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.

Membrane Vesicles of Pectobacterium as an Effective Protein Secretion System

“…Our results were similar to those previously reported for Pectobacterium, where crude MVs were obtained with different isolation methods and different growth media [29,30]. Several outer membrane markers were found, including Omp proteins, the major outer membrane lipoprotein Lpp, and elongation factors.…”

“…Many of the observed large (100-300 nm) vesicles in the TEM images were similar to O-IMVs. O-IMVs were also observed in [29,30], which may suggest that they are commonly produced by bacteria.…”

“…First characterisations of the MVs of Pectobacterium were published in 2020 and 2021 [29,30]. To date, the MVs production was reported for P. atrosepticum [34], P. betavasculorum [29], and P. brasiliense [30]. In our work, we characterise MVs of three other Pectobacterium species: P. zantedeschiae, P. ordoriferum and P. versatile.…”

“…MVs production in Pectobacterium was first reported in 1992 [34]. First characterisations of the MVs of Pectobacterium were published in 2020 and 2021 [29,30]. To date, the MVs production was reported for P. atrosepticum [34], P. betavasculorum [29], and P. brasiliense [30].…”

“…MVs also seem to be the alternate delivery route for extracellular enzymes excreted through T2SS and possibly type III (T3SS) secretion systems [25,26,28]. Recently, the protein content of Pectobacterium MVs has been described for P. betavasculorum [29] and P. brasiliense [30]. The analyses revealed a presence of several proteins involved in a host-pathogen interplay, including proteases, pectate lyases and oligoglycan transporter (KdgM).…”