Molecular characterization of megaplasmids encoding the type VI secretion system in Campylobacter jejuni isolated from chicken livers and gizzards

Abstract

Megaplasmids in Campylobacter spp. likely play important roles in antibiotic resistance, virulence, and horizontal gene transfer. In this study, megaplasmids pCJDM202 (119 kb) and pCJDM67L (116 kb) from C. jejuni strains WP2-202 and OD2-67, respectively, were sequenced and characterized. These megaplasmids contained genes for tetracycline resistance [tet(O)], the Type IV secretion system, conjugative transfer and the Type VI secretion system (T6SS). The T6SS genes in Campylobacter plasmids encoded genes and proteins that were similar to those identified in Campylobacter chromosomal DNA. When the megaplasmid pCJDM202 from C. jejuni WP2-202 was transferred via conjugation to C. jejuni NCTC11168 Nal + , transconconjugants acquired tetracycline resistance and enhanced cytotoxicity towards red blood cells. A T6SS mutant of strain WP2-202 was generated and designated Δhcp3; the mutant was significantly impaired in its ability to lyse red blood cells and survive in defibrinated blood. The cytotoxicity of Campylobacter strains towards the human embryonic kidney cell line HEK 293 was not impacted by the T6SS. In summary, the T6SS encoded by Campylobacter megaplasmids mediates lysis of RBcs and likely contributes to survival on retail meats where blood cells are abundant. The high prevalence of Campylobacter spp. in retail chicken products is associated with campylobacteriosis outbreaks worldwide 1 and reflects the challenges in preventing Campylobacter contamination in retail meat production and storage 2,3. The consumption of undercooked liver dishes prepared from contaminated chicken liver products was responsible for the multistate outbreaks of campylobacteriosis in the USA 4-6. The antimicrobial resistance and virulence genes in Campylobacter have substantially increased the difficulty in controlling campylobacteriosis 7 , and the transmission of drug-resistant foodborne pathogens through livestock, food products and humans creates a huge public health burden 8. Various aspects of Campylobacter contamination, transmission, and pathogenicity remain unexplored 9. The genetic transfer of antimicrobial resistance and virulence genes between organisms in environmental niches is a common route of acquisition by foodborne pathogens 8. Plasmids in foodborne pathogens such as Campylobacter often encode antimicrobial resistance genes such as tetracycline resistance (tetO); furthermore, pVir plasmids in Campylobacter are frequently involved in virulence 10,11. Many Campylobacter plasmid sequences have been deposited in GenBank 12,13 , and these plasmids have been categorized according to size and genomic composition 14. The complete sequences for several C. jejuni megaplasmids are available but the potential role of these genes in virulence and survival is not clear 13. The Type VI secretion system (T6SS) has been identified in diverse species of Gram-negative bacteria and functions to kill competing bacteria via a bacteriophage-like invasion and injection mechanism 15,16. Various foodborne pathogens, including Salmonella spp., ...

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

“…Interestingly, several C. jejuni megaplasmids also carry T6SS genes (Gunther et al, 2016;Fakhr, 2016, 2017). Most recently, megaplasmids pCJDM202 and pCJDM67L from C. jejuni WP2-202 and OD2-67 strains, respectively, were found to harbour the T6SS cluster, along with the tetracycline resistance gene tetO, and T4SS conjugative DNA transfer systems (Marasini et al, 2020). The authors demonstrated that the presence of the T6SS on the megaplasmids contributed to enhanced haemolysis, suggested to support the survival of C. jejuni in retail meats (Marasini et al, 2020).…”

“…Homologues of TraG and transfer coupling protein VirD4, a component of the Agrobacterium tumefaciens Type IVa secretion system (T4SSa), have been previously identified in the chromosomes of C. jejuni 81-176 and ATCC 43431, as well as in plasmids pCC31 and pTet (Batchelor et al, 2004;Poly et al, 2005;Chandran Darbari and Waksman, 2015). T4SS DNA conjugation systems were recently found harboured on megaplasmids in C. jejuni (Grohmann et al, 2018;Marasini et al, 2020). The T6SS-positive and TraGcontaining megaplasmid pCJDM202 from C. jejuni WP2-202 strain was successfully transferred by conjugation to a T6SSnegative mutant NCTC11168 Nal + recipient cell, with the hcp gene observed in the transconjugants, thus demonstrating a role in the transfer of T6SS-containing megaplasmids to transconjugants (Marasini et al, 2020).…”

Mobile Type VI secretion system loci of the gut Bacteroidales display extensive intra-ecosystem transfer, multi-species spread and geographical clustering

“…The GA1 and GA2 T6SS loci are contained on Integrative and Conjugative Elements (ICE) and are present in diverse Bacteroidales species [ 3 ]. In other bacterial lineages, T6SS loci are typically contained on non-core genomic islands, but, with few exceptions [ 8 ], are rarely found on conjugative elements. Some T6SS-associated genes, such as immunity genes [ 9 ], reside on mobile elements, and a full T6SS locus is present on a mobile prophage-like element in environmental Vibrio cholerae strains [ 10 ].…”

Type VI secretion system killing by commensal Neisseria is influenced by the spatial dynamics of bacteria

“…Effectors Nte1 to 5 are also encoded in the same locus, but with Nte6 encoded elsewhere on the plasmid. To date, plasmid encoded T6SS have only been described in Campylobacter species (Marasini and Fakhr, 2016), with this plasmid T6SS mobilised via conjugation (Marasini et al, 2020). Although other small plasmids have been reported in N. cinerea (Knapp et al, 1984; Roberts, 1989) and N. cinerea can be a recipient of N. gonorrhoeae plasmids (Genco et al, 1984), it is not yet known whether T6SS plasmids are widespread among Neisseria , or whether the plasmid can be mobilised by conjugation or transformation.…”