The Periplasmic HrpB1 Protein from Xanthomonas spp. Binds to Peptidoglycan and to Components of the Type III Secretion System

Abstract

The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate bacterial effector proteins into eukaryotic host cells. The membrane-spanning secretion apparatus consists of 11 core components and several associated proteins with yet unknown functions. In this study, we analyzed the role of HrpB1, which was previously shown to be essential for T3S and the formation of the extracellular T3S pilus. We provide experimental evidence that HrpB1 localizes to the bacterial periplasm and binds to peptidoglycan, which is in agreement with its predicted structural similarity to the putative peptidoglycan-binding domain of the lytic transglycosylase Slt70 from Escherichia coli. Interaction studies revealed that HrpB1 forms protein complexes and binds to T3S system components, including the inner membrane protein HrcD, the secretin HrcC, the pilus protein HrpE, and the putative inner rod protein HrpB2. The analysis of deletion and point mutant derivatives of HrpB1 led to the identification of amino acid residues that contribute to the interaction of HrpB1 with itself and HrcD and/or to protein function. The finding that HrpB1 and HrpB2 colocalize to the periplasm and both interact with HrcD suggests that they are part of a periplasmic substructure of the T3S system. Bacteria of the genus Xanthomonas cause devastating diseases of a variety of mono-and dicotyledonous plants worldwide, including important crop plants (1, 2). The successful interaction with the host often depends on protein secretion systems which transport bacterial virulence factors into the extracellular milieu or directly into the host cell. The type III secretion (T3S) system, which is often essential for pathogenicity, is a complex molecular nanomachine dedicated to the transkingdom delivery of bacterial effector proteins into eukaryotic cells (3). T3S systems are conserved in many Gram-negative plant-and animal-pathogenic bacteria and presumably share a similar architecture consisting of membrane-associated ring structures that enclose an inner transport channel (4-9). The inner membrane (IM) and outer membrane (OM) rings are connected via a predicted periplasmic inner rod structure (7,10,11). The IM ring is presumably associated with members of the conserved YscR, -S,-T, -U, and -V families of IM proteins that form the export apparatus. The nomenclature of these proteins refers to the Ysc proteins from the animal-pathogenic bacterium Yersinia (12). Members of the export apparatus are linked to the predicted cytoplasmic (C) ring of the T3S system, which presumably harbors docking sites for T3S substrates and interacts with the cytoplasmic ATPase (YscN family members) and its predicted regulator (YscL family members) (13-16). The ATPase provides the energy for T3S and/or mediates the unfolding of T3S substrates (17). As the inner diameter of the secretion channel is too narrow to allow the transport of fully folded proteins, T3S substrates presumably travel the secretion apparatus in a partial...

The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation

“…2D). As described previously, a similar localization was observed for HrpB1 and HrpB2 (34,35) (Fig. 2D).…”

“…S4 in the supplemental material. It was previously shown that HrpB1, HrpB2, HrcD, and HrpE do not unspecifically interact with T3S-associated proteins (24,35).…”

Xanthomonas campestris pv. vesicatoria Secretes Proteases and Xylanases via the Xps Type II Secretion System and Outer Membrane Vesicles

“…1B and D) (45,46). Furthermore, the detection of proteins in the culture supernatants did not result from unspecific binding of the c-Myc epitope-specific antibody to bacterial proteins (see Fig.…”

“…TE and SN were analyzed by immunoblotting using an HrpB1-specific antibody. HrpB1 and HrpB1-c-Myc have the expected molecular masses of approximately 16 and 21 kDa, respectively, and formed oligomeric complexes as described previously (45). HrpB1-specific signals are indicated by white asterisks.…”

The Cpx Envelope Stress Response Modifies Peptidoglycan Cross-Linking via the l,d-Transpeptidase LdtD and the Novel Protein YgaU

“…The induction of the Cpx response changes the expression of a set of genes that are enriched for inner membrane proteins, including a large group of transporter proteins, and also downregulates the production of envelope-spanning protein complexes involved in motility, secretion, and transport (11,32,51). In this light, it is interesting that a number of these complexes depend upon direct interactions with the peptidoglycan for assembly and function (52)(53)(54)(55). These observations may suggest that cell wall alterations could impact the activity and stability of secretion and transport complexes, potentially generating a Cpx-inducing signal.…”