Potentiation of the cytotoxic activity of copper by polyphosphate on biofilm-producing bacteria: a bioinspired approach

Abstract

Adhesion and accumulation of organic molecules represent an ecologically and economically massive problem. Adhesion of organic molecules is followed by microorganisms, unicellular organisms and plants together with their secreted soluble and structure-associated byproducts, which damage unprotected surfaces of submerged marine structures, including ship hulls and heat exchangers of power plants. This is termed biofouling. The search for less toxic anti-biofilm strategies has intensified since the ban of efficient and cost-effective anti-fouling paints, enriched with the organotin compound tributyltin, not least because of our finding of the ubiquitous toxic/pro-apoptotic effects displayed by this compound. Our proposed bio-inspired approach for controlling, suppressing and interfluencing the dynamic biofouling complex uses copper as one component in an alternative anti-fouling system. In order to avoid and overcome the potential resistance against copper acquired by microorganisms we are using the biopolymer polyphosphate (polyP) as a further component. Prior to being functionally active, polyP has to be hydrolyzed to ortho-phosphate which in turn can bind to copper and export the toxic compound out of the cell. It is shown here that inhibition of the hydrolysis of polyP by the bisphosphonate DMDP strongly increases the toxic effect of copper towards the biofilm-producing Streptococcus mutans in a synergistic manner. This bisphosphonate not only increases the copper-caused inhibition of cell growth but also of biofilm production by the bacteria. The defensin-related ASABF, a marine toxin produced by the sponge Suberites domuncula, caused only an additive inhibitory effect in combination with copper. We conclude that the new strategy, described here, has a superior anti-biofilm potential and can be considered as a novel principle for developing bio-inspired antifouling compounds, or cocktails of different compounds, in the future.

Figure 1

Growth kinetics of S. mutans in the absence of any compound (■), in the presence of 3 µM CuSO₄ (●) or in cultures with 100 µM CuSO₄ (▲). The growth of the bacteria was monitored optically at 600 nm.

Figure 2

Growth inhibitory effect of the inhibitory compounds used in this study, added alone to the cultures: (A) CuSO₄, (B) biopolymer polyphosphate (polyP), (C) bisphosphonate dichloromethylene diphosphonic acid (DMDP) and (D) the marine toxin (the defensin-related Ascaris suum antibacterial factor (ASABF)) produced by the sponge S. domuncula. The results are expressed as means (10 experiments each) ± SD of the mean; * P < 0.01.

Figure 3

Isobolograms showing the synergistic effect (A) of polyP and (B) of the bisphosphonate DMDP on copper-caused toxicity on S. mutans. (C) The marine toxin (defensin-related ASABF) from S. domuncula caused only an additive effect on copper toxicity.

Figure 4

Effect of CuSO₄ in the culture medium of S. mutans on the intracellular polyP content. The cells were exposed to different concentrations of CuSO₄ for 25 h. Then the cells were broken and assayed for polyP level using the two-step enzymatic conversion of polyP into ATP. The polyP level is correlated with the bacterial protein content from which polyP was quantitatively determined. Data are means (6 experiments each) ± SD of the mean; * P < 0.01.

Figure 5

Efflux of P_i from S. mutans during incubation with the bisphosphonate DMDP. At first the cells were labeled with H₃[³²P]O₄ and then transferred to medium without H₃[³²P]O₄. Then the cells were continued to be incubated either in the absence (0 µM) or presence (10 µM or 30 µM) of DMDP for 0 to 24 h, as indicated. At the time indicated the cell free supernatants were collected by centrifugation and counted for radioactivity. Means (6 experiments each) ± SD are given; * P < 0.01.

Figure 6

Inhibition of biofilm production by polyP/bisphosphonate-DMDP. S. mutans had been grown on microscope slides for 5 days (A and B) in the absence of any inhibitory compound, in the presence of 1 µg/mL of polyP (C and D), or with 1 µg/mL of polyP together with 1 µg/mL bisphosphonate-DMDP (E and F). After incubation the specimens were reacted with WGA-labeled with Alexa Fluor 555 and DAPI. Then the samples were analyzed for fluorescence of the labeled WGA-lectin (A, C, E), or for DAPI (B, D, F).

Figure 7

Schematic outline of the synergistic effect caused by copper in combination with polyP/bisphosphonate on bacteria cells. This bioinspired concept implies that copper (Cu), taken up from the solid phase onto which the bacteria are in the process to attach, is imported into the cells and displays it toxicity. In a parallel de-toxification process polyP that is either formed intracellularly or taken up by the polyP-3-hydroxybutyrate-Ca²⁺ pump undergoes enzymatic hydrolysis by the exopolyphosphatases (ExoP’ase) under formation of ortho-phosphate. In turn ortho-phosphate forms with copper an intracellular salt that is exported from the bacterial cells. In a parallel mode of action polyP, extracellularly attaching to the bacterial membranes removes Ca²⁺ from the cell membrane and by that causes death of the microorganisms.