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The type VI secretion system (T6SS) is a secretion apparatus widely found in pathogenic Gram-negative bacteria and is important for competition among various bacteria and host cell pathogenesis. Hcp is a core component of functional T6SS and transports toxic effectors into target cells by assembling to form tube-like structures. Studies have shown that Hcp simultaneously acts as an effector to influence cellular physiological activities; however, the mechanism of its activity in host cells remains unclear. To investigate the target of effector protein Hcp2a in a chicken fibroblast cell line, we first detected the subcellular localization of Hcp2a in DF-1 cells by indirect immunofluorescence assay. The results showed that Hcp2a protein was localized in the endoplasmic reticulum of DF-1 cells. We also used a streptavidin–biotin affinity pull-down assay combined with LC–MS/MS to screen DF-1 cell lysates for proteins that interact with Hcp2a and analyze the cellular functional pathways affected by them. The results showed that Hcp2a interacted with 52 DF-1 cellular proteins that are involved in multiple intracellular pathways. To further explore the mechanism of Hcp2a protein targeting the endoplasmic reticulum of DF-1 cells, we screened three endoplasmic reticulum-associated proteins (RSL1D1, RPS3A, and RPL23) from 52 prey proteins of Hcp2a for protein–protein molecular docking analysis. The docking analysis showed that the effector protein Hcp2a and the RPL23 protein had good complementarity. Overall, we propose that Hcp2a has strong binding activity to the RPL23 protein in DF-1 cells and this may help Hcp2a anchor to the endoplasmic reticulum in DF-1 cells. [ABSTRACT FROM AUTHOR]

Background: Bacteria deliver effector proteins into the host cell via a secretory system that can directly act on the target to cause disease. As an important pipeline structural protein of the type VI secretion system (T6SS) complex, Hcp acts together with other virulence factors in the target cell. There is growing evidence that T6SS plays a key role in the pathogenic mechanism of APEC. However, the regulatory function played by the effector protein Hcp during its interaction with host cells is not clear. Here, tandem mass tag (TMT) analysis was used to quantify the proteins affected by increased expression of Hcp2a in DF-1 cells. Results: The host response was significantly different between the overexpression and null groups at the protein level. A total of 195 differentially expressed proteins (DEPs) were detected in the overexpression group (upregulated, n = 144, downregulated, n = 51). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the biological functions and pathways of differentially expressed proteins. The results showed that these DEPs were mainly enriched in RNA degradation, spliceosome, and mRNA surveillance pathways. Conclusions: This study suggests that Hcp2a, the effector protein of APEC, plays an important role in regulating mRNA splicing and protein quality control in DF-1 cells. These findings provide useful clues to elucidate the pathogenic mechanism of effector protein Hcp2a on host target cells. [ABSTRACT FROM AUTHOR]

Background: The type VI protein secretion system (T6SS) is important in diverse cellular processes in Gram-negative bacteria, including interactions with other bacteria and with eukaryotic hosts. In this study we analyze the evolution of the T6SS in the genus Xanthomonas and evaluate its importance of the T6SS for virulence and in vitro motility in Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of bacterial blight in cassava (Manihot esculenta). We delineate the organization of the T6SS gene clusters in Xanthomonas and then characterize proteins of this secretion system in Xpm strain CIO151. Results: We describe the presence of three different clusters in the genus Xanthomonas that vary in their organization and degree of synteny between species. Using a gene knockout strategy, we also found that vgrG and hcp are required for maximal aggressiveness of Xpm on cassava plants while clpV is important for both motility and maximal aggressiveness. Conclusion: We characterized the T6SS in 15 different strains in Xanthomonas and our phylogenetic analyses suggest that the T6SS might have been acquired by a very ancient event of horizontal gene transfer and maintained through evolution, hinting at their importance for the adaptation of Xanthomonas to their hosts. Finally, we demonstrated that the T6SS of Xpm is functional, and significantly contributes to virulence and motility. This is the first experimental study that demonstrates the role of the T6SS in the Xpm-cassava interaction and the T6SS organization in the genus Xanthomonas. [ABSTRACT FROM AUTHOR]

Background: Campylobacter jejuni (C. jejuni) is one of the most common bacteria responsible for human gastroenteritis worldwide. The mode of human transmission is foodborne infections due to consumption of contaminated food, especially poultry. Type 6 secretion systems (T6SS) were described recently as Campylobacter virulence mechanisms. Furthermore, infection sequelae associated with neurological disorders like Guillain–Barré (GBS) and Miller Fisher (MF) syndromes can become serious health problems in some patients after Campylobacter gastroenteritis. Our objective was to determine the distribution of these virulence genes among C. jejuni isolated from stool of human diarrhea. Methods: A total of 524 C. jejuni strains from travelers and pediatric cases of acute diarrhea in Thailand were selected for this study. All isolates belonged to one of 20 known capsule types and all were assayed by PCR for T6SS, a hemolysin co-regulated protein (hcp) gene, and GBS-associated genes (cgtA, cgtB, cstIIHS19 and cstIIHS2) which are involved in sialic acid production in the lipooligosaccharide (LOS) cores of C. jejuni. The distribution of these genes are summarized and discussed. Results: Of all isolates with these 20 capsule types identified, 328 (62.6%) were positive for hcp, ranging from 29.2 to 100% among 10 capsule types. The GBS-associated LOS genes were detected among 14 capsule type isolates with 24.4% and 23.3% of C. jejuni isolates possessed either cstIIHS19 or all three genes (cgtA, cgtB and cstIIHS19), which were classified as LOS classes A and B whereas 9.2% of C. jejuni isolates possessing cstIIHS2 were classified as LOS class C. The C. jejuni isolates of LOS A, B, and C together accounted for 56.9% of the isolates among 14 different capsule types while 31.1% of all C. jejuni isolates did not possess any GBS-associated genes. No significant difference was detected from C. jejuni isolates possessing GBS-associated LOS genes among travelers and children, but changes between those with hcp were significant (p < 0.05). Conclusions: Our results suggested a high diversity of hcp and GBS-associated LOS genes among capsule types of C. jejuni isolated from Thailand. [ABSTRACT FROM AUTHOR]

Background: Many Gram-negative bacteria rely on a type VI secretion system (T6SS) to infect eukaryotic cells or to compete against other microbes. Common to these systems is the presence of two conserved proteins, in Vibrio cholerae denoted VipA and VipB, which have been shown to interact in many clinically relevant pathogens. In this study, mutagenesis of a defined region within the VipA protein was used to identify residues important for VipB binding in V. cholerae O1 strain A1552. Results: A dramatically diminished interaction was shown to correlate with a decrease in VipB stability and a loss of hemolysin co-regulated protein (Hcp) secretion and rendered the bacterium unable to compete with Escherichia coli in a competition assay. Conclusions: This confirms the biological relevance of the VipA-VipB interaction, which is essential for the T6SS activity of many important human pathogens. [ABSTRACT FROM AUTHOR]