Hence YdaA was identified to be a zinc binding ATPase which might play some yet unidentified role in lipid A biosynthesis pathway. Chapter 4 describes the attempts made towards understanding the functional role of YnaF, a single domain USP from S. typhimurium. A description of the expression, purification, crystallisation and X ray diffraction techniques used for structure determination of YnaF and its single site mutant have been provided in detail. Gel filtration, dynamic light scattering studies and the crystal structure determination of YnaF showed a tetrameric organisation of four USP protomers stabilised in the centre by chloride ions. Additionally, YnaF crystallised with a bound ATP even though ATP was not included in the crystallisation cocktail. Biochemical assays on YnaF with radiolabelled ATP showed that it was inactive with respect to ATP hydrolysis. When selected mutations that disrupt chloride binding were made, YnaF was converted to an active ATPase. The crystal structure of the mutant complexed with an ATP analogue revealed key differences at the active site in comparison with that of the wild type and allowed identification of residues that might be important for ATP hydrolysis in this group of proteins. Hence YnaF might play the role of a sensor protein in some signal transduction pathway involving chloride ions in bacteria. A structure based analysis and comparison of USPs from the Protein Data Bank with the structures of YnaF and YdaA is summarised at the end of this chapter. Chapter 5 describes the efforts carried out towards determination of mechanism of isomerisation catalysed by phosphomannose isomerise (PMI). Earlier reports suggest that the enzyme catalyses the reversible isomerisation of mannose-6-phosphate and fructose-6-phosphate via formation of a cis-enediol intermediate. The structure of phosphomannose isomerase from S. typhimurium has been reported by our laboratory. The enzyme is a monomer with three domains; a catalytic domain, a carboxy terminal domain and an α-helical domain. Residues from the catalytic domain were found to coordinate a zinc ion. Overexpression, purification, co crystallisation experiments and soaking studies carried out on crystals of PMI and its single site mutants are outlined in this chapter. The structure of a complex of PMI with mannose-6-phosphate at pH 7.0 revealed the presence of a blob of density close to the zinc binding site which was confirmed to be the active site by analysis of conservation of residues in the site. Based on site directed mutagenesis, activity studies and analysis of structure of PMI, zinc was identified to play an important role in maintaining the structural integrity of the active site. Electrostatic surface analysis of the structure of PMI revealed that the zinc ion might also play the role of anchoring phosphate moiety of the substrate in a highly negatively charged active site pocket. Activity assays following site directed mutagenesis studies eliminated the role of Glu264 in catalysis and implicated two lysines, Lys86 and Lys132 as the possible base in the reaction. The plausible role of a highly conserved residue Arg274 was also proposed based on comparison of structures of wild type and mutant PMIs. The future prospects of the work are briefly discussed towards the end of the thesis. Further experiments and analysis required to obtain better understanding of the functions of these proteins have been discussed. The Appendix section describes extensive crystallisation attempts that were carried out on the enzyme sorbitol-6-phosphate-dehydrogenase from S. typhimurium which catalyses the isomerisation reaction between sorbitol-6-phosphate and glucose-6-phosphate using NADPH as the cofactor. Needle shaped crystals were obtained which diffracted to a poor resolution of 7-8 Å at our in house X ray facility. Attempts to improve the quality of the crystals like co crystallisation with substrate and its analogues, soaking in various compounds and seeding are briefly described. The following manuscripts based on work described in this thesis have been published or will be communicated for publication. 1. Structural and functional analysis of two universal stress proteins YdaA and YnaF from Salmonella typhimurium: possible roles in microbial stress tolerance. Bangera M., Panigrahi R., Sagurthi S.R., Savithri H.S., Murthy M.R.N. Journal of Structural Biology, 2015 Mar; 189 (3): 238-50. 2. Structural and functional insights into phosphomannose isomerise: role of zinc and catalytic residues. Bangera M., Savithri H.S., Murthy M.R.N. Manuscript under preparation