Action of Dicumarol on Glucosamine-1-Phosphate Acetyltransferase of GlmU and Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis is one of most pathogenic microorganisms in the world. Previously, the bifunctional enzyme GlmU with glucosamine-1-phosphate acetyltransferase activity and N-acetylglucosamine-1-phosphate uridyltransferase activity has been suggested as a potential drug target; therefore, discovering compounds targeting GlmU acetyltransferase is necessary. The natural products were tested for inhibition of GlmU acetyltransferase activity. We found that dicumarol exhibited inhibitory effects on GlmU acetyltransferase, with a concentration achieving a 50% inhibition (IC₅₀) value of 4.608 μg/ml (13.7 μM). The inhibition kinetics indicated that dicumarol uncompetitively inhibited acetyl CoA and showed mixed-type inhibition for glucosamine-1-phosphate (GlcN-1-P). The activity of dicumarol against M. tuberculosis H37Ra was evaluated with a minimum inhibitory concentration (MIC) value of 6.25 μg/ml (18.55 μM) in the Alamar blue assay. Dicumarol also exhibited inhibitory effects on several clinically sensitive M. tuberculosis strains and drug-resistant strains, with a range of MIC value of 6.25 to >100 μg/ml. Dicumarol increased the sensitivity of anti-tuberculosis drugs (isoniazid and rifampicin) when dicumarol was present at a low concentration. The transcriptome and proteome data of M. tuberculosis H37Ra treated by dicumarol showed that the affected genes were associated with cell wall synthesis, DNA damage and repair, metabolic processes, and signal transduction. These results provided the mechanism of dicumarol inhibition against GlmU acetyltransferase and M. tuberculosis and also suggested that dicumarol is a potential candidate for TB treatment.

Keywords: GlmU; Mycobacterium tuberculosis; acetyltransferase; cell wall; dicumarol; inhibitor.

FIGURE 1

GlmU acetyltransferase assay and inhibitor screening. The first reaction is catalyzed by the acetyltransferase activity at the C-terminal domain, and the second reaction is catalyzed by uridylyltransferase at the N-terminal domain of GlmU (A). The compounds were tested to screen the inhibitors of GlmU acetyltransferase activity (B). The structure of dicumarol (C). The best hit was compound 22 (dicumarol) with an inhibition >80%. The inhibitory effect of dicumarol on the growth of M. tuberculosis H37Ra (D).

FIGURE 2

Mode of action of dicumarol inhibition. Parallel lines on a Lineweaver–Burke plot represented different dicumarol concentrations. The inhibition type was uncompetitive inhibition for acetyl CoA (A). The lines of the different dicumarol concentrations merged at the second quadrant, suggesting a mixed type of inhibition for GlcN-1-P (B).

FIGURE 3

Susceptibility testing of dicumarol on bacterial growth. The inhibitory effect of dicumarol on the growth of M. tuberculosis H37Ra/pVV2 (∙) and M. tuberculosis H37Ra/pVV2-glmU (■) (A). The absorbance was measured for the combination treatment of dicumarol and INH (B) or RFP (C) against M. tuberculosis H37Ra. INH/RFP treated alone (●), and INH/RFP combined with dicumarol (■).

FIGURE 4

Anti-M. tuberculosis activity of dicumarol in M. tuberculosis-infected macrophages. Fluorescence microscope images of M. tuberculosis GFP-H37Ra-infected RAW264.7 cells (MOI = 10) following treatment with dicumarol (a = 0 μg/ml, b = 6.25 μg/ml, c = 12.5 μg/ml) for 24 h (A). Blue: nuclei, Green: green fluorescent protein overexpressed in M. tuberculosis H37Ra/pCG-GFP. The percentage of bacteria in living cells was calculated by fluorescence microscope images (B). Following dicumarol treatment to the infected-RAW264.7 cell, the RAW264.7 cells were lysed, and intracellular M. tuberculosis burden was determined by a CFU assay (C). Data represent the mean ± SEM of triplicate samples (n = 3). ^*p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^****p < 0.0001 compared to that of untreated cell.

FIGURE 5

Fluorescence microscope images of membrane permeabilization. M. tuberculosis H37Ra was observed with red fluorescence of permeable PI when the integrity of the cell walls was destroyed. Untreated M. tuberculosis H37Ra (A) showed obvious red fluorescence than treated with dicumarol (12.5 μg/ml) (B).

FIGURE 6

Transcriptional changes in M. tuberculosis H37Ra in response to dicumarol treatment. Differentially expressed genes (DEGs) were in M. tuberculosis H37Ra in the absence and presence of dicumarol (12 μg/ml) (A). Gene Ontology (GO) analyzed the dicumarol-regulated genes (B). The histogram sets for each functional category generated from DEGs according to the enriched gene counts. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyzed the dicumarol-regulated genes (C). The list of DEGs is shown in Supplementary Tables S1, S2. WT, non-treatment; DLC, dicumarol treatment.