Recognition of a Translocation Motif in the Regulator HpaA from is Controlled by the Type III Secretion Chaperone HpaB

Overview

Journal Front Plant Sci

Date 2022 Aug 15

PMID 35968103

Authors

Sabine Drehkopf

Christian Otten

Daniela Buttner

Affiliations

Soon will be listed here.

Abstract

The Gram-negative plant-pathogenic bacterium is the causal agent of bacterial spot disease in pepper and tomato plants. Pathogenicity of depends on a type III secretion () system which translocates effector proteins into plant cells and is associated with an extracellular pilus and a translocon in the plant plasma membrane. Effector protein translocation is activated by the cytoplasmic chaperone HpaB which presumably targets effectors to the system. We previously reported that HpaB is controlled by the translocated regulator HpaA which binds to and inactivates HpaB during the assembly of the system. In the present study, we show that translocation of HpaA depends on the substrate specificity switch protein HpaC and likely occurs after pilus and translocon assembly. Translocation of HpaA requires the presence of a translocation motif (TrM) in the N-terminal region. The TrM consists of an arginine-and proline-rich amino acid sequence and is also essential for the function of HpaA. Mutation of the TrM allowed the translocation of HpaA in mutant strains but not in the wild-type strain, suggesting that the recognition of the TrM depends on HpaB. Strikingly, the contribution of HpaB to the TrM-dependent translocation of HpaA was independent of the presence of the C-terminal HpaB-binding site in HpaA. We propose that HpaB generates a recognition site for the TrM at the system and thus restricts the access to the secretion channel to effector proteins. Possible docking sites for HpaA at the system were identified by and interaction studies and include the ATPase HrcN and components of the predicted cytoplasmic sorting platform of the system. Notably, the TrM interfered with the efficient interaction of HpaA with several system components, suggesting that it prevents premature binding of HpaA. Taken together, our data highlight a yet unknown contribution of the TrM and HpaB to substrate recognition and suggest that the TrM increases the binding specificity between HpaA and system components.

References

Hu B, Morado D, Margolin W, Rohde J, Arizmendi O, Picking W . Visualization of the type III secretion sorting platform of Shigella flexneri. Proc Natl Acad Sci U S A. 2015; 112(4):1047-52. PMC: 4313800. DOI: 10.1073/pnas.1411610112. View

Berger C, Ravelli R, Lopez-Iglesias C, Kudryashev M, Diepold A, Peters P . Structure of the Yersinia injectisome in intracellular host cell phagosomes revealed by cryo FIB electron tomography. J Struct Biol. 2021; 213(1):107701. DOI: 10.1016/j.jsb.2021.107701. View

Ngou B, Jones J, Ding P . Plant immune networks. Trends Plant Sci. 2021; 27(3):255-273. DOI: 10.1016/j.tplants.2021.08.012. View

Schulz S, Buttner D . Functional characterization of the type III secretion substrate specificity switch protein HpaC from Xanthomonas campestris pv. vesicatoria. Infect Immun. 2011; 79(8):2998-3011. PMC: 3147569. DOI: 10.1128/IAI.00180-11. View

Wang Y, Sun M, Bao H, White A . T3_MM: a Markov model effectively classifies bacterial type III secretion signals. PLoS One. 2013; 8(3):e58173. PMC: 3589343. DOI: 10.1371/journal.pone.0058173. View

Cheng L, Kay O, Schneewind O . Regulated secretion of YopN by the type III machinery of Yersinia enterocolitica. J Bacteriol. 2001; 183(18):5293-301. PMC: 95411. DOI: 10.1128/JB.183.18.5293-5301.2001. View

Russmann H, Kubori T, Sauer J, Galan J . Molecular and functional analysis of the type III secretion signal of the Salmonella enterica InvJ protein. Mol Microbiol. 2002; 46(3):769-79. DOI: 10.1046/j.1365-2958.2002.03196.x. View

Zilkenat S, Franz-Wachtel M, Stierhof Y, Galan J, Macek B, Wagner S . Determination of the Stoichiometry of the Complete Bacterial Type III Secretion Needle Complex Using a Combined Quantitative Proteomic Approach. Mol Cell Proteomics. 2016; 15(5):1598-609. PMC: 4858942. DOI: 10.1074/mcp.M115.056598. View

Spaeth K, Chen Y, Valdivia R . The Chlamydia type III secretion system C-ring engages a chaperone-effector protein complex. PLoS Pathog. 2009; 5(9):e1000579. PMC: 2734247. DOI: 10.1371/journal.ppat.1000579. View

10.

Schesser K, Wolf-Watz H . Delineation and mutational analysis of the Yersinia pseudotuberculosis YopE domains which mediate translocation across bacterial and eukaryotic cellular membranes. J Bacteriol. 1996; 178(24):7227-33. PMC: 178637. DOI: 10.1128/jb.178.24.7227-7233.1996. View

11.

Buttner D, Bonas U . Getting across--bacterial type III effector proteins on their way to the plant cell. EMBO J. 2002; 21(20):5313-22. PMC: 129068. DOI: 10.1093/emboj/cdf536. View

12.

Samudrala R, Heffron F, McDermott J . Accurate prediction of secreted substrates and identification of a conserved putative secretion signal for type III secretion systems. PLoS Pathog. 2009; 5(4):e1000375. PMC: 2668754. DOI: 10.1371/journal.ppat.1000375. View

13.

Lara-Tejero M, Galan J . The Injectisome, a Complex Nanomachine for Protein Injection into Mammalian Cells. EcoSal Plus. 2019; 8(2). PMC: 6450406. DOI: 10.1128/ecosalplus.ESP-0039-2018. View

14.

An S, Potnis N, Dow M, Vorholter F, He Y, Becker A . Mechanistic insights into host adaptation, virulence and epidemiology of the phytopathogen Xanthomonas. FEMS Microbiol Rev. 2019; 44(1):1-32. PMC: 8042644. DOI: 10.1093/femsre/fuz024. View

15.

Sory M, Boland A, Lambermont I, Cornelis G . Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. Proc Natl Acad Sci U S A. 1995; 92(26):11998-2002. PMC: 40283. DOI: 10.1073/pnas.92.26.11998. View

16.

Worrall L, Hong C, Vuckovic M, Deng W, Bergeron J, Majewski D . Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body. Nature. 2016; 540(7634):597-601. DOI: 10.1038/nature20576. View

17.

Hu J, Worrall L, Vuckovic M, Hong C, Deng W, Atkinson C . T3S injectisome needle complex structures in four distinct states reveal the basis of membrane coupling and assembly. Nat Microbiol. 2019; 4(11):2010-2019. DOI: 10.1038/s41564-019-0545-z. View

18.

Gan Y, Yang L, Yang L, Li W, Liang X, Jiang W . The C-terminal domain of the type III secretion chaperone HpaB contributes to dissociation of chaperone-effector complex in Xanthomonas campestris pv. campestris. PLoS One. 2021; 16(1):e0246033. PMC: 7842900. DOI: 10.1371/journal.pone.0246033. View

19.

Hu B, Lara-Tejero M, Kong Q, Galan J, Liu J . In Situ Molecular Architecture of the Salmonella Type III Secretion Machine. Cell. 2017; 168(6):1065-1074.e10. PMC: 5393631. DOI: 10.1016/j.cell.2017.02.022. View

20.

Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S . A modular cloning system for standardized assembly of multigene constructs. PLoS One. 2011; 6(2):e16765. PMC: 3041749. DOI: 10.1371/journal.pone.0016765. View