Peptide linkage mapping of the Agrobacterium tumefaciens vir -encoded type IV secretion system reveals protein subassemblies

Abstract

Numerous bacterial pathogens use type IV secretion systems (T4SS) to deliver virulence factors directly to the cytoplasm of plant, animal, and human host cells. Here, evidence for interactions among components of the Agrobacterium tumefaciens virencoded T4SS is presented. The results derive from a high-resolution yeast two-hybrid assay, in which a library of small peptide domains of T4SS components was screened for interactions. The use of small peptides overcomes problems associated with assaying for interactions involving membrane-associated proteins. We established interactions between VirB11 (an inner membrane poreforming protein), VirB9 (a periplasmic protein), and VirB7 (an outer membrane-associated lipoprotein and putative pilus component). We provide evidence for an interaction pathway, among conserved members of a T4SS, spanning the A. tumefaciens envelope and including a potential pore protein. In addition, we have determined interactions between VirB1 (a lytic transglycosylase likely involved in the local remodeling of the peptidoglycan) and primarily VirB8, but also VirB4, VirB10, and VirB11 (proteins likely to assemble the core structure of the T4SS). VirB4 interacts with VirB8, VirB10, and VirB11, also establishing a connection to the core components. The identification of these interactions suggests a model for assembly of the T4SS.

The versatile bacterial type IV secretion systems

“…Further investigations exploring these stabilizing effects, as well as recent cell biology studies, have led to the development of a biogenesis pathway for this transfer apparatus 22,53 .A crucial intermediate in this pathway is a 'core' structure that is composed of VirB4, VirB7-VirB10 and, probably, VirB6. The existence of this structure is now supported by data from dihybrid screens and complementary biochemical assays [54][55][56][57][58][59] (Table 2). Additionally, some of the interactions required for assembly of the putative core are conserved in the B. pertussis Ptl system.…”

“…Additionally, some of the interactions required for assembly of the putative core are conserved in the B. pertussis Ptl system. For example, it has been postulated that the A. tumefaciens VirB8 protein functions in part by recruiting the VirB1 transglycosylase for localized lysis of the PEPTIDOGLYCAN at the site of machine assembly 57 . B. pertussis PtlE is the VirB8 homologue, but PtlE itself shows transglycosylase activity, apparently eliminating the requirement for a VirB1 homologue in the Ptl system 60 .…”

“…Recently, evidence was presented for an interaction between VirB4 and VirB11 (REF. 57). An intriguing area for further study is how these two ATPases functionally interact.…”

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…However, the VirB8-like protein of this secretion system (PtlE) contains an N-terminal domain mediating a VirB1-like peptidoglycanase activity (Rambow-Larsen and Weiss, 2002), suggesting that VirB1 and VirB8 may closely interact in other Mpf systems. Indeed, interactions between VirB1 and VirB8 of A. tumefaciens have been detected in a yeast two-hybrid screen (Ward et al, 2002). The other VirB1 interactions detected in the same screen (Table 2) await validation by biochemical data.…”

“…These interactions were initially detected in two-hybrid screens (Malek et al, 2004;Rain et al, 2001;Ward et al, 2002) and, in case of VirB10, VirB11, and VirD4, have been validated by in vitro methods (Atmakuri et al, 2004;Terradot et al, 2004). Of special importance are the interactions between the three nucleotide-binding/hydrolyzing components VirB4, VirB11, and VirD4, since these components seem to conjointly energize the transport of the substrate from the cytosol into the membrane-spanning Mpf channel (see below).…”