Antibacterial activity of alkyl gallates is a combination of direct targeting of FtsZ and permeabilization of bacterial membranes

Abstract

Alkyl gallates are compounds with reported antibacterial activity. One of the modes of action is binding of the alkyl gallates to the bacterial membrane and interference with membrane integrity. However, alkyl gallates also cause cell elongation and disruption of cell division in the important plant pathogen Xanthomonas citri subsp. citri, suggesting that cell division proteins may be targeted by alkyl gallates. Here, we use Bacillus subtilis and purified B. subtilis FtsZ to demonstrate that FtsZ is a direct target of alkyl gallates. Alkyl gallates disrupt the FtsZ-ring in vivo, and cause cell elongation. In vitro, alkyl gallates bind with high affinity to FtsZ, causing it to cluster and lose its capacity to polymerize. The activities of a homologous series of alkyl gallates with alkyl side chain lengths ranging from five to eight carbons (C5–C8) were compared and heptyl gallate was found to be the most potent FtsZ inhibitor. Next to the direct effect on FtsZ, alkyl gallates also target B. subtilis membrane integrity—however the observed anti-FtsZ activity is not a secondary effect of the disruption of membrane integrity. We propose that both modes of action, membrane disruption and anti-FtsZ activity, contribute to the antibacterial activity of the alkyl gallates. We propose that heptyl gallate is a promising hit for the further development of antibacterials that specifically target FtsZ.

A simplified curcumin targets the membrane of Bacillus subtilis

“…FtsZ is a GTPase that polymerizes into protofilaments in the presence of magnesium and both nucleotide cofactors GTP or GDP (Erickson, Anderson, & Osawa, ), and is the main scaffold protein of the bacterial septum. The possible interference of 2 was investigated using B. subtilis FtsZ and a polymerization/associated GTP hydrolysis assay (Król et al., ). Incubation of FtsZ with a concentration of 2 equivalent to the MIC90 (12.7 μg/mL) led to 16% inhibition of the GTPase activity (Figure a; compare the black bars at NC and 12.5 μg/mL).…”

“…As a control, 1% DMSO was used. FtsZ sedimentation assays were performed as described in (Król et al., ). Ten μM FtsZ and BSA (control) were incubated with 10 mM MgCl 2 in the presence of 50 μg/mL of 2 0.5% DMSO (control) in 50 mM HEPES pH 7.5, 50 mM KCl at 30°C and the reaction was started with 2 mM GTP or GDP (or a corresponding volume of buffer added where no nucleotide was present).…”

Identification of Agents Targeting Ftsz Assembly

“…Although a modest inhibitor of B. subtilis 168 proliferation (MIC50 20 μg/ml), the most potent analogue 29 (n = 6) bound FtsZ with high affinity (Kd 80 nM) and inhibited GTPase activity without causing aggregation, while also affecting membrane integrity [117].…”

Purification and characterization of FtsZ from the citrus canker pathogen Xanthomonas citri subsp. citri

“…The cell division protein that has been most widely investigated in this respect is the extremely well‐conserved protein FtsZ, a (distant) homolog of tubulin that is absolutely critical for cell division (Den Blaauwen, Andreu, & Monasterio, ; Li & Ma, ; Panda et al., ). We have recently characterized the mode of action of alkyl gallates, antimicrobial agents that target both FtsZ and membrane integrity in the Gram‐positive model organism Bacillus subtilis (Król et al., ). Our ultimate aim, however, was to investigate the activity of alkyl gallates against FtsZ from the Gram‐negative plant pathogen Xanthomonas citri subsp.…”

“…Earlier, we and others identified alkyl gallates as broad‐spectrum antimicrobial agents that can kill Xac, Salmonella sp., Staphylococcus aureus including MRSA, B. subtilis, and other bacteria (Król et al., ; Kubo, Fujita, Ken‐ichi Nihei, & Nihei, ; Kubo, Fujita, & Nihei, ; Kubo, Fujita, Nihei, & Masuoka, ; Kubo, Xiao, & Fujita, ; Shibata et al., ; Silva et al., ). In an important manner, these compounds are not mutagenic or cytotoxic and therefore safe to use (Silva, Polaquini, Regasini, Ferreira, & Pavan, ).…”