HpaC Controls Substrate Specificity of the Xanthomonas Type III Secretion System

Abstract

The Gram-negative bacterial plant pathogen Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject bacterial effector proteins into the host cell cytoplasm. One essential pathogenicity factor is HrpB2, which is secreted by the T3S system. We show that secretion of HrpB2 is suppressed by HpaC, which was previously identified as a T3S control protein. Since HpaC promotes secretion of translocon and effector proteins but inhibits secretion of HrpB2, HpaC presumably acts as a T3S substrate specificity switch protein. Protein–protein interaction studies revealed that HpaC interacts with HrpB2 and the C-terminal domain of HrcU, a conserved inner membrane component of the T3S system. However, no interaction was observed between HpaC and the full-length HrcU protein. Analysis of HpaC deletion derivatives revealed that the binding site for the C-terminal domain of HrcU is essential for HpaC function. This suggests that HpaC binding to the HrcU C terminus is key for the control of T3S. The C terminus of HrcU also provides a binding site for HrpB2; however, no interaction was observed with other T3S substrates including pilus, translocon and effector proteins. This is in contrast to HrcU homologs from animal pathogenic bacteria suggesting evolution of distinct mechanisms in plant and animal pathogenic bacteria for T3S substrate recognition.

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

“…T3S4 proteins share little amino acid sequence identity with each other but contain a structurally conserved C-terminal domain (termed the T3S4 domain) that is probably essential for the substrate specificity switch and harbors a P-X-L-G amino acid motif (2). T3S4 proteins from both translocation-associated and flagellar T3S systems interact with the C-terminal cytoplasmic domains of members of the conserved YscU/FlhB family of IM proteins, as shown for the T3S4 proteins FliK, Spa32, and HpaC (50,332,381,392) (Tables 2 and 6). Binding of T3S4 proteins to the C-terminal domains of FlhB, YscU, and homologs might induce a conformational change in these domains that alters the substrate specificity of the T3S system.…”

“…Functional studies of T3S4 proteins have so far been performed only with X. campestris pv. vesicatoria (332). In contrast to T3S4 proteins from animal pathogens, HpaC from X. campestris pv.…”

“…Furthermore, an interaction was reported between the C-terminal domain of the YscU/ FlhB homolog HrcU, from the plant-pathogenic bacteria Xanthomonas campestris pv. vesicatoria and P. syringae, and secreted proteins (332,550). The contribution of YscU/FlhB family members to the substrate specificity switch is discussed below.…”

Type III Protein Secretion in Plant Pathogenic Bacteria

“…Furthermore, HpaC does not control secretion of the pilus protein HrpE, suggesting that HpaC is not involved in length determination of the pilus (Lorenz et al, 2008b). T3S4 proteins are not highly conserved among different plant and animal pathogenic bacteria, and no significant sequence identity is detected between HpaC from X. campestris pv vesicatoria and the predicted T3S4 protein HrpP from P. syringae (Fig.…”

“…In X. campestris pv vesicatoria, the predicted T3S4 protein HpaC was shown to switch the substrate specificity of the T3SS from secretion of the putative inner rod protein HrpB2 to secretion of translocators and effector proteins. HpaC interacts with and presumably induces a conformational change in the C-terminal domain of HrcU, which is a member of the YscU/FlhB protein family (Lorenz et al, 2008b; Fig. 3).…”