Niclosamide Blocks Rice Leaf Blight by Inhibiting Biofilm Formation of Xanthomonas oryzae

Abstract

Rice (Oryza sativa) is the leading source of nutrition for more than half of the world's population, and by far it is the most important commercial food crop. But, its growth and production are significantly hampered by the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) which causes leaf blight disease. Earlier studies have reported the antibacterial ability of FDA-approved niclosamide drug against Xoo. However, the underlying mechanism by which niclosamide blocks the growth of Xoo remained elusive. In the present study, by employing the microbiological, microscopical, molecular, bioinformatics and analytical tools we found that niclosamide can directly inhibit the growth of the Xoo by hampering the biofilm formation and the production of xanthomonadin and exopolysaccharide substances (EPS) required for relentless growth and virulence of Xoo. Interestingly, niclosamide was found to specifically suppress the growth of Xoo without affecting other bacteria like Escherichia coli. Our electron microscopic observations disclosed that niclosamide disrupts the membrane permeability of Xoo and causes the release of intracellular components. Similarly, the molecular docking analysis disclosed the molecular interaction of niclosamide with the biofilm, virulence and quorum sensing related proteins, which was further substantiated by relative gene expression analysis where niclosamide was found to significantly downregulate the expression of these key regulatory genes. In addition, considerable changes in chemical structures were detected by Fourier Transform Infrared Spectroscopy (FTIR) in response to niclosamide treatment. Overall, our findings advocate the utilization of niclosamide as a safe and potent alternative antibacterial compound to control bacterial blight disease in rice.

Keywords: Xanthomonas oryzae; bacterial blight disease; biofilm; extracellular polysaccharide; niclosamide; plant–pathogen interaction; rice.

FIGURE 1

Niclosamide perturbs the growth of Xanthomonas oryzae pv. oryzae (Xoo) and blocks the progression of bacterial blight disease in rice. (A,B) Inhibition rate of niclosamide against Xoo. Firstly, the Xoo was grown up to the logarithmic phase in nutrient medium (NM), and then it was treated with increasing concentration of niclosamide and incubated at 28°C at 180 rpm in shaking incubator for 24–48 h and the growth of the cultures were monitored on a microplate reader by measuring the optical density at 595 nm. Results are means of three technical replicates and the bar indicates the standard deviation. The experiment was repeated three times with similar results. (C) Microscopic observation of crystal violet stained Xoo grown in the presence or absence of 5 µg/ml niclosamide. (40X, scale bar = 100 µm). (D) Assessment of cell viability and ROS production by XTT assay. The log phase grown bacterial dispersions were treated with 5 µg/ml niclosamide and immediately mixed with 1 mL of 0.4 mM XTT. The changes in absorbance at 470 nm were monitored with a microplate reader at the indicated time points. (E,F) Significant reduction in bacterial blight disease by niclosamide. The fully expanded 4th and 5th leaves staged rice seedlings were inoculated by Xoo bacterial suspensions (OD600 = 0.5) by leaf-tip-clipping method. The rice plants were sprayed with 10 µg/ml niclosamide at 4-day intervals (Kim et al., 2016b). Plants inoculated with the 0.02% Tween-20 solution were used as mock-inoculated controls. The lesion length was measured after 14 days of inoculation. Values are means ± SD from triplicate biological repeats. Significant differences were determined by a Post Hoc Test (p < 0.05) using different letters. Bar = 1 cm.

FIGURE 2

Niclosamide restricts the developmental of biofilm in Xoo. (A) Upper panel shows the growth of bacteria (Xoo and E. coli) in nutrient agar medium after 72 h of incubation in the presence or absence of 5 µg/ml niclosamide. Turbid and opaque tubes indicate bacterial growth while translucent ones represent the growth inhibition. The bottom panel depicts the crystal violet stained adherent biofilms on glass test tubes. (B) The percentage of biofilm inhibition by niclosamide drug. (C) Reduction in xanthomonadin activity after niclosamide treatment. (D) Inhibition rate of exopolysaccharide substance (EPS) production by niclosamide. (E) Biofilm formation by Xoo is hampered by Niclosamide. Xoo were grown on cover slides with or without 5 µg/ml niclosamide, and were stained with fluorescein diacetate (FDA) prior to observation under confocal laser scanning microscopy. The left panel shows the Xoo cells while right panel shows the 2.5D stacked image of biofilm growth at the indicated time points. The height represents the FDA signal intensity representing the bacterial density and biofilm thickness. Scale bar = 2 µm. All the experiments were repeated thrice using independent samples with similar results. Values are means ± SD from triplicate biological repeats. Significant differences were determined by a Post Hoc Test (^∗p < 0.05 and ^∗∗p < 0.01).

FIGURE 3

Niclosamide causes ultrastructural modifications in Xoo. Scanning electron microphotographs of Xoo treated with sterile distilled water (control) and with 5 µg/ml niclosamide. Black arrow indicates the damage caused by niclosamide drug. Bar = 1 µm.

FIGURE 4

Niclosamide disrupts the membrane permeability of Xoo, and causes the release of intracellular components. Transmission electron microphotographs of Xoo treated with 5 µg/ml niclosamide or sterile distilled water (control). Bar = 500 nm.

FIGURE 5

Molecular docking reveals the physical interaction of niclosamide with the biofilm and quorum sensing related proteins (A) GumB, (B) RpfB 3D and (C) XanA. Left panel shows the 3D interaction pose of niclosamide in the active site of the respective proteins. While, right panel shows the 2D view of intermolecular H bonding interaction and neighboring residues.

FIGURE 6

Niclosamide downregulates the expression of biofilm, EPS and quorum sensing related genes, and causes substantial changes in chemical structures of Xoo. (A) qRT-PCR analysis showing the differential gene expression in Xoo in the presence or absence of 5 µg/ml niclosamide. Only “Xoo” served as control while “Xoo+ niclosamide” represents the treatment in the figure. gyrB was used as an endogenous control. Gene expression values are presented relative to untreated Xoo (set as 1). Results are means of three technical replicates and the bar indicates the standard deviation. Asterisk indicates ^∗∗P < 0.01 and ^∗P < 0.05. (B) Fourier transform infrared spectroscopy (FTIR) showing the chemical modifications in Xoo after 12 h of niclosamide (5 µg/ml) treatment. Dashed red line indicates the shift in the IR spectra compared to untreated samples (solid black line).

FIGURE 7

DNA cleavage analysis in response to niclosamide treatment. DNA was extracted from log-phase grown PXO99 and E. coli. After quantification the concentration was adjusted to 140 ng/µl, and 5 µl of DNA was mixed with 5 µg/ml or 5 mg/ml of niclosamide and incubated at 28°C for 30 min. The bands were visualized in 1% agarose gel electrophoresis buffered with 1X TAE buffer.

FIGURE 8

The proposed model depicting the mechanism of Xoo’s biofilm disruption by niclosamide drug and its utility to protect rice plants from highly destructive bacterial blight disease caused by Xoo. The spherical red dot denotes the niclosamide drug. (This illustrative model was drawn in Edraw Max software, Version 9.0).