XatA, an AT-1 autotransporter important for the virulence of Xylella fastidiosa Temecula1

Abstract

Xylella fastidiosa Temecula1 is the causative agent of Pierce's disease of grapevine, which is spread by xylem-feeding insects. An important feature of the infection cycle is the ability of X. fastidiosa to colonize and interact with two distinct environments, the xylem of susceptible plants and the insect foregut. Here, we describe our characterization of XatA, the X. fastidiosa autotransporter protein encoded by PD0528. XatA, which is classified as an AT-1 (classical) autotransporter, has a C-terminal β-barrel domain and a passenger domain composed of six tandem repeats of approximately 50 amino acids. Localization studies indicate that XatA is present in both the outer membrane and membrane vesicles and its passenger domain can be found in the supernatant. Moreover, XatA is important for X. fastidiosa autoaggregation and biofilm formation based on mutational analysis and the discovery that Escherichia coli expressing XatA acquire these traits. The xatA mutant also shows a significant decrease in Pierce's disease symptoms when inoculated into grapevines. Finally, X. fastidiosa homologs to XatA, which can be divided into three distinct groups based on synteny, form a single, well-supported clade, suggesting that they arose from a common ancestor.

Keywords: Adhesin; biofilm; sharpshooter; type V secretion; virulence factor.

Figure 1

Structural features of XatA. (A). Diagram giving an overview of the structural features in XatA: the signal peptide (black), the six tandem repeats (vertical bars), and the β-barrel domain (white). The numbers identify the different tandem repeats, which are located at the following positions: 69–117 (PD0528-1), 120–169 (PD0528-2), 170–219 (PD0528-3), 236–285 (PD0528-4), 294–343 (PD0528-5), 344–393 (PD0528-6). (B). Alignment of the six repeats in the passenger domain. The six repeats were aligned by ClustalW2 (Chenna et al. 2003) and visualized by Multalin (Corpet 1988). Colors are used to identify the amount of conservation: high (red), low (blue), and neutral (black). In the consensus sequence, two additional symbols are used: # (D or N) and ! (I or V). The black bar above the alignment indicates the location of the LGxL motif.

Figure 2

Subcellular localization of XatA and the XatA passenger domain. Proteins from the different fractions were separated on a 7.5% SDS-PAGE and subjected to Western analysis using antibodies against the XatA passenger domain (αRXatA): outer membrane (OM) (lane 1), OM vesicles (OMV) (lane 2), the secreted protein fraction (1 μl; lane 3), the secreted protein fraction (7 μl; lane 4). To determine the susceptibility of XatA to protease digestion, intact Xylella fastidiosa cells were treated with proteinase K and OM proteins (OMPs) were then isolated and subjected to Western analysis: OM from untreated cells (lane 5), OM from cells after proteinase K treatment (lane 6).

Figure 3

OM profile of the xatA3 mutant. The OM proteins were separated on an 8% SDS-PAGE and then stained with SyproRuby: xatA⁺ (X. fastidiosa Temecula1), the xatA3 mutant, (TAM103), and the xatA 3/p- xatA⁺ complementation strain (TAM103/pAM61). Lane M shows the molecular weight standard labeled in KDa. The identification of the bands indicated by the arrows as the XatA protein was confirmed by MALDI-TOF mass spectrometry (data not shown).

Figure 4

Impact of XatA on biofilm formation. Cells were grown in PD3 in 18-mm glass tubes for seven days without agitation: xatA⁺ (Temecula1), xatA3 (TAM103), and xatA 3/p- xatA⁺ (TAM103/pAM61). Biofilms were stained with crystal violet and resuspended in 95% ethanol. The absorbance at 550 nm was then measured to quantify biofilm production.

Figure 5

Heterologous expression of XatA in Escherichia coli. (A). Aggregation of XatA-expressing cells. Cells were vortexed for 10 sec and incubated statically for 3.5 h at room temperature. Samples were taken from the top of the tube at 30-min intervals and the absorbance at 550 nm was determined for UT5600 containing either the vector (pBBR1MCS-5) or p- xatA⁺ (pAM61). (B). Biofilm formation by XatA-expressing cells. Cells were grown in LB in 18-mm tubes for two days without agitation. Biofilms were stained with crystal violet and resuspended in 95% ethanol. The absorbance at 570 nm was then measured to quantify biofilm production.

Figure 6

The xatA3 mutant impacts X. fastidiosa virulence in grapevines. Greenhouse-grown grapevines (cv. Thompson seedless) were inoculated using the needle puncture method with one of the following: xatA⁺ (X. fastidiosa Temecula1), xatA3 (TAM103), or water (mock infection). These photographs show representative vines 16 weeks after infection.

Figure 7

The impact of XatA on insect-mediated infection. Individual blue-green sharpshooters were allowed access to a sachet containing a diet solution and the indicated strain for 3 h: xatA⁺ (X. fastidiosa Temecula1), xatA⁺ Cm^r (TAM22), xatA3 (TAM103), and xatA 3/p- xatA⁺ (TAM103/pAM61). The insects were then individually transferred to a single leaf on a grape host and allowed to feed for four days. After 12 weeks, the leaves were collected and the petioles were examined for the presence or absence of X. fastidiosa. The results with the xatA3 mutant (diagonal bar) showed a significant difference (P < 0.05) when compared to those for other three strains (gray bars).