An investigation of helicobacter pylori integrative and conjugative elements
Publication Date
July 19, 2023
Creator
Abstract
Several key Helicobacter pylori virulence factors are encoded within three major genomic islands namely, the cag pathogenicity island (cagPAI), tfs3 and tfs4. Although the cagPAI is fairly well characterised, understanding the functional role played by tfs3 and tfs4 in disease outcomes remains unknown. Also, the possibility of functional interchangeability between the components of both tfs3 and tfs4 subtypes by interacting with one another to form a functional type IV secretion system (T4SS) remains to be investigated.
The main aim of this study was to investigate interactions between proteins encoded by tfs3, tfs4 and tfs4 variant types that are important for T4SS assembly and function. A second aim of this study was to evaluate approaches for virulence typing, comparing genomics with PCR typing using an available rich dataset including cagA and cagE PCR typing data, and phenotypic data. Additionally, the study aimed to explore the association of tfs T4SS genes in H. pylori strains isolated from patients with their disease status.
Initially, the yeast two-hybrid assay (Y2H) was used to establish a network of interactions between tfs3, tfs4 and tfs4 variant encoded proteins comprising the T4SS structure. Other interactions involving proteins that are not part of the T4SS assembly, tfs4 VirD2 and orphan VirB11 together with proteins of tfs3, tfs4 and tfs4 variant were also investigated using Y2H assay to assess whether the tfs T4SSs might be functional for export/transfer of any tfs integrative and conjugative elements (ICEs). The ABRicate tool was configured with custom scripts of both tfs3 and tfs4 genes from reference H. pylori strains. Then, the association between disease status and tfs3 and tfs4 clusters of genes in genomic data from clinical isolates was examined using the ABRicate bioinformatics tool to identify virulence genes. Prior to using ABRicate to search for tfs3 and tfs4 genes in isolates, its efficacy was assessed by comparing its output on the presence of cagA and cagE genes with PCR genotype and phenotypic data available from clinical isolates from patients.
The Y2H analyses identified several novel interactions between paired proteins of tfs3, tfs4 and tfs4 variant type (tfs4VirB11/tfs4VirB11v, tfs4VirB11/tfs4VirB4v, tfs4VirB11v/tfs4VirB4, tfs4VirB4/tfs4VirB4v, tfs4VirB4/tfs4VirB11v, tfs3VirB11/tfs4VirB8v, tfs3VirB11/tfs4VirB4v) suggesting the possibility of functional complementation between tfs ICE proteins in the assembly of a T4SS. Other interactions involving tfs4 VirD2, identified novel interactions with components of tfs3 T4SS (tfs4VirD2/tfs3VirB4), which could suggest some functional flexibility of the tfs T4SSs for inter ICE transfer especially when both ICEs are present within a single strain.
The ABRicate tool detected the cagA gene in the genome of 17 out of 18 clinical isolates that were previously found to be cagA-positive by PCR. Also, the tool found the cagE gene in the genome of all 15 isolates that were previously reported to be cagE-positive by PCR. Additionally, PCR typing, and genomic data were in agreement for 18 dupA-negative isolates and for 6 dupA-positive isolates analysed.
The presence of dupA, a virB4 homolog in tfs4 and some tfs4 vir genes (virB2, virB4, virB8 and virB10) that form the “dupA cluster”, in clinical isolates was compared with disease status in the patients infected with them. Although the ABRicate tool detected a dupA cluster in four strains isolated from two patients, who presented with duodenal ulcer and gastric ulcer disease, genome sequencing of isolates from many more patients would be required to establish associations between disease status and cluster of tfs4 genes in isolates.
The Y2H results provided an insight of system specificity in protein-protein interactions of tfs3 and tfs4 and, variable cross-complementation between tfs3 and tfs4 VirB proteins to form a functional T4SS. Also, the comparison between genomic and PCR typing data in this study, provides some support in the reliability of both PCR typing and genomics analyses in the detection of virulence factors in H. pylori. Lastly, genomic analysis could not be used to establish association between disease status and tfs T4SS genes in isolates due to the small sample size but it indicated the feasibility for using genomics in future studies. Therefore, genomics analysis approaches can readily be used to investigate the genes present in tfs ICEs.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Medicine
eprints ID
73968
UoN Repository URI
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