Characterization of periplasmic components of the type III secretion system from Xanthomonas
Plant Biochemistry and Biophysics
Final Report Abstract
Pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which translocates effector proteins into plant cells. T3S systems are conserved in plant- and animal-pathogenic bacteria and consist of a membrane-spanning secretion apparatus, which is associated with an extracellular pilus-like appendage and a channel-like translocon in the eukaryotic plasma membrane. The secretion apparatus is anchored to the inner and outer bacterial membrane by ring structures, which are presumably connected by a predicted periplasmic inner rod. We previously identified HrpB1 and the secreted HrpB2 protein as putative inner rod proteins from X. campestris pv. vesicatoria. Both proteins interact with each other and with inner and outer membrane ring components of the T3S system. Furthermore, HrpB1 interacts with the pilus protein HrpE and colocalizes with HrpB2 to the bacterial periplasm. We, therefore, assumed that HrpB1 and HrpB2 provide an assembly platform for the extracellular pilus in the periplasm. Aim of the present project was the functional characterization of HrpB1 and HrpB2 by in vivo localization and interaction studies as well as the identification of functionally important protein regions. We showed that the function of HrpB1 depends on N-terminal and central protein regions and possibly on its interaction with periplasmic components of the T3S system. Furthermore, our interaction studies revealed that HrpB1 binds to peptidoglycan and to the predicted lytic transglycosylase HpaH. These results are in agreement with the predicted function of HrpB1 as a periplasmic inner rod protein. The association of HrpB1 with HpaH might facilitate the local degradation of peptidoglycan by HpaH and thus the assembly of the T3S system. In the second part of the project, we analysed T3S signals in HrpB2 as well as the interaction of HrpB2 with components of the T3S system. HrpB2 is essential for pilus formation and itself secreted by the T3S system, suggesting that it is an early substrate. Secretion of HrpB2 is suppressed by the T3S substrate specificity switch protein HpaC, which promotes secretion of translocon and effector proteins. The HpaC-mediated T3S substrate specificity switch presumably depends on the cytoplasmic domain of HrcU, which provides a docking site for HrpB2 and HpaC. In the present project, we analysed the interaction between HrpB2 and HrcU and showed that the linker region of HrcU, which connects the transmembrane with the cytoplasmic domain, as well as a conserved C-terminal amino acid motif are essential for the interaction with HrpB2. Furthermore, we provide evidence that the N-terminal region of HrpB2 is required and sufficient for the interaction with HrcU. The results of secretion and in vivo translocation assays revealed that the region between amino acids 10 – 40 of HrpB2 contains a joint signal for T3S and translocation. Translocation of HrpB2 is suppressed by the switch protein HpaC as well as by the T3S chaperone HpaB and its secreted regulator HpaA, which are required for efficient effector protein translocation. Deletion of hpaA, hpaB and hpaC leads to a loss of pathogenicity but allows the translocation of HrpB2 reporter fusions. Our data suggest a potential use of the X. campestris pv. vesicatoria hpaABC mutant as delivery tool for the translocation of fusion proteins which contain the HrpB2 T3S and translocation signal.
Publications
- (2013) The inner membrane protein HrcV from Xanthomonas is involved in substrate docking during type III secretion. Mol Plant Microbe Interact 26: 1176-1189
Hartmann N, Büttner D
(See online at https://doi.org/10.1094/MPMI-01-13-0019-R) - (2013) The periplasmic HrpB1 protein from Xanthomonas spp. binds to peptidoglycan and to components of the type III secretion system. Appl Environ Microbiol 79: 6312-6324
Hausner J, Hartmann N, Lorenz C, Büttner D
(See online at https://doi.org/10.1128/AEM.01226-13) - (2014) The YscU/FlhB homolog HrcU from Xanthomonas controls type III secretion and translocation of early and late substrates. Microbiol 160: 576- 588
Hausner J, Büttner D
(See online at https://doi.org/10.1099/mic.0.075176-0) - (2016) Type III-dependent translocation of HrpB2 by a non-pathogenic hpaABC mutant of the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria. Appl Environ Microbiol 82: 3331-3347
Scheibner F, Schulz S, Hausner J, Marillonnet S, Büttner D
(See online at https://doi.org/10.1128/AEM.00537-16) - (2017) The predicted lytic transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria associates with the type III secretion system and promotes effector protein translocation. Infect Immunol 85:e00788-16
Hausner J, Hartmann N, Jordan M, Büttner D
(See online at https://doi.org/10.1128/IAI.00788-16)
