Title: The use of recombinase-based in vivo expression technology to identify genes of avian pathogenic Escherichia coli induced during infection in chicken
Other Titles: Identificatie van aviaire pathogene Escherichia coli genen specifiek geïnduceerd gedurende kippeninfecties via recombinase-gebaseerde in vivo expressie technologie
Authors: Tuntufye, Huruma
Issue Date: 27-Sep-2012
Abstract: Avian pathogenic Escherichia coli (APEC) belong to the extra-intestinal pathogenic Escherichia coli (ExPEC) that can cause a variety of extra-intestinal infections in humans and animals. APEC are the causative agent of localized colibacillosis or systemic infection in poultry. Systemic infection starts as an infection of the upper respiratory tract and develops into a systemic infection. ExPEC are generally characterized by a broad variety of virulence-associated factors which may contribute to pathogenesis. However the major virulence factors which clearly define ExPEC including APEC have not been identified. Additionally, the pathogenesis of APEC in poultry is also poorly understood. ExPEC could not be clearly distinguished by molecular epidemiology, and this lead to difficulties in distinguishing avian ExPEC from human ExPEC hence pose a hypothetical zoonotic risk.The main objective of this study was to identify APEC strain genes which are specifically induced during infection in chicken. Recombinase-based in vivo expression technology (RIVET) was selected for this purpose. For the functional RIVET, a promoterless recombinase cre was chosen for recombination. An APEC strain containing loxP sites flanking the antibiotic marker nptII for kanamycin/ neomycin resistance and rpsL for streptomycin sensitivity (LoxP cassette) was constructed. Kanamycin was used to select for the presence of the LoxP cassette in vitro and streptomycin was used to select for the loss of the cassette after isolating the bacteria from the chicken (in vivo). A promoter-trap plasmid containing a promoterless cre and ampicillin resistance gene (bla) for selection was also constructed. The whole system was tested for its suitability for gene identification. It was demonstrated that the Cre/lox system worked very well in APEC and the whole system worked as required when it was tested in vitro and in vivo when the chicken were infected with the bacteria. On this background, the second task in this thesis focused on the identification of the APEC genes that were induced in vivo during infection in chicken. A genomic library of APEC was constructed by cloning random genomic DNA fragments upstream of a promoterless cre in the promoter-trap plasmid. The library was grown in vitro in the presence of the antibiotics kanamycin and ampicillin to counter-select against clones displaying cre expression in vitro. The grown cultures were used for intratracheal administration to chickens. After 24 hours chickens were euthanized and APEC were isolated from liver and spleen for the systemic infection. Clones which were resistant to streptomycin but sensitive to kanamycin were selected, subjected to plasmid isolation and these plasmids were introduced into the APEC strains with an intact LoxP cassette for a second round of infection. The streptomycin resistant clones were isolated and plasmidswere isolated, purified and the insert in these plasmids were sequenced. After performing BLASTx, the screening resulted into 21 unique clones twelve of which carried E. coli conserved genes, six of the genes were bacteriophage-associated, and three (tkt1, irp2 and eitD) were pathogen-specific genes. Various cellular functions such as metabolism, adaptation and stress response, cell membrane and integrity, transport systems, virulence, phage-related or unknown functions were assigned to the identified genes. The approach demonstrates a little overlap between the APEC genes identified from signature-tagged mutagenesis (STM) study, selective capture of transcribed sequences (SCOTS) study and those identified by our approach. The genes identified provide a highlight on the APEC genes that are induced inside the chicken host during infection and that are likely to be involved in the survival/fitness and successful colonization of this pathogen in the stressful host environment. Among the genes identified by our RIVET approach, was a gene coding for a putative transketolase 1 (tkt1). Due to the fact that there is a growing attention to the sugar metabolism of ExPEC, and the high prevalence of this gene in avian and human ExPEC, we decided to further analyse tkt1. We performed virulence studies in order to evaluate whether the tkt1 played a role in the APEC CH2 virulence in chickens. APEC CH2 with deleted tkt1 was constructed, inoculated subcutaneously into 1 day old chicks and intratracheally into 4 weeks old chickens and compared with the parent APEC CH2. The evaluation revealed that the mutantÂ’s virulence was slightly reduced when compared to the wild type during infection of chicken indicating that this gene may be contributing to its fitness in different environments encountered by the bacteria as the literature suggests. The gene was sequenced from APEC CH2 and an in silico analysis was performed. The BLASTn analysis revealed that tkt1 has 68% identity with tktA and tktB, of ExPEC and E. coli MG1655. The BLASTx revealed that tkt1 shared 69% and 68% identity with TktA and TktB proteins, respectively of ExPEC and E. coli MG1655. The phylogenetic analysis demonstrated that three genes (tktA, tktB and tkt1) cluster in three different clades, suggesting that Tkt1 proteins have originated from a different ancestor and may serve a different function compared to other transketolases. Interestingly, when a gene tree of tkt1 was compared to the species tree, the analysis suggested a horizontal gene transfer (HGT). This was further analysed using HGT detection program T-REX revealing that there is an evidence that tkt1 may have been horizontally acquired by ExPEC from plant associated bacteria within the family Enterobacteriaceae. In conclusion, the RIVET approach was developed and used in this study for the identification of genes which were specifically induced when the bacteria were inside the host (chicken). The role of some of the genes identified in this study for the pathogenesis of APEC in chicken remains to be determined as this may contribute to the understanding of the APEC pathogenesis in poultry. This may even be important for the identification of APEC pathotype in an attempt to reveal their zoonotic risks.
Table of Contents: Acknowledgement iii
Summary v
Samenvatting ix
List of abbreviations xiii
Chapter 1 General introduction on avian pathogenic Escherichia coli 1
Objective of the study 24
Chapter 2: Construction of avian pathogenic Escherichia coli strains with a chromosomally integrated loxP cassette for site-specific recombination 27
Chapter 3: Identification of avian pathogenic Escherichia coli genes that are induced in vivo during infection in chickens 41
Chapter 4: Virulence evaluation of tkt1 from avian pathogenic Escherichia coli in chickens and it’s in silico analysis 59
Chapter 5: General discussion and future perspectives 75
References 82
Publications in international peer reviewed journals 100
ISBN: 978-90-8826-263-0
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Division of Gene Technology (-)

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