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Isolation and Characterization of Outer Membrane Vesicles of Pectobacterium brasiliense 1692.

ABSTRACT: Pectobacterium brasiliense (Pbr) 1692 is an aggressive phytopathogen affecting a broad host range of crops and ornamental plants, including potatoes. Previous research on animal pathogens, and a few plant pathogens, revealed that Outer Membrane Vesicles (OMVs) are part of Gram-negative bacteria's (GNB) adaptive toolkit. For this reason, OMV production and subsequent release from bacteria is a conserved process. Therefore, we hypothesized that OMVs might transport proteins that play a critical role in causing soft rot disease and in the survival and fitness of Pbr1692. Here, we show that the potato pathogen, Pbr1692, releases OMVs of various morphologies in Luria Bertani media at 31 °C. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) confirmed the production of OMVs by Pbr1692 cells. Transmission Electron Microscopy showed that these exist as chain-, single-, and double-membrane morphologies. Mass spectrometry followed by Gene Ontology, Clusters of Orthologous Groups, Virulence Factor, CAZymes, Antibiotic Resistance Ontology, and Bastion6 T6SE annotations identified 129 OMV-associated proteins with diverse annotated roles, including antibiotic stress response, virulence, and competition. Pbr1692 OMVs contributed to virulence in potato tubers and elicited a hypersensitive response in Nicotiana benthamiana leaves. Furthermore, Pbr1692 OMVs demonstrated antibacterial activity against Dickeya dadantii.

SUBMITTER: Maphosa S

PROVIDER: S-EPMC8469291 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Among bacterial cell envelopes, the Borrelia burgdorferi outer membrane (OM) is structurally unique in that the identities of many protein complexes remain unknown; however, their characterization is the first step toward our understanding of membrane protein interactions and potential functions. Here, we used two-dimensional blue native/SDS-PAGE/mass spectrometric analysis for a global characterization of protein-protein interactions as well as to identify protein complexes in OM vesicles isolated from multiple infectious sensu stricto isolates of B. burgdorferi. Although we uncovered the existence of at least 10 distinct OM complexes harboring several unique subunits, the complexome is dominated by the frequent occurrence of a limited diversity of membrane proteins, most notably P13, outer surface protein (Osp) A, -B, -C, and -D and Lp6.6. The occurrence of these complexes and specificity of subunit interaction were further supported by independent two-dimensional immunoblotting and coimmunoprecipitation assays as well as by mutagenesis studies, where targeted depletion of a subunit member (P66) selectively abolished a specific complex. Although a comparable profile of the OM complexome was detected in two major infectious isolates, such as B31 and 297, certain complexes are likely to occur in an isolate-specific manner. Further assessment of protein complexes in multiple Osp-deficient isolates showed loss of several protein complexes but revealed the existence of additional complex/subunits that are undetectable in wild-type cells. Together, these observations uncovered borrelial antigens involved in membrane protein interactions. The study also suggests that the assembly process of OM complexes is specific and that the core or stabilizing subunits vary between complexes. Further characterization of these protein complexes including elucidation of their biological significance may shed new light on the mechanism of pathogen persistence and the development of preventative measures against the infection.

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Isolation and Characterization of Outer Membrane Vesicles of Pectobacterium brasiliense 1692.

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