Deciphering the functions of type II and type IV secretion systems from the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria

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The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease on pepper and tomato plants and one of the model organisms to study secretion of bacterial virulence factors. Essential for pathogenicity of Xcv is the type III secretion system, which translocates bacterial effector proteins into eukaryotic cells. Furthermore, the efficient interaction of Xcv with its host plants depends on the Xps-type II secretion (T2S) system, which secretes degradative enzymes into the extracellular milieu. We previously showed that the T2S secretomes vary in different pathovars of X. campestris, which is in contrast to the previously anticipated conservation of bacterial T2S substrate specificities. The Xps-T2S system from Xcv secretes proteases, xylanases and a lipase, which contribute to bacterial virulence. In the absence of functional T2S systems, secretion of T2S substrates from Xcv was not completely abolished, suggesting that they are targeted to alternative transport routes. In agreement with this observation, we detected T2S substrates in outer membrane vesicles (OMVs) of Xcv. In the present project, we want to characterize the contribution of OMVs to the secretion of virulence factors from Xcv. Furthermore, we propose to investigate possible interactions between T2S substrates and components of the secretion apparatus and to identify protein regions, which are involved in substrate docking. In addition to the analysis of the T2S system and OMVs, we aim at the functional characterization of the two type IV secretion (T4S) systems of Xcv, which resemble the VirB/D4-T4S system from Agrobacterium tumefaciens and the Icm/Dot-T4S system from the animal-pathogenic bacterium Legionella pneumophila, respectively. Xcv is the only known plant-pathogenic bacterium, which contains an Icm/Dot-like T4S system. Our recent mutant studies revealed that both T4S systems contribute to the interaction of X. campestris pv. vesicatoria with susceptible pepper plants. Furthermore, bioinformatic approaches led to the identification of candidate T4S substrates, which are homologous to known T4S effectors and/or contain conserved C-terminal amino acid motifs, which presumably serve as secretion signals. In the present project, we will study secretion and translocation of candidate T4S substrates from Xcv and their potential contribution to bacterial virulence. Furthermore, given the recent report on the role of the VirB/D4-like T4S system from the related pathogen Xanthomonas axonopodis pv. citri in bacterial killing, we will investigate a possible role of T4S systems in the interaction of Xcv with other bacterial cells.