The roles of pilG in cell growth, attachment, biofilm formation, and pathogenicity pilG-knock-out strain XfΔpilG and its complemented strain XfΔpilG-C were obtained as described previously (14). The expression of pilG was not detected in XfΔpilG but was in complemented XfΔpilG-C (Data not shown). No significant difference in cell growth was observed between wild-type, XfΔpilG and XfΔpilG-C strains grown in liquid culture. The growth curves of the XfΔpilG mutant and
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complemented XfΔpilG-C strains paralleled wild-type, all three strains showed similar growth curves suggesting that deletion of pilG does not affect cell growth under rich cultural media. However, bacterial populations of mutant XfΔpilG strain are significantly lower than that of wild type and complemented strains in infected grapevines, indicating mutation causes the reduction of fitness in host. In vitro study showed that X. fastidiosa and XfΔpilG-C strains attached to the inner surface of walls of the tubes and formed a wide ring whereas no cell-attached ring was observed in XfΔpilG cells (Fig. 1A). The biofilm formation of XfΔpilG was about 5-6 fold less than that of wild-type and XfΔpilG-C strain (P<0.01) (Fig. 1B).
In planta pathogenicity assessment further confirmed that grapevines inoculated with XfΔpilG-C developed typical PD symptoms with a severity comparable to wild type. In contrast, grapevines inoculated with XfΔpilG exhibited no visible symptoms in greenhouse experiments (Fig. 2). The titer of X. fastidiosa was well correlated with the severity of disease symptoms. Grapevines inoculated by mutant strain had significantly lower of Xf populations than those inoculated by wt while complemented strains showed similar levels to wt (data not shown). Twitching motility is one of the important virulence factors. Several X. fastidiosa twitching motility-associated mutants have been reported (7, 9). Most of these were found only in partial reduction in virulence and PD symptoms (1, 9). In this study, however, we found that the pathogenicity was completely knocked-out in XfΔpilG. To this regard, based on our in vitro and in planta data we conclude that pilG could have critical roles involving multiple regulatory functions and pathogenicity, therefore, it is a central virulence factor in mediating PD development.
Effect of anti-virulence molecules on virulence
Many pathogenic bacteria use a conserved membrane histidine sensor kinase (QseC) to respond to external signals or stimulus in order to promote the expression of virulence factors. Rasko et al (11) identified a small molecule that inhibits the binding of signals to QseC and prevented its autophosphorylation and consequently inhibited QseC-mediated activation of virulence gene expression. This type of small molecule usually is not toxic but specifically disrupt functional domain of the target genes. In addition, such small molecules (< 500 Daltons) are ready to be delivered into the cells.
Since molecules only target to functional domains of virulence genes they don’t likely impose selection pressure on cell growth. In addition, such molecules usually do not
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repress pathogen growth but selectively inhibit the target virulence of pathogens in vitro and in vivo in animal (11). Similarly, several studies demonstrated that small molecules inhibitors had functional roles in inhibiting the pilus assembly and suppressing bacterial motility (8, 13, 17). For example, the inhibition of motility with phenamil in V. cholera has been shown to have effects on virulence gene expression and mitigation of the disease development (17). Consequently, these findings suggest that small molecules inhibitors could exert anti-virulence action on virulence traits of pathogens. Results from our study demonstrated that pilG mutant exhibited deficiency in twitching motility, reduction in biofilm formation, and virulence (14). To examine whether antivirulence molecules are capable of disrupting twitching motility and therefore disarm pathogenicity of X. fastidiosa, we constructed a custom chemical library (ChemBridge Corp, San Diego, USA) consisting of two thousand putatively small molecule inhibitors.
We examined the peripheral fringe of cell morphology, an indication of the capability of type IV pilus-mediated twitching motility (14), and assessed the inhibitory effect of small molecules on the peripheral fringe morphologies of X. fastidiosa. From chemical library screening, we have identified several compounds that showed promising inhibitory effects on bacterial twitching motility. For example, one of the compounds, DL-3-Amino butyric acid exerts notable effective inhibition on peripheral fringes at a concentration of as low as 5µM. (Fig. 3). A microfluidic chamber time-lapse recording system further confirmed the suppression of twitching motility treated by selected anti-virulence molecules (data not shown).
To further confirm the effect of small molecular inhibitors on the pathogenicity of X. fastidiosa, greenhouse-grown X. fastidiosa-infected tobacco plants were used for pathogenicity assay. Plants were mechanically inoculated with X. fastidiosa. Two weeks post-inoculation, plants in treatment group were foliar-sprayed with selected inhibitor compounds while plants in control group were sprayed with water. Spraying continued once a week for 4 weeks. Our results indicated that leaves of tobacco plants developed chlorosis and necrosis five weeks post inoculation with X. fastidiosa and symptoms continued up to 12 weeks while plants treated with DL-3-Amino butyric acid showed significantly alleviated symptoms (Fig. 4A) indicating treatment of virulence inhibitor mitigate X. fastidiosa infection and disease development. Anti-virulence treatment also resulted in lower bacterial titers compared to those of untreated tobacco
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plants (Figure. 4B). These results are consistent with the data obtained from in vitro evaluations.
CONCLUSIONS
X. fastidiosa possesses a multitude of virulence factors that enable the pathogen to invade to and disseminate of pathogens in host and subsequently cause systemic infections. The virulence factors including cell-to-cell auto-aggregation, biofilm formation, and twitching motility are critical requirements for pathogenicity of X.
fastidiosa (5, 10, 15) (Purcell and Saunders 1999; Hopkins 1989; Simpson et al. 2000). A conventional strategy to combat bacterium-associated diseases is to kill bacteria by antibiotics or bactericides which directly disrupt essential metabolic pathways such as preventing protein synthesis, cell wall synthesis and DNA replication (12). While these approaches are usually effective, they impose strong selection pressure on bacteria which facilitates the selection for development of antibiotic resistance populations (12). Anti-virulence molecular approach specifically targets bacterial virulence genes or factors and selectively disarm of bacterial pathogenicity without affecting bacterial survival. This approach is unlikely to impose strong selection pressure on bacteria for the development of resistance (12). Taking these results together, the application of virulence-target-based strategy provides a novel means for controlling X. fastidiosa or other bacterium-associated economically significant crop diseases.
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2. Cursino, L., Cheryl, D., Galvani, D. A., Zaini, P. A., Li, Y., De La Fuente, L., Hoch, H. C., Burr, T. J., & Mowery, P. 2011. Identification of an operon, Pil-Chp,that controls twitching motility and virulence in Xylella fastidiosa. Mol. Plant Microbe In. 24(10):1198-1206.
3. Ferandez, A., Hawkins, A. C., Summerfield, D. T., & Harwood, C. S. 2002. Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response. J. Bacteriol. 184(16):4374-4383.
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The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulatine adenylate cyclase activity. Mol. Microbiol. 76(4):889-904.
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8. Mehta, A. S., Snigdha, K., Potukuchi, M. S., and Tsonis, P. A. 2015. Comparative sequence- and structure-inspired drug design for PilF protein of Neisseria meningitides. Hum Genomics. 9(1):5.
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10. Purcell, A.H. and Saunders, S. R. 1999. Glassy-winged sharpshooters expected to increase plant disease. Calif. Agr. 53(2):26-27.
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K., Williams, N., Taussig, R., Wei, S., Roth, M., Hughes, D. T., Huntley, J. F., Fina, M. W., Falck, J. R., Sperandio V. 2008. Targeting QseC signaling and virulence for antibiotic development. Science 5892:1078-1080.
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ACKNOWLEDGMENTS
This work was supported by the United States Department of Agriculture, Agricultural Research Service. “Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer”.
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Fig. 1. A. Representative cell attachment of X. fastidiosa wild type, XfΔpilG and XfΔpilG-C in PD2 broth. X. fastidiosa wt cells showed an attached ring inside wall of the glass tube, no ring was formed in XfΔpilG cells under the same cultural conditions. Complemented strain XfΔpilG-C restored ring formation. Xf-free PD2 medium was served as a negative control. B. Quantitative measurement of biofilm formation of X. fastidiosa wild type, XfΔpilG and XfΔpilG-C trains. Data are the average of three replications with error bars indicating standard deviation. Bars with the same lowercase letter are not significantly different (P< 0.01). The experiments were repeated three times.
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Fig. 2. Pathogenicity assays on Chardonnay grapevines inoculated with PBS (negative control), X. fastidiosa wild type, XfΔpilG and XfΔpilG-C in the greenhouse, respectively. Twenty weeks post-inoculation grapevines inoculated with wt and XfΔpilG-C developed typical PD systems while vines infected with XfΔpilG showed very mild or no symptoms. The experiments were repeated three times with at least six plants per treatment group.
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Fig. 3. The peripheral fringes were observed in X. fastidiosa colonies grown on PD2 agar medium while pilG mutant Xf∆pilG showed smooth colony morphologies. When PD2 medium was supplemented with 5µM, 10µM, 20µM and 25 µM of small molecular inhibitor SM01 (DL-3-Amino butyric acid), no peripheral fringes were observed on X.
fastidiosa colonies. In contrast, the effective concentrations on suppression of peripheral fringe structure were observed on PD medium supplemented with at least 25µM Kanamycin. The experiments were repeated three times.
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Fig. 4. Pathogenicity evaluation on tobacco plants inoculated with X. fastidious. A. Progressive development of leaf symptoms on the experimental tobacco plants 5 weeks and 12 weeks after inoculation with X. fastidiosa and foliar-sprayed with SM01(DL-3-Amino butyric acid), water or Kanamycin, respectively, Foliar spray was conducted once a week at 50μM for four weeks in the greenhouse. Tobacco plants developed disease symptoms from mild to severe while treatment groups showed alleviated symptoms. Greenhouse experiments were repeated three times. B) X. fastidiosa titers of tobacco leaves were estimated by ELISA two months post-inoculation. Data were means from five replications. Different letters indicate statistical significance (P < 0.05).
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