The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

Abstract

eThe type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells. Citrobacter freundii strain CF74 has a complete T6SS genomic island (GI) that contains clpV, hcp-2, and vgr T6SS genes. We constructed clpV, hcp-2, vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74⌬T6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased in clpV, hcp-2, vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74⌬T6SS, CF74⌬clpV, and CF74⌬hcp-2 mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role in C. freundii.

Type VI secretion system MIX‐effectors carry both antibacterial and anti‐eukaryotic activities

“…While we do not yet know what mechanism leads to this apparent down‐regulation of the flagellar machinery and other non‐T6SS proteins, others have previously reported similar findings. For example, the T6SS in Citrobacter was reported to modulate flagellar gene expression and secretion , as did the T6SS in Ralstonia . Therefore, it is possible that a common cross talk mechanism exists between T6SS activity and motility, and perhaps also between the T6SS and siderophore or heme transport.…”

Functional Characterization and Conditional Regulation of the Type VI Secretion System in Vibrio fluvialis

“…In P. aeruginosa , HSI-I T6SS mediated the export of Hcp1, and evoked an immune response in cystic fibrosis (Mougous et al, 2006). Subsequent to these two landmark publications, numerous follow-up studies have demonstrated that T6SS is involved in the virulence and environmental competitive fitness of various bacterial species, such as Burkholderia species (Schell et al, 2007; Shalom et al, 2007), Edwardsiella tarda (Zheng and Leung, 2007), Agrobacterium tumefaciens (Wu et al, 2012), Vibrio parahaemolyticus (Yu et al, 2012), Aeromonas hydrophila (Suarez et al, 2008), Citrobacter freundii (Liu et al, 2015), Vibrio alginolyticus (Sheng et al, 2012; Salomon et al, 2015), and Burkholderia cenocepacia (Aubert et al, 2016). A genome-level survey revealed that more than 25% of genome-sequenced Gram-negative species contain T6SS gene clusters (Bingle et al, 2008).…”

The Type VI Secretion System of Xanthomonas Phaseoli pv. Manihotis is Involved in Virulence and in vitro Motility

“…The T6SS has been implicated in pathogenicity 15,51,52 , motility [53][54][55] and interaction with other bacteria 21,56 . Motility contributes to virulence in the genus Xanthomonas [57][58][59] .…”