Proteomic and Metabolomic Analyses of Xylella fastidiosa OMV-Enriched Fractions Reveal Association with Virulence Factors and Signaling Molecules of the DSF Family

Abstract

Xylella fastidiosa releases outer membrane vesicles (OMVs) known to play a role in the systemic dissemination of this pathogen. OMVs inhibit bacterial attachment to xylem wall and traffic lipases/esterases that act on the degradation of plant cell wall. Here, we extended the characterization of X. fastidiosa OMVs by identifying proteins and metabolites potentially associated with OMVs produced by Temecula1, a Pierce’s disease strain, and by 9a5c and Fb7, two citrus variegated chlorosis strains. These results strengthen that one of the OMVs multiple functions is to carry determinants of virulence, such as lipases/esterases, adhesins, proteases, porins, and a pectin lyase-like protein. For the first time, we show that the two citrus variegated chlorosis strains produce X. fastidiosa diffusible signaling factor 2 (DSF2) and citrus variegated chlorosis-DSF (likewise, Temecula1) and most importantly, that these compounds of the DSF (X. fastidiosa DSF) family are associated with OMV-enriched fractions. Altogether, our findings widen the potential functions of X. fastidiosa OMVs in intercellular signaling and host–pathogen interactions.

Xylella fastidiosa subsp. pauca Strains Fb7 and 9a5c from Citrus Display Differential Behavior, Secretome, and Plant Virulence

“…The serine protease PspB, XF9a_00245/XF0267, and the cysteine/serine protease XF9a_00488/XF0531 were also more abundant in the secretome of strain 9a5c. XF0267 was not detected previously in the OMV-enriched fractions [49] and might be exclusive to the OMV-independent fraction. Elongation factor Tu encoded by XF9a_02482/XF2628 is also worth mentioning as, although it displayed similar levels between both strains, it is highly abundant in their secretome, possibly triggering responses to pathogen-associated molecular patterns (PAMP) in the plant host.…”

“…Most of the proteins found in the secreted samples (Table 3) were also detected in samples enriched with outer membrane vesicles of X. fastidiosa [49]. This secretion strategy is known to also interfere with adherence to surfaces besides delivering virulence factors to sites within the plant that are distant from the producing bacterial cells [39,73].…”

“…Perhaps the most intriguing result in the secretome of strain 9a5c is the 8-fold higher content of the conserved protein of unknown function XF9a_01552/XF1577 compared to strain Fb7. This protein has recently been detected in OMV-enriched samples of 9a5c, but not in Fb7 ones [49]. Another protein of unknown function with higher levels in the 9a5c secretome is (XF9a_01177/XF1287).…”

“…Surface fimbrial structures that mediate twitching motility and adhesion also rely on porins and usher proteins that promote protein export across the outer membrane during pili assembly, influencing plant colonization and biofilm formation [41,[45][46][47]. Outer membrane vesicles (OMVs) are yet another protein export system, also contributing to X. fastidiosa pathogenesis [41,[48][49][50]. Being much smaller than cells, averaging~100 nm, some virulence factors can thus be delivered by OMVs to a greater range within the host [39,47].…”

“…Being much smaller than cells, averaging~100 nm, some virulence factors can thus be delivered by OMVs to a greater range within the host [39,47]. Among those found in X. fastidiosa, OMVs are lipases/esterases, adhesins, proteases, and toxins/antitoxins [39,41,49,50]. Given the importance of these proteins to host-plant interaction, analytical methods such as mass spectrometry can provide a non-biased proteome assessment and improve our understanding of their major functions and specific players.…”