Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum

Abstract

Ralstonia solanacearum is one of the most lethal phytopathogens in the world. Due to its broad host range, it can cause wilting disease in many plant species of economic interest. In this work, we identified the O-oligosaccharyltransferase (O-OTase) responsible for protein O-glycosylation in R. solanacearum. An analysis of the glycoproteome revealed that 20 proteins, including type IV pilins are substrates of this general glycosylation system. Although multiple glycan forms were identified, the majority of the glycopeptides were modified with a pentasaccharide composed of HexNAc-(Pen)-dHex(3), similar to the O antigen subunit present in the lipopolysaccharide of multiple R. solanacearum strains. Disruption of the O-OTase led to the total loss of protein glycosylation, together with a defect in biofilm formation and reduced pathogenicity towards tomato plants. Comparative proteomic analysis revealed that the loss of glycosylation is not associated with widespread proteome changes. Only the levels of a single glycoprotein, the type IV pilin, were diminished in the absence of glycosylation. In parallel, disruption of glycosylation triggered an increase in the levels of a surface lectin homologous to Pseudomonas PA-IIL. These results reveal the important role of glycosylation in the pathogenesis of R. solanacearum.

Keywords: Type IV pili; biofilm; protein O-glycosylation.

Fig. 1.

Glycosylation of Neisseria protein (DsbA) by Ralstonia O-OTase (PglL_Rs) in E. coli. Whole-cell lysates of E. coli CLM24 cells expressing DsbA as an acceptor protein, together with bacillosamine and different O-OTases, were separated by SDS–PAGE and analyzed by western blotting. His-tagged DsbA was detected using the monoclonal anti-His antibody (green). The bacillosamine sugar was detected using specific antibody (red). The overlapping signals are shown in yellow (merge). Both O-OTases from Ralstonia (PglL_Rs) and Neisseria (PglL_Nm) were able to glycosylate DsbA (Lanes 2 and 3, respectively).

Fig. 2.

Major O-glycan structures identified in R. solanacearum glycoproteins. O-Glycan structures were identified using ITMS-CID fragmentation of Ralstonia glycopeptides. (A) A pentamer glycan of HexNAc-(Pen)-dHex₃ attached to ⁴⁵QAVDSASNAASQAADTAK⁶² of Q8XVC9_RALSO. (B) A heptamer glycan of HexNAc-(Pen₃)-dHex₃ attached to ¹⁴⁴AQAASSVAPSGTM(+16)SLAAK¹⁶¹ of Q8Y1X9_RALSO. Sugar monomers were represented graphically as follows: rectangles for N-acetyl hexosamine (HexNAc) units, triangles for deoxyhexose (dHex) units and stars for pentose (Pen) units.

Fig. 3.

Loss of O-glycosylation altered the levels of pilin in R. solanacearum. Whole-cell lysates of different R. solanacearum strains were run on SDS–PAGE gel followed by immunoblotting using rabbit polyclonal anti-pilin and mouse monoclonal anti-RNA polymerase (1:2500, RNAP ɑ-subunit; Neoclone). Membranes were then probed with IRDye conjugated anti-mouse and anti-rabbit antibodies and visualized on an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE).

Fig. 4.

Ralstonia O-OTase mutant is defective in biofilm formation. Different R. solanacearum strains were grown for 48 h in 96-well plates at 30°C. The formed biofilms were washed and stained with 0.1% crystal violet, then dissolved in 95% ethanol. Biofilm formation was measured spectrophotometrically by measuring absorbance at λ = 590 nm. Readings were normalized by the OD₆₀₀ values of the corresponding strains after 48 h. Biofilm production in O-OTase⁻ and complemented strains was presented relative to WT. The data shown were obtained from three independent experiments (n = 3). *P < 0.01, **P < 0.001.

Fig. 5.

O-Glycosylation may be involved in the virulence of Ralstonia towards tomato plants. Different Ralstonia strains were grown overnight at 30°C, then diluted to 10⁶ CFU/mL using sterile distilled water. Inocula concentrations were checked using serial dilution plating on BG agar. Leaf petioles were inoculated with 3 µL of the normalized inocula from the three Ralstonia strains. A set of six tomato plants were inoculated with each strain and placed in separated trays in order to prevent cross contamination. After 16 h photoperiod incubation at 28°C, only plants infected with WT and the complemented strains caused wilting symptoms. The experiment was repeated twice under the same conditions to a total of three experiments.