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A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism

Abstract

Pathogenic bacteria often use effector molecules to increase virulence. In most cases, the mode of action of effectors remains unknown. Strains of Pseudomonas syringae pv. syringae (Pss) secrete syringolin A (SylA), a product of a mixed non-ribosomal peptide/polyketide synthetase, in planta1. Here we identify SylA as a virulence factor because a SylA-negative mutant in Pss strain B728a obtained by gene disruption was markedly less virulent on its host, Phaseolus vulgaris (bean). We show that SylA irreversibly inhibits all three catalytic activities of eukaryotic proteasomes, thus adding proteasome inhibition to the repertoire of modes of action of virulence factors. The crystal structure of the yeast proteasome in complex with SylA revealed a novel mechanism of covalent binding to the catalytic subunits. Thus, SylA defines a new class of proteasome inhibitors that includes glidobactin A (GlbA), a structurally related compound from an unknown species of the order Burkholderiales2, for which we demonstrate a similar proteasome inhibition mechanism. As proteasome inhibitors are a promising class of anti-tumour agents, the discovery of a novel family of inhibitory natural products, which we refer to as syrbactins, may also have implications for the development of anti-cancer drugs3. Homologues of SylA and GlbA synthetase genes are found in some other pathogenic bacteria, including the human pathogen Burkholderia pseudomallei, the causative agent of melioidosis4. It is thus possible that these bacteria are capable of producing proteasome inhibitors of the syrbactin class.

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Figure 1: Syringolin-negative mutant exhibits reduced virulence.
Figure 2: SylA inhibits the eukaryotic proteasome.
Figure 3: Structural basis for proteasome inhibition by syrbactins.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates have been deposited in the RCSB Protein Data Bank (http://www.rcsb.org/pdb) under the accession code 2ZCY (yeast 20S proteasome–syringolin A complex) and 3BDM (yeast 20S proteasome–glidobactin A complex).

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Acknowledgements

We thank U. Grossniklaus and J. Celenza (Boston University) for the CycB1;1-GUS line. U. Grossniklaus and H. Gehring are thanked for reading the manuscript. We also thank D. Albert, H. Gehring, Z. Hasenkamp, R. Go, D. Koomoa, J. Molnar, A. Niewienda and C. Wallick for technical advice and assistance. We are grateful to L. Eberl for use of equipment. C.R.A was supported by a graduate student research assistantship from the Cell and Molecular Biology Graduate Program, University of Hawaii. Support by grants from the Swiss National Science Foundation to R.D. is acknowledged.

Author Contributions M.K., M.G., S.L., R.D. and A.S.B. designed experiments, analysed results and wrote the manuscript. R.D. constructed the sylA-negative mutant, T.K.P. performed the bean inoculation experiments, B.S. isolated SylA and GlbA, and performed in vitro proteasome and protease inhibition assays. M.G., M.K and R.H. performed and analysed crystallization experiments. C.R.A. performed the mammalian cell-based proteasome inhibition assays and immunoblot studies.

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Correspondence to André S. Bachmann, Markus Kaiser or Robert Dudler.

Supplementary information

The file contains Supplementary Figures 1 - 4 with Legends, Supplementary Table 1 and additional references.

The Supplementary Figure 1 shows results confirming that SylA inhibits proteasomes irreversibly. The Supplementary Figure 2 shows that SylA inhibits bean proteasomes in crude extracts of etiolated bean seedlings. The Supplementary Figure 3 exhibits root tips of Arabidopsis seedlings homozygous for a CyclinB1;1-uidA (GUS) reporter fusion gene stained for GUS activity, showing that SylA treatment leads to enhanced staining of dividing cells. This indicates that the fusion gene product, which is expressed only in the G2/M transition of the cell cycle and then is degraded by the proteasome, accumulates upon SylA treatment. The Supplementary Figure 4 shows the results of in vivo proteasome inhibition assays in cultured human cells using the known proteasome inhibitor epoxomicin and the apoptosis-inducing agent cerulenin as positive and negative control agents, respectively. The Supplementary Table 1 lists statistical parameters of crystallographic data collection and refinement. (PDF 484 kb)

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Groll, M., Schellenberg, B., Bachmann, A. et al. A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism. Nature 452, 755–758 (2008). https://doi.org/10.1038/nature06782

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