Agrobacterium tumefaciens Small Lipoprotein Atu8019 Is Involved in Selective Outer Membrane Vesicle (OMV) Docking to Bacterial Cells

Abstract

Outer membrane vesicles (OMVs), released from Gram-negative bacteria, have been attributed to intra-and interspecies communication and pathogenicity in diverse bacteria. OMVs carry various components including genetic material, toxins, signaling molecules, or proteins. Although the molecular mechanism(s) of cargo delivery is not fully understood, recent studies showed that transfer of the OMV content to surrounding cells is mediated by selective interactions. Here, we show that the phytopathogen Agrobacterium tumefaciens, the causative agent of crown gall disease, releases OMVs, which attach to the cell surface of various Gram-negative bacteria. The OMVs contain the conserved small lipoprotein Atu8019. An atu8019-deletion mutant produced wildtype-like amounts of OMVs with a subtle but reproducible reduction in cell-attachment. Otherwise, loss of atu8019 did not alter growth, susceptibility against cations or antibiotics, attachment to plant cells, virulence, motility, or biofilm formation. In contrast, overproduction of Atu8019 in A. tumefaciens triggered cell aggregation and biofilm formation. Localization studies revealed that Atu8019 is surface exposed in Agrobacterium cells and in OMVs supporting a role in cell adhesion. Purified Atu8019 protein reconstituted into liposomes interacted with model membranes and with the surface of several Gram-negative bacteria. Collectively, our data suggest that the small lipoprotein Atu8019 is involved in OMV docking to specific bacteria.

Biophysical and proteomic analyses suggest functions ofPseudomonas syringaepvtomatoDC3000 extracellular vesicles in bacterial growth during plant infection

“…Bacterial EVs contain immunogenic molecules such as EF-Tu and LPS, and can contain digestive enzymes and effectors that undermine host defences (Bahar et al , 2016, Feitosa-Junior et al , 2019, Knoke et al , 2020, Rybak & Robatzek, 2019). To determine the effect of EVs from Pto DC3000 on plant cells, we first examined the ability of the Pto DC3000 EVs to modulate the outcome of bacterial infection.…”

“…Previous studies revealed a number of molecular cargoes present in EVs from phytopathogenic bacteria of the Agrobacterium tumefaciens, P. syringae, Xanthomonas campestris and Xylella fastidiosa species. These EV-associated proteins include degradative enzymes, Type-II-secreted virulence-associated proteins, components of the Type-III secretion system and its secreted proteins (Feitosa-Junior et al , 2019, Chowdhury & Jagannadham, 2013, Knoke et al , 2020, Nascimento et al , 2016, Sidhu et al , 2008, Sole et al , 2015). While respective genetic deletions of EV-associated degradative enzymes reduced bacterial virulence, the role of EVs in their delivery remained unanswered (Nascimento et al , 2016, Sidhu et al , 2008, Sole et al , 2015).…”

Membrane Vesicle Production as a Bacterial Defense Against Stress

“…pathway (Kaplan et al, 2018), after which it remains at the periplasmic site or is transported to the cell surface (Knoke et al, 2020). Stress can alter the structure of peptidoglycan or Lpp, leading to membrane blebbing due to the loss of membrane integrity; the balance between peptidoglycan turnover and elements that preserve membrane integrity through lipoprotein crosslinking determines the degree of membrane vesiculation (Martins et al, 2019;Ultee et al, 2019).…”

“…After their export through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory (Sec) pathway, the proteins are modified with lipids at the cytoplasmic membrane in a multistep process ( Zückert, 2014 ). Depending on N -acylation and specific sorting signals, Lpp (the best known crosslinked lipoprotein) either remains in the inner membrane or is translocated to the inner leaflet of the outer membrane via the Lpp outer membrane localization pathway ( Kaplan et al, 2018 ), after which it remains at the periplasmic site or is transported to the cell surface ( Knoke et al, 2020 ). Stress can alter the structure of peptidoglycan or Lpp, leading to membrane blebbing due to the loss of membrane integrity; the balance between peptidoglycan turnover and elements that preserve membrane integrity through lipoprotein crosslinking determines the degree of membrane vesiculation ( Martins et al, 2019 ; Ultee et al, 2019 ).…”

“…Stress can alter the structure of peptidoglycan or Lpp, leading to membrane blebbing due to the loss of membrane integrity; the balance between peptidoglycan turnover and elements that preserve membrane integrity through lipoprotein crosslinking determines the degree of membrane vesiculation ( Martins et al, 2019 ; Ultee et al, 2019 ). Moreover, the presence of diacylated Lpp induces specific attachment and internalization of OMVs by target bacteria ( Knoke et al, 2020 ).…”