Secretome Analysis Uncovers an Hcp-Family Protein Secreted via a Type VI Secretion System inAgrobacterium tumefaciens

Abstract

Agrobacterium tumefaciens is a plant-pathogenic bacterium capable of secreting several virulence factors into extracellular space or the host cell. In this study, we used shotgun proteomics analysis to investigate the secretome of A. tumefaciens, which resulted in identification of 12 proteins, including 1 known secretory protein (VirB1*) and 11 potential secretory proteins. Interestingly, one unknown protein, which we designated hemolysin-coregulated protein (Hcp), is a predicted soluble protein without a recognizable N-terminal signal peptide. Western blot analysis revealed that A. tumefaciens Hcp is expressed and secreted when cells are grown in both minimal and rich media. Further biochemical and immunoelectron microscopy analysis demonstrated that intracellular Hcp is localized mainly in the cytosol, with a small portion in the membrane system. To investigate the mechanism of secretion of Hcp in A. tumefaciens, we generated mutants with deletions of a conserved gene, icmF, or the entire putative operon encoding a recently identified type VI secretion system (T6SS). Western blot analysis indicated that Hcp was expressed but not secreted into the culture medium in mutants with deletions of icmF or the t6ss operon. The secretion deficiency of Hcp in the icmF mutant was complemented by heterologous trans expression of icmF, suggesting that icmF is required for Hcp secretion. In tumor assays with potato tuber disks, deletion of hcp resulted in approximately 20 to 30% reductions in tumorigenesis efficiency, while no consistent difference was observed when icmF or the t6ss operon was deleted. These results increase our understanding of the conserved T6SS used by both plant-and animal-pathogenic bacteria.

VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex

“…Agrobacterium tumefaceins C58 harbors one T6SS that is activated transcriptionally by an ExoR-ChvG/ChvI signaling cascade and posttranslationally via threonine phosphorylation when sensing acidity (34)(35)(36). Three type VI effectors, namely type VI amidase effector (Tae) and type VI DNase effectors (Tde1 and Tde2), confer antibacterial activity of this bacterium.…”

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…In addition, the affinity of the ChvE protein for sugar acids is increased at low pH, suggesting a link between the sugar and acidity response in A. tumefaciens (Hu et al, 2012). A. tumefaciens utilizes the ChvG/ChvI two-component system to activate the transcription of virG and induces expression of several other bacterial genes (Mantis and Winans, 1992;Chang and Winans, 1996), including genes involved in chemotaxis and motility, several chromosome-encoded virulence genes, succinoglycan biosynthesis genes, and the gene cluster encoding the type VI secretion system (T6SS) (Yuan et al, 2007a;Wu et al, 2008;Wu et al, 2012). Recent studies further identified ExoR as a periplasmic negative regulator acting upstream of the ChvG sensor kinase to repress acid-inducible gene expression (Wu et al, 2012;Heckel et al, 2014) and uncovered a role for the T6SS in interbacterial competition activity during the plant colonization process (Ma et al, 2014).…”

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

“…The cytosolic part harbors a conserved Walker A motif (phosphate-binding loop), implying that IcmF functions as an ATPase during type VI protein secretion. icmF mutants have been shown to prevent Hcp secretion (2,(8)(9)(10). ClpV is a member of the AAA ϩ (ATPases associated with various cellular activities) protein family (8,11).…”