A simplified curcumin targets the membrane of Bacillus subtilis

Abstract

Curcumin is the main constituent of turmeric, a seasoning popularized around the world with Indian cuisine. Among the benefits attributed to curcumin are anti-inflammatory, antimicrobial, antitumoral, and chemopreventive effects. Besides, curcumin inhibits the growth of the gram-positive bacterium Bacillus subtilis. The anti-B. subtilis action happens by interference with the division protein FtsZ, an ancestral tubulin widespread in Bacteria. FtsZ forms protofilaments in a GTP-dependent manner, with the concomitant recruitment of essential factors to operate cell division. By stimulating the GTPase activity of FtsZ, curcumin destabilizes its function. Recently, curcumin was shown to promote membrane permeabilization in B. subtilis. Here, we used molecular simplification to dissect the functionalities of curcumin. A simplified form, in which a monocarbonyl group substituted the β-diketone moiety, showed antibacterial action against gram-positive and gram-negative bacteria of clinical interest. The simplified curcumin also disrupted the divisional septum of B. subtilis; however, subsequent biochemical analysis did not support a direct action on FtsZ. Our results suggest that the simplified curcumin exerted its function mainly through membrane permeabilization, with disruption of the membrane potential necessary for FtsZ intra-cellular localization. Finally, we show here experimental evidence for the requirement of the β-diketone group of curcumin for its interaction with FtsZ.

Investigating the Modes of Action of the Antimicrobial Chalcones BC1 and T9A

“…FT-IR of B. subtilis cells treated with BC1 and T9A was measured, together with a negative control and a positive control using nisin ( Figure 5 ). FT-IR is a technique that can provide a fingerprint of different components of the bacterial cell, and thus can also report on the disruption of cellular structures [ 14 ]. The peaks shown in the spectrum represent perturbations of diverse chemical structures from compounds present in B. subtilis [ 21 ].…”

“…The MIC was defined as the lowest concentration of compound that prevented bacterial growth after incubating cultures for 24 h at 30 °C [ 35 ]. MICs were also determined using the Resazurin Microtiter Assay Plate (REMA) essentially as described by [ 14 ]. The antibiotic kanamycin at 20 µg/mL was used as positive control in both evaluations.…”

“…Fourier-transform infrared (FT-IR) spectrophotometry was done on B. subtilis cells in a FT-IR spectrophotometer (Shimadzu, model 8300, Shimadzu, Kyoto, Japan), as described by [ 14 ]. Cells were cultivated in LB medium (29 °C) up to the concentration of 10 5 cells/mL, then treated with BC1 (50 µg/mL), T9A (40 µg/mL), nisin (5 µg/mL), or a negative control without the addition of any compound for 30 min.…”

“…In our continuing search for new antibacterial phenolic compounds, we have synthesized and evaluated hundreds of compounds using antibacterial phenotypic assays against gram-positive and gram-negative species [ 13 , 14 , 15 , 16 ]. The structural design of our library of curcumin analogs has been based on phenolic functionality, because most of bacterial division inhibitors display at least one phenol ring [ 17 , 18 ].…”

“…Here, we tested the influence of T9A and BC1 on macromolecular synthesis (DNA, RNA, protein, and peptidoglycan), ATP concentration, and membrane permeability using X. citri and B. subtilis . We chose B. subtilis as a gram-positive model organism, as this is commonly used as a model for mode of action studies, and several methods are established in our labs, including FtsZ purification and FT-IR spectrophotometry [ 14 ].…”