Barbara Schellenberg

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

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 planta. 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 Burkholderiales, 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 drugs. Homologues of SylA and GlbA synthetase genes are found in some other pathogenic bacteria, including the human pathogen Burkholderia pseudomallei, the causative agent of melioidosis. It is thus possible that these bacteria are capable of producing proteasome inhibitors of the syrbactin class.

Pseudomonas syringae Virulence Factor Syringolin A Counteracts Stomatal Immunity by Proteasome Inhibition

The peptide derivative syringolin A, a product of a mixed nonribosomal peptide and polyketide synthetase, is secreted by certain strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Syringolin A was shown to be a virulence factor for P. syringae pv. syringae B728a because disease symptoms on its host Phaseolus vulgaris (bean) were greatly reduced upon inoculation with syringolin A-negative mutants. Syringolin As mode of action was recently shown to be irreversible proteasome inhibition. Here, we report that syringolin A-producing bacteria are able to open stomata and, thus, counteract stomatal innate immunity in bean and Arabidopsis. Syringolin A-negative mutants, which induce stomatal closure, can be complemented by exogenous addition of not only syringolin A but also MG132, a well-characterized and structurally unrelated proteasome inhibitor. This demonstrates that proteasome activity is crucial for guard cell function. In Arabidopsis, stomatal immunity was salicylic acid (SA)-dependent and required NPR1, a key regulator of the SA-dependent defense pathway whose proteasome-dependent turnover has been reported to be essential for its function. Thus, elimination of NPR1 turnover through proteasome inhibition by syringolin A is an attractive hypothesis to explain the observed inhibition of stomatal immunity by syringolin A.

Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

Syrbactins, a family of natural products belonging either to the syringolin or glidobactin class, are highly potent proteasome inhibitors. Although sharing similar structural features, they differ in their macrocyclic lactam core structure and exocyclic side chain. These structural variations critically influence inhibitory potency and proteasome subsite selectivity. Here, we describe the total synthesis of syringolin A and B, which together with enzyme kinetic and structural studies, allowed us to elucidate the structural determinants underlying the proteasomal subsite selectivity and binding affinity of syrbactins. These findings were used successfully in the rational design and synthesis of a syringolin A-based lipophilic derivative, which proved to be the most potent syrbactin-based proteasome inhibitor described so far. With a Ki′ of 8.65 ± 1.13 nM for the chymotryptic activity, this syringolin A derivative displays a 100-fold higher potency than the parent compound syringolin A. In light of the medicinal relevance of proteasome inhibitors as anticancer compounds, the present findings may assist in the rational design and development of syrbactin-based chemotherapeutics.

Syrbactin class proteasome inhibitor-induced apoptosis and autophagy occurs in association with p53 accumulation and Akt/PKB activation in neuroblastoma.

Syrbactins belong to a new class of proteasome inhibitors which include syringolins and glidobactins. These small molecules are structurally distinct from other, well-established proteasome inhibitors, and bind the eukaryotic 20S proteasome by a novel mechanism. In this study, we examined the effects of syringolin A (SylA) and glidobactin A (GlbA) as well as two synthetic SylA-analogs (SylA-PEG and SylA-LIP) in human neuroblastoma (SK-N-SH), human multiple myeloma (MM1.S, MM1.RL, and U266), and human ovarian cancer (SKOV-3) cells. While all four syrbactins inhibited cell proliferation in a dose-dependent manner, GlbA was most potent in both dexamethasone-sensitive MM1.S cells (IC(50): 0.004microM) and dexamethasone-resistant MM1.RL cells (IC(50): 0.005microM). Syrbactins also inhibited the chymotrypsin-like proteasome activity in a dose-dependent fashion, and GlbA was most effective in SK-N-SH cells (IC(50): 0.015microM). The GlbA-promoted inhibition of proteasomal activity in SK-N-SH cells resulted in the accumulation of ubiquitinated proteins and tumor suppressor protein p53 and led to apoptotic cell death in a time-dependent manner. GlbA treatment also promoted the activation of Akt/PKB via phosphorylation at residue Ser(473) and induced autophagy as judged by the presence of the lipidated form of microtubule-associated protein 1 light chain 3 (LC3) and autophagosomes. Collectively, our data suggest that syrbactins belong to a new and effective proteasome inhibitor class which promotes cell death. Proteasome inhibition is a promising strategy for targeted anticancer therapy and syrbactins are a new class of inhibitors which provide a structural platform for the development of novel, proteasome inhibitor-based drug therapeutics.

Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate

BackgroundSyringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium Pseudomonas syringae pv. syringae B728a with its host plant Phaseolus vulgaris (bean), was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a second valine via a very unusual ureido group. Analysis of sequence and architecture of the syringolin A synthetase gene cluster with the five open reading frames sylA-sylE allowed to formulate a biosynthesis model that explained all structural features of the tripeptide part of syringolin A but left the biosynthesis of the unusual ureido group unaccounted for.ResultsWe have cloned a 22 kb genomic fragment containing the sylA-sylE gene cluster but no other complete gene into the broad host range cosmid pLAFR3. Transfer of the recombinant cosmid into Pseudomonas putida and P. syringae pv. syringae SM was sufficient to direct the biosynthesis of bona fide syringolin A in these heterologous organisms whose genomes do not contain homologous genes. NMR analysis of syringolin A isolated from cultures grown in the presence of NaH13CO3 revealed preferential 13C-labeling at the ureido carbonyl position.ConclusionThe results show that no additional syringolin A-specific genes were needed for the biosynthesis of the enigmatic ureido group joining two amino acids. They reveal the source of the ureido carbonyl group to be bicarbonate/carbon dioxide, which we hypothesize is incorporated by carbamylation of valine mediated by the sylC gene product(s). A similar mechanism may also play a role in the biosynthesis of other ureido-group-containing NRPS products known largely from cyanobacteria.

Syringolin A: Action on Plants, Regulation of Biosynthesis and Phylogenetic Occurrence of Structurally Related Compounds

Syringolin A, the product of a mixed non-ribosomal peptide/polyketide synthetase, is secreted by Pseudomonas syringae pv. syringae under in planta conditions and is one of the molecular determinants recognized by nonhost plant species. Spray application of syringolin A onto powdery mildew-infected wheat and Arabidopsis has the remarkable effect of reprogramming epidermal cells that are colonized by the powdery mildew fungi Blumeria graminis f. sp. tritici and Erysiphe cichoracearum, respectively, in a compatible interaction to undergo hypersensitive cell death. No hypersensitive cell death is observed if the compound is applied onto uninfected plants. Transcriptome analyses in wheat and Arabidopsis with regard to powdery mildew inoculation and/or syringolin A spraying lead to a hypothesis about how syringolin A may accomplish to induce the hypersensitive reaction (HR) in colonized cells. The model is supported by transcriptome analyis of an Arabidopsis mutant in which HR is not induced upon syringolin A spraying of powdery mildew-infected plants. Cloning of the syringolin A synthetase genes has allowed us to build a detailed model of syringolin A synthesis based on the gene structure. This model in turn enabled us to clone the genes responsible for the synthesis of glidobactins (syn. cepafungins), antibiotics with a structure related to syringolin A that were reported to have antitumor activity, from an unknown species belonging to the order Burkholderiales. Comparisons to the approximately 700 complete eubacterial genomic sequences known resulted in the identification of a small but very intriguing group of pathogenic bacteria postulated to produce glidobacting-like molecules.

Abstract 2632: Syrbactin, a novel proteasome inhibitor class, induces apoptosis and autophagy in neuroblastoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Syrbactins belong to a new class of proteasome inhibitors which include syringolins and glidobactins. These small molecules are structurally distinct from other, well-established proteasome inhibitors, and bind the eukaryotic 20S proteasome by a novel mechanism. In this study, we examined the effects of syringolin A (SylA) and glidobactin A (GlbA) as well as two synthetic SylA-analogs (SylA-PEG and SylA-LIP) in human neuroblastoma (SK-N-SH), human multiple myeloma (MM1.S, MM1.RL, and U266), and human ovarian cancer (SKOV3) cells. While all four syrbactins inhibited cell proliferation in a dose-dependent manner, GlbA was most potent in both dexamethasone-sensitive MM1.S cells (IC50: 0.004 µM) and dexamethasone-resistant MM1.RL cells (IC50: 0.005 µM). Syrbactins also inhibited the chymotrypsin-like proteasome activity in a dose-dependent fashion, and GlbA was most effective in SK-N-SH cells (IC50: 0.015 µM). The GlbA-promoted inhibition of proteasomal activity in SK-N-SH cells resulted in the accumulation of ubiquitinated proteins and tumor suppressor protein p53, and led to apoptotic cell death. Strikingly, GlbA treatment also promoted the activation of Akt/PKB via phosphorylation at residue Ser473 and induced autophagy as judged by the presence of the lipidated form of microtubule-associated protein 1 light chain 3 (LC3) and autophagosomes. Collectively, our data suggest that syrbactins belong to a new and effective proteasome inhibitor class which promotes cell death. Proteasome inhibition is a promising strategy for targeted anticancer therapy and syrbactins are a new class of inhibitors which provide a structural platform for the development of novel, proteasome inhibitor-based drug therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2632.