Bioinformatic Analysis of the Campylobacter jejuni Type VI Secretion System and Effector Prediction

Abstract

The Type VI Secretion System (T6SS) has important roles relating to bacterial antagonism, subversion of host cells, and niche colonisation. Campylobacter jejuni is one of the leading bacterial causes of human gastroenteritis worldwide and is a commensal coloniser of birds. Although recently discovered, the T6SS biological functions and identities of its effectors are still poorly defined in C. jejuni. Here, we perform a comprehensive bioinformatic analysis of the C. jejuni T6SS by investigating the prevalence and genetic architecture of the T6SS in 513 publicly available genomes using C. jejuni 488 strain as reference. A unique and conserved T6SS cluster associated with the Campylobacter jejuni Integrated Element 3 (CJIE3) was identified in the genomes of 117 strains. Analyses of the T6SS-positive 488 strain against the T6SS-negative C. jejuni RM1221 strain and the T6SS-positive plasmid pCJDM202 carried by C. jejuni WP2-202 strain defined the "T6SS-containing CJIE3" as a pathogenicity island, thus renamed as Campylobacter jejuni Pathogenicity Island-1 (CJPI-1). Analysis of CJPI-1 revealed two canonical VgrG homologues, CJ488_0978 and CJ488_0998, harbouring distinct C-termini in a genetically variable region downstream of the T6SS operon. CJPI-1 was also found to carry a putative DinJ-YafQ Type II toxin-antitoxin (TA) module, conserved across pCJDM202 and the genomic island CJIE3, as well as several open reading frames functionally predicted to encode for nucleases, lipases, and peptidoglycan hydrolases. This comprehensive in silico study provides a framework for experimental characterisation of T6SS-related effectors and TA modules in C. jejuni.

Keywords: Campylobacter jejuni; T6SS effectors; T6SS immunity proteins; Type VI Secretion System; pathogenicity island; toxin-antitoxin.

FIGURE 1

Representation of the C. jejuni T6SS in extended state [adapted from Cherrak et al. (2019)]. Proteins are labelled according to the nomenclature used for C. jejuni T6SS components. Tss, Type six secretion.

FIGURE 2

Comparative analyses of CJPI-1 (middle) from T6SS-positive C. jejuni 488 strain to CJIE3 from T6SS-negative strain RM1221 (top) and T6SS-positive megaplasmid pCJDM202 (bottom) from C. jejuni WP2-202 strain. Homologous genes across the three strains share the same arrow colour and are connected by shaded bands corresponding to sequence identity (black = 100%, white = 0%). Red arrows represent major T6SS components. Sequences found in only one strain possess a grey arrow. Only regions of the megaplasmid associated with the T6SS and CJIE3/CJPI-1 regions have been included. The figure was constructed using Clinker.

FIGURE 3

Genomic architecture of the PAI CJPI-1 in T6SS-positive C. jejuni 488 strain. Coloured coding domain sequences (CDS) represent proteins with inferred functions and are labelled. The scale under the CDS represents the nucleotide position [kilobase pair (kbp)] of the pathogenicity island in the genome of C. jejuni 488 strain. The location of the T6SS and genetically variable region are also denoted. The positive (+) symbol on the left-hand side indicates the sense strand in the genome of C. jejuni 488 strain, whilst the negative (–) symbol indicates the antisense strand. Genes sizes are not to scale. Genes are coloured according to predicted function: integrases are shaded magenta, regulators are light green, TA components are grey, conjugative systems are red, putative effectors are purple, PAAR-like proteins are light blue, T6SS components are green, VgrGs are dark blue, putative immunity proteins are pink, and putative chaperone proteins are olive.

FIGURE 4

Full amino acid sequence alignment of the two VgrG homologues present in T6SS-positive C. jejuni 488 strain using ClustalO. Conserved residues found in both sequences correspond to letters in uppercase below the aligned sequences. Protein locus tag and amino acid position are provided for each respective sequence. The N-terminal VgrG domain (COG3501) in both sequences is highlighted black. The Jag domain (COG1847) is highlighted light grey for VgrG1, while letters in bold represent the Phyre2 structural homology match to the C-terminal lysozyme domain of Gp5 (PDB: 1K28). The Gp5_C domain (PF06715) is highlighted dark grey for VgrG2.

FIGURE 5

Phylogenetic tree derived from a Maximum likelihood analysis on a MUSCLE alignment of VgrG amino acid sequences from T6SS-positive C. jejuni of assembly level “complete” or higher (bootstrap n = 500, partial deletion). The value of each node is given. Top bracket = 838 amino acids (aa), bottom bracket = 883 aa.

FIGURE 6

Genomic architecture of the genetically variable region downstream of the T6SS operon in five T6SS-positive C. jejuni strains; 488, 14980A, ATCC33560, 00-1597, and AR-0412. Genes found within the variable region possessing a domain by NCBI CDD (Marchler-Bauer et al., 2017) are coloured according to the domains identified: Ankyrin domains are pink, Tox-REase-7 are purple, AHH are yellow, TNT are red, DUF4299 are sky blue, Imm70 are dark green, PoNe are light green, DUF1911 are brown, five superfamily domain are lavender, and PAAR-like are dark blue. Variable region genes with no predicted function or identifiable domains are shaded grey and genes neighbouring the variable region are orange. Gene sizes are not to scale. The corresponding locus tags for selected genes is labelled above accordingly.

FIGURE 7

Partial amino acid sequence alignment of Tox-REase-7 and AHH domain-containing representative amino acids against putative effectors downstream of the T6SS from C. jejuni 488 strain. (A) Alignment of CJ488_0980 and CJ488_0982 against Tox-REase-7 representative amino acids. (B) Alignment of CJ488_0994 against AHH representative amino acids. Catalytic residues for each domain are highlighted in yellow, and the hash (#) indicates the position of the residue in the C. jejuni 488 proteins. Red letters indicate amino acids with greater than 90% homology across all sequences for the alignment. Protein locus tag, organism name, and amino acid position are to the left and either side of the respective sequences.