Letter Cite This: Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/journal/estlcu
Perfluoroalkyl Substances Increase the Membrane Permeability and Quorum Sensing Response in Aliivibrio f ischeri Nicole J. M. Fitzgerald,†,§ Matt F. Simcik,‡ and Paige J. Novak*,† †
Department of Civil, Environmental, and Geo-Engineering, University of Minnesota, 500 Pillsbury Drive Southeast, Minneapolis, Minnesota 55455, United States ‡ School of Public Health, University of Minnesota, 420 Delaware Street Southeast, Minneapolis, Minnesota 55455, United States S Supporting Information *
ABSTRACT: Perfluoroalkyl substances (PFAS) are used in a variety of products and are ubiquitous in the environment. They have been found to associate with eukaryotic cell membranes and alter membrane properties. Bacteria are exposed to elevated concentrations of PFAS in some environments; nevertheless, the effect of PFAS exposure on microbial membranes has not yet been studied. Some quorum sensing pathways require the passive diffusion of signaling molecules through cell membranes. Quorum sensing initiates a variety of bacterial processes, such as biofilm formation and antibiotic production. If PFAS exposure increased the microbial quorum sensing response, these processes could be initiated at lower population densities, with wide-ranging ramifications for PFAS-impacted environments. This study examined the effect of perfluorinated alkyl sulfonates and carboxylates on quorum sensing in a model bacterium, Aliivibrio fischeri. Results showed that cultures exposed to PFAS were brighter after they received the signaling molecule. The observed increase in luminescence was dose-dependent and increased with the fluorinated carbon number. Specifically, perfluorooctanesulfonate increased luminescence at levels as low as 10 μg/L. PFASexposed bacteria were also more permeable to a semi-membrane permeable dye. Therefore, it is likely that increased permeability was, at least in part, the cause of increased luminescence.
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INTRODUCTION Perfluoroalkyl substances (PFAS) are found in the environment at a wide range of concentrations (e.g., refs 1 and 2). PFAS are present at the highest concentrations, up to milligram per liter levels, in areas contaminated with aqueous film-forming foams.3,4 They are also detected in wastewater and landfill leachate at microgram per liter levels.5−9 PFAS in surface waters are typically at the nanogram per liter level, but their levels can be greater in areas effected by industrial activity.10−13 Bacteria are present in each of these environments where we depend on them for nutrient cycling and contaminant degradation. It is therefore important to understand the effect that PFAS have on bacteria at a range of concentrations. PFAS have been shown to alter eukaryotic cell membrane properties and increase membrane permeability. In mitochondria, perfluorooctane (PFOA) and perfluorooctanesulfonate (PFOS) exposure increased membrane fluidity and permeability to protons, increasing the proton leak at PFOA and PFOS concentrations of 41 and 5 mg/L, respectively.14 Likewise, the response of breast cancer cells to estradiol increased in the presence of 0.1 mg/L PFOS,15 presumably as a result of increased cell permeability. Exposure to 41 mg/L PFOA or 50 mg/L PFOS increased the membrane permeability and toxicity of pentachlorophenol in liver cells,16 and exposure to 12 mg/L PFOS increased the uptake and genotoxicity of cyclophosphamide in lung cells.17 Though PFOA and PFOS increased eukaryotic cell membrane permeability, it is not clear © XXXX American Chemical Society
whether bacterial membranes respond similarly to PFAS exposure and which membrane-related cell functions may be vulnerable to such changes. The quorum sensing response in bacteria relies, in part, upon membrane integrity. Quorum sensing can be described as a process by which bacteria communicate, with many quorum sensing pathways requiring that signaling molecules diffuse passively through cell membranes to reach a receptor within the cell (e.g., refs 18−21). The activated receptor induces a signaling cascade, producing more acyl homoserine lactone (AHL) and triggering a population-wide response (e.g., refs 18−21). Quorum sensing initiates a variety of processes, such as biofilm formation, pathogenicity, antibiotic production, and bioluminescence (e.g., refs 18−21). A PFAS-induced increase in membrane permeability and a subsequent increase in the quorum sensing response could therefore initiate these processes at lower microbial population densities, causing increased fouling, disease susceptibility, and antibiotic resistance. This study was designed to screen for the potential of PFAS to affect bacterial cell membrane permeability and quorum sensing. To better interpret experimental results, a well-studied Received: Revised: Accepted: Published: A
November 16, 2017 December 8, 2017 December 12, 2017 December 12, 2017 DOI: 10.1021/acs.estlett.7b00518 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
Letter
Environmental Science & Technology Letters
μg/mL, chosen on the basis of previous studies27). The fluorescent DAPI signal was measured 10 min after exposure. Bacteria were then lysed via five freeze−thaw cycles, releasing all of the DNA for DAPI binding, and exposed to DAPI for 10 min, after which the maximum fluorescence in each sample was measured for the purpose of normalization. Details can be found in the Supporting Information. Data Analysis. Luminescence and fluorescence in each well were normalized by the optical density of the culture in that well. Values were averaged, and 95% confidence intervals were determined using a Student’s t test. These values were normalized by the value obtained for the PFAS-free control in the same 96-well plate, and the error was propagated. Significance was tested using one-way analysis of variance (ANOVA) with post hoc comparisons using the Fischer LSD test (α = 0.05). For the permeability experiment, live cell fluorescence was normalized by the fluorescence in that sample after cell lysis. Values were averaged for each treatment, and t tests, assuming unequal variances, were performed between each treatment and the control. Additional details regarding the data analysis can be found in the Supporting Information.
quorum-sensing bacterial strain, Aliivibvrio f ischeri, was used. In this strain, quorum sensing initiates luminescence. Because the quorum sensing pathway utilized by A. f ischeri is homologous to pathways that initiate biofilm formation and virulence factor and antibiotic production in other bacteria,18,20,22,23 this organism serves as a model for environmentally relevant microbial processes initiated via quorum sensing.
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METHODS Chemicals. Six PFAS were used to study effects of fluorinated chain length and functional group: perfluorobutanoate (PFBA, 3C), PFOA (7C), perfluorononanoate (PFNA, 8C), perfluorobutanesulfonate (PFBS, 4C), perfluorohexanesulfonate (PFHxS, 6C), and PFOS (8C). The signaling molecule used in the experiments was N-(β-ketocaproyl)-Lhomoserine lactone. Additional details regarding the chemicals used are provided in the Supporting Information. Cell Cultures. A. fischeri mutant DC43, the sole species used in this study, was obtained from E. Stabb.24 A. f ischeri DC43 is active in quorum sensing and responds to the diffusion of AHL molecules into the cell by luminescing.24 This mutant was selected because, while it is able to respond to the signaling molecule N-(β-ketocaproyl)-L-homoserine lactone, it is unable to produce it.24 This allowed for tighter control over the concentration of the signaling molecule in experiments. A. f ischeri DC43 was cultured at room temperature (20−22 °C) in sterile photobacterium broth.25 Cultures were inoculated via transfer [1% (v/v)] of a 24 h culture of A. f ischeri DC43. Luminescence and Metabolism. A. f ischeri DC43 used in experiments was grown in 1 mL of broth in 10 mL glass tubes in the presence of 10 μg/L to 50 mg/L PFBS, PFBA, PFHxS, PFOA, PFOS, or PFNA for 30 h at room temperature (20−22 °C) while being shaken at 150 rpm. A negative control (PFASfree) was grown and tested with each experiment. PFAS were added via a methanol stock, and all treatments contained equal amounts of methanol, with control cultures receiving methanol only. There were between 13 and 15 replicates for each treatment. Bacteria were aliquoted into white or black clear-bottom 96well plates. Each well received 200 μL of culture. Metabolism was measured in the black plate via the reduction of resazurin dye to the fluorescent resorufin.26 Resazurin was added to a final concentration of 40 μM, and plates were incubated for 45 min at room temperature. Luminescence and optical density were measured in the white plates. AHL was added to a final concentration of 1000 nM based on previous research,24 and bacteria were incubated at room temperature for 1 h. The optical density was measured at 600 nm in the white plates for normalization. Details can be found in the Supporting Information. Dye Permeability. Cell permeability was measured by use of the dye 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). DAPI is a membrane semipermeable dye that fluoresces when it binds to DNA. The diffusion of DAPI into PFAS-exposed bacteria or PFAS-free control bacteria was monitored via its fluorescent signal. Bacteria were grown in the presence of each PFAS at 50 mg/L, washed in phosphatebuffered saline (PBS), and resuspended in PFAS-amended PBS, as described in the Supporting Information. PFAS were added via a methanol stock, and all treatments contained equivalent amounts of methanol, with PFAS-free controls receiving methanol only. The bacterial suspension (2 mL) was combined with 1 mL of PBS containing DAPI (final concentration of 4
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RESULTS AND DISCUSSION Effect of PFAS Exposure on A. f ischeri Metabolic Activity and Growth. Metabolic activity was monitored to assess toxicity or activity indicative of uncoupling as a result of PFAS exposure. PFAS have been shown to increase membrane permeability to protons.14,15,28 During respiration, bacteria rely on a proton gradient across the cell membrane to generate energy.29 If membrane permeability is increased and protons can leak across the membrane, respiration becomes uncoupled from energy generation and bacteria must increase their rate of respiration to generate the same amount of energy.30 High concentrations (milligrams per liter) of the 4C and 6C sulfonate compounds increased the metabolic rate (Figure 1), perhaps as a result of proton leakage, as seen with mitochondrial membranes.14,15,28 4C PFBS appeared to increase the metabolic rate at a concentration lower than that of 6C PFHxS (Figure 1); the reasons for this are unknown. Exposure to 50 mg/L 8C PFOS, however, significantly decreased the metabolic rate, suggesting that PFOS was toxic to A. f ischeri at this high concentration. For the carboxylated PFAS, there was no metabolic indication of toxicity, but increases in the rate of metabolism, again, as a result of possible uncoupling, were observed for the 8C PFNA at 0.3−50 mg/L (Figure 1). Growth of A. fischeri was slightly but variably affected by PFAS exposure (Figure S1). The largest changes in growth were observed for 50 mg/L PFBS and PFNA (18 and 12%, respectively); all other changes were 100% represent increases in the rate of respiration in a given treatment as compared to the control. Error bars are 95% confidence intervals, as determined by the Student’s t test. Asterisks above or below the data indicate the treatments that were statistically different from the control via one-way ANOVA with post hoc comparisons using the Fischer LSD test (α = 0.05).
Figure 2. Luminescence after addition of AHL in A. f ischeri DC43 exposed to varying concentrations of PFAS for (A) fluorinated sulfonates and (B) fluorinated carboxylates. The y-axis shows the normalized luminescence, with values of >100% representing increases in the quorum sensing response of a given treatment as compared to the control. Error bars are 95% confidence intervals, as determined by the Student’s t test. Asterisks above or below the data indicate the treatments that were statistically different from the control via one-way ANOVA with post hoc comparisons using the Fischer LSD test (α = 0.05).
cence) increased as PFAS fluorinated chain length and exposure concentration increased. This was especially evident with the carboxylates (Figure 2). This effect was also observed with the sulfonates, with 50 mg/L 6C PFHxS causing more luminescence than 50 mg/L 4C PFBS did; nevertheless, 1 mg/ L PFBS did increase luminescence, while 1 mg/L PFHxS did not. One explanation for this could be the enhanced rate of respiration upon exposure to 1 mg/L PFBS, which was not observed at 1 mg/L PFHxS (Figure 1). Exposure to the 8C PFOS resulted in greater luminescence at concentrations of
