Dr Jai Mehat

Campylobacter jejuni and Campylobacter coli are recognised as the principal causative agents of bacterial gastroenteritis in the developed world. However, despite the identification of factors integral to infection, characterisation of the virulence strategies employed by Campylobacter remains a significant challenge. Bacterial autotransporter proteins comprise the largest and most diverse class of secretory proteins in Gram-negative bacteria; notably almost all previously characterised autotransporter proteins contribute to bacterial virulence to some extent. The aim of this study was to characterise CapC, a newly identified, strain-specific gene predicted to encode an autotransporter protein, and to examine the contribution of this factor to the virulence of Campylobacter jejuni.
The capC gene was initially confirmed as being encoded by approximately 60% of C. jejuni and C. coli human clinical and poultry associated isolates. Moreover, CapC was confirmed as a member of the autotransporter family through the use of bioinformatic prediction tools and the localisation site of this protein was determined as the outer membrane of C. jejuni. Targeted mutagenesis of the capC gene in C. jejuni 81116 and C. jejuni M1 and subsequent comparison of wild-type and isogenic mutant strains demonstrated that CapC contributes directly to virulence in the Galleria mellonella invertebrate model (p=0.00017; p=0.002323). Furthermore, tissue culture assays using non-polarised, partially differentiated Caco-2 and T84 intestinal epithelial cells indicate that deletion of CapC resulted in significantly decreased adhesion and invasion efficiency. Additional analyses indicated that CapC primarily contributes to adhesion to intestinal epithelial cells. Additional assays showed that deletion of the capC gene has no significant phenotypic effect on cytotoxicity in a Caco-2 cell model.
A secondary aim of this study was to examine the distribution of capC amongst campylobacters and to establish any potential genetic associations of this virulence determinant. Using publically
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available genome sequences, capC was established to be highly prevalent in C. jejuni (67.9%) and C. coli (84%). Campylobacter autotransporter proteins were also shown to be present in truncated and full length forms. Interestingly, full length CapC was shown to be primarily associated with the ST-45, ST-283 and ST-573 clonal complexes in C. jejuni and the ST-828 clonal complex in C. coli. Furthermore, this study detailed the identification of a novel autotransporter in Campylobacter species, tentatively designated as CapD. This autotransporter was found to be genetically distinct from CapC and is the most prevalent autotransporter identified in Campylobacter species.
The studies presented in this thesis indicate that CapC is a strain-specific virulence determinant in Campylobacter species that is associated with select lineages of C. jejuni and C. coli. CapC contributes to the integral infection process of adhesion however further studies are required to fully elucidate the exact nature of this interaction. Additionally, it can be concluded that possession of Campylobacter autotransporter proteins is dependent on genetic background.

Campylobacter jejuni is recognized as an important causative agent of bacterial gastroenteritis in the developed world. Despite the identification of several factors contributing to infection, characterization of the virulence strategies employed by C. jejuni remains a significant challenge. Bacterial autotransporter proteins are a major class of secretory proteins in Gram-negative bacteria and notably many autotransporter proteins contribute to bacterial virulence. The aim of this study was to characterise the C. jejuni 81116 C8J_1278 gene (capC), predicted to encode an autotransporter protein, and examine the contribution of this factor to virulence of Campylobacter jejuni. The predicted CapC protein has a number of features that are consistent with autotransporters including the N-terminal signal sequence and the C-terminal ²-barrel domain and was determined to localise to the outer membrane. Inactivation of the capC gene in C. jejuni 81116 and C. jejuni M1 resulted in reduced insecticidal activity in Galleria mellonella larvae. Furthermore, C. jejuni capC mutants displayed significantly reduced adherence to and invasion of non-polarized, partially differentiated Caco-2 and T84 intestinal epithelial cells. Gentamicin treatment showed that the reduced invasion of the capC mutant is primarily caused by reduced adherence to intestinal epithelial cells, not by reduced invasion capability. C. jejuni capC mutants caused reduced IL-8 secretion from intestinal epithelial cells and elicited a significantly diminished immune reaction in Galleria larvae indicating that CapC functions as an immunogen. In conclusion, CapC is a new virulence determinant of C. jejuni that contributes to the integral infection process of adhesion to human intestinal epithelial cells.

In recent years, several plasmids harbouring genes encoding phosphoethanolamine transferases conferring colistin resistance have been described in multiple Enterobacteriaceae species. Avian Pathogenic E. coli (APEC) causes colibacillosis and is responsible for a considerable proportion of the disease burden in commercial poultry flocks, and may be linked to zoonotic infections in humans. Here, we describe the genotypic and phenotypic characteristics of a multidrug-resistant APEC ST69 isolate (APECA2), recovered in 2016 from a diseased broiler at post-mortem examination in Germany. The isolate was resistant to several antibiotics of human and veterinary importance, including colistin. The mcr-1 gene was detected on a mobile genetic element located on an IncHI2/ST4 plasmid, which was characterized using long-read Nanopore and short-read Illumina sequencing of purified plasmid. Isolate APECA2 displayed resistance to chicken serum and harbours numerous virulence genes. This study highlights the public health importance of enhanced antimicrobial resistance surveillance and strict antimicrobial stewardship in human and veterinary healthcare.

Background:
Campylobacter jejuni and Campylobacter coli are major global causes of bacterial gastroenteritis. Whilst
several individual colonisation and virulence factors have been identified, our understanding of their role in the
transmission, pathogenesis and ecology of Campylobacter has been hampered by the genotypic and phenotypic
diversity within C. jejuni and C. coli. Autotransporter proteins are a family of outer membrane or secreted proteins in Gram-negative bacteria such as Campylobacter, which are associated with virulence functions. In this study we have
examined the distribution and predicted functionality of the previously described capC and the newly identified,
related capD autotransporter gene families in Campylobacter.

Results:
Two capC-like autotransporter families, designated capC and capD, were identified by homology searches
of genomes of the genus Campylobacter. Each family contained four distinct orthologs of CapC and CapD. The
distribution of these autotransporter genes was determined in 5829 C. jejuni and 1347 C. coli genomes.
Autotransporter genes were found as intact, complete copies and inactive formats due to premature stop codons
and frameshift mutations. Presence of inactive and intact autotransporter genes was associated with C. jejuni and C.
coli multi-locus sequence types, but for capC, inactivation was independent from the length of homopolymeric
tracts in the region upstream of the capC gene. Inactivation of capC or capD genes appears to represent lineagespecific
gene decay of autotransporter genes. Intact capC genes were predominantly associated with the C. jejuni
ST-45 and C. coli ST-828 generalist lineages. The capD3 gene was only found in the environmental C. coli Clade 3
lineage. These combined data support a scenario of inter-lineage and interspecies exchange of capC and subsets of
capD autotransporters.

Conclusions:
In this study we have identified two novel, related autotransporter gene families in the genus
Campylobacter, which are not uniformly present and exhibit lineage-specific associations and gene decay. The
distribution and decay of the capC and capD genes exemplifies the erosion of species barriers between certain
lineages of C. jejuni and C. coli, probably arising through co-habitation. This may have implications for the
phenotypic variability of these two pathogens and provide opportunity for new, hybrid genotypes to emerge.