Bacterial ClpP Protease Is a Potential Target for Methyl Gallate

Abstract

Methyl gallate (MG) is an effective microbicide with great potential application in the integrated management of plant diseases and an important potential drug for clinical application. However, its target remains unknown. This study conducted a transposon sequencing (Tn-seq) under MG treatment in plant pathogenic bacterium Ralstonia solanacearum. Tn-seq identified that the mutation of caseinolytic protease proteolytic subunit gene clpP significantly increased the resistance of R. solanacearum to MG, which was validated by the in-frame gene deletion. iTRAQ (isobaric tags for relative and absolute quantitation) proteomics analysis revealed that chemotaxis and flagella associated proteins were the major substrates degraded by ClpP under the tested condition. Moreover, sulfur metabolism-associated proteins were potential substrates of ClpP and were upregulated by MG treatment in wild-type R. solanacearum but not in clpP mutant. Furthermore, molecular docking confirmed the possible interaction between MG and ClpP. Collectively, this study revealed that MG might target bacterial ClpP, inhibit the activity of ClpP, and consequently disturb bacterial proteostasis, providing a theoretical basis for the application of MG.

Keywords: Ralstonia solanacearum; drug target; methyl gallate; protease ClpP; transposon sequencing.

FIGURE 1

Transposon sequencing (Tn-seq) analysis of gene essentiality for methyl gallate (MG). (A) Schematic diagram of Tn-seq for the identification of resistance genes and target genes for MG. (B) Coefficient of reads coverages for Tn-seq biological replicates (scatter plot) and for all Tn-seq samples (heatmap).

FIGURE 2

ClpP is necessary for the antibacterial activity of methyl gallate (MG) against R. solanacearum. (A) Transposon insertion distribution of clpP and its flanking sequence. (B) Gradient diluted bacterial cultures were inoculated on BG agar medium with or without 25 μg/ml of MG. The growth of R. solanacearum strains was observed and photographed at certain times. (C) Growth of R. solanacearum strains cultured in BG liquid medium with or with 25 μg/ml of MG. Growth differences between mutants and the wild-type strain GMI1000 with or without treatments at 23 and 31 h post inoculation are marked with asterisks, indicating P < 0.01 (**) or P < 0.05 (*), statistically analyzed by analysis of variance (ANOVA).

FIGURE 3

Possible substrates of ClpP identified by iTRAQ (isobaric tags for relative and absolute quantitation). (A) Relative expression of proteins identified in ΔclpP compared with the wild-type (WT) strain GMI1000. The proteins with >1.5-fold expression difference between ΔclpP and WT and with P < 0.05 were defined as differentially expressed proteins. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of differentially expressed proteins of ΔclpP versus WT. The number of differentially expressed proteins is indicated by the size of the bubbles. P-values of enrichment are indicated by the color of bubbles. The ratio represents the number of proteins identified in this pathway divided by the number of differentially expressed proteins mapped to this pathway. The top six over enriched KEGG pathways were shown. (C) Relative expression of differentially expressed proteins mapped to bacterial chemotaxis, flagellar assembly, and sulfur metabolism pathways.

FIGURE 4

Differentially expressed proteins shared by ΔclpP versus wild-type (WT) and WT_MG versus WT.

FIGURE 5

Structures of ClpP binding with (4S)-2-methylpentane-2,4-diol (MPD) (PDB ID: 1YG6), benzyloxycarbonyl-leucyltyrosine chloromethyl ketone (Z-LY-CMK) (PDB ID: 2FZS), and methyl gallate (MG) (the best docking pose). Ligands were indicated by green arrows.