Article pubs.acs.org/biochemistry
Exploration of the Innate Immune System of Styela clava: Zn2+ Binding Enhances the Antimicrobial Activity of the Tunicate Peptide Clavanin A Samuel A. Juliano,† Scott Pierce,† James A. deMayo,‡ Marcy J. Balunas,‡ and Alfredo M. Angeles-Boza*,† †
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269-3060, United States Division of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269, United States
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‡
S Supporting Information *
ABSTRACT: Tunicates have been used as primitive models for understanding cellmediated and humoral immunity. Clavanin A (ClavA) is one member of a family of antimicrobial peptides produced by the solitary tunicate Styela clava. In this work, we demonstrate that ClavA utilizes Zn2+ ions to potentiate its antimicrobial activity not only by reducing the concentration at which the peptide inhibits the growth of bacteria but also by increasing the rate of killing. Membrane depolarization, β-galactosidase leakage, and potassium leakage assays indicate that ClavA is membrane active, forms small pores, but induces cell death by targeting an intracellular component. ClavA and ClavA-Zn2+ added to Escherichia coli and imaged by confocal microscopy translocate across the cell membrane. E. coli mutants lacking the functional Zn2+ import system are less susceptible to ClavA, suggesting that the synergistic activity between ClavA and Zn2+ has a cytoplasmic target, which is further supported by its nucleolytic activity. Overall, these studies identify a remarkable new mechanism by which zinc contributes to the immune response in the tunicate S. clava.
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are found in all complex living organisms. In addition to their ability to directly kill microorganisms, AMPs exert immunomodulatory activities, which has led researchers in recent years to refer to AMPs by the more general term host defense peptides (HDPs).14,15 The chemical characteristics of AMPs (i.e., generally cationic and the presence of ∼50% hydrophobic amino acids) allow them to strongly interact with cellular membranes, particularly those of bacteria. Therefore, many AMPs were suggested to kill bacteria through fatal interaction with the components of the bacterial membrane.16−21 More recently, internalization of AMPs with the concomitant damage of an intracellular target has been uncovered for some peptides.22−25 Our group has been particularly interested in the effect of the binding of metal ions on the activity of AMPs.26,27 Because zinc and copper ions colocalize with antimicrobial peptides during immune responses,28−35 we have hypothesized that AMPs with metal-binding motifs can form either transient or stable complexes with these metal ions to improve their antimicrobial activity. Herein, we investigate the effect of zinc ions on the activity of an AMP isolated from the tunicate Styela clava. These investigations are strongly motivated by the search for molecular models to characterize fundamental immunological roles of zinc.
he importance of zinc in immunity is well-known; however, despite the considerable advances in recent years, many fundamental questions about the immunomodulatory and antimicrobial properties of zinc at the cellular and molecular levels have remained unanswered.1,2 Zinc exists as the divalent cation Zn2+ and together with iron and copper is one of the most abundant transition metals in humans.3 Zinc plays a multitude of roles in human immunology, but not surprisingly, the complexity of the human immune system has not allowed researchers to fully unravel all of the biochemical roles of zinc.4−6 Because the complex role of zinc ions in the human immune system probably evolved from the innate immune system of ancestors on the taxonomic scale, understanding the role of zinc in the immune response of these ancestors could provide explanations about its role in humans. Tunicates, like humans, are members of the phylum Chordata, which primarily includes vertebrates, and have the capacity for cell-mediated immunity and humoral immunity.7−9 Importantly, tunicates have been used as primitive models for understanding fundamental immunological mechanisms by analyzing their processes either in vivo or in vitro.10,11 Thus, it can be hypothesized that by tracing the evolutionary history of zinc interactions at the molecular level we should be able to elucidate mechanisms of zinc contributions to the immunological response in the complex human immune system. Key participants in the immune response are also the molecules known as antimicrobial peptides (AMPs) that can be expressed constitutively or induced by the presence of pathogens.12,13 AMPs are evolutionarily ancient weapons and © 2017 American Chemical Society
Received: October 11, 2016 Revised: February 18, 2017 Published: February 22, 2017 1403
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Biochemistry S. clava, a solitary tunicate, has been shown to express several AMPs that are important components of its innate immune system.36−39 Extracts from S. clava hemocytes have produced two families of AMPs, styelins and clavanins.40−42 Immunolocalization studies found clavanins in the cytoplasm of macrophages and in eosinophilic granulocytes.39 In vitro assays have shown that one member of the clavanin family, clavanin A (ClavA, VFQFLGKIIHHVGNFVHGFSHVF-NH2), is active against a broad spectrum of pathogens, including methicillinresistant Staphylococcus aureus, and its activity is greater at pH 5.5 than at pH 7.4.36 The activity at low pH is biologically relevant because the vacuoles of phagocytic hemocytes from S. clava are likely to have a pH as low as 5 as found in the tunicate Ascidia ceratodes.43 Such conditions should support optimal antimicrobial activity. Unlike most AMPs, ClavA remains active at elevated NaCl concentrations (≤0.3 M).36 In addition, ClavA displays a negligible toxicity toward mammalian cells. Because of these properties, ClavA has been tested in vitro in wound and sepsis murine models and was shown to have potent activity in both models.44 Because of its similarity to the magainins in size, primary sequence, and antimicrobial activity, ClavA is thought to act by permeabilizing bacterial membranes.16,45,46 In the α-helical conformation, ClavA places His17 and His21 on one face of the helix, creating a putative Zn2+-binding site.47 The i, i + 4 spacing between two amino acids within an α-helix has been previously exploited to design synthetic metal-binding motifs.47−49 Specifically, two histidines at positions 7 and 11 in a 16-residue peptide were used to create a synthetic zincbased nuclease.47 Coincidentally, hemocytes of aquatic invertebrates contain large amounts of metal ions, particularly Zn2+, at concentrations higher than in the surrounding plasma.50−52 For example, in Ostrea edulis, specialized mechanisms allow for hemocyte uptake of Zn2+ ions up to a 1.2 M concentration.53 Taken together, the presence of a potential Zn2+-binding site and the high concentrations of this metal ion in marine invertebrate hemocytes beg the question of whether a relationship exists between metal binding and antimicrobial ability in the activities of ClavA. In this work, we examine the antimicrobial activity of ClavA in the presence of Zn2+ ions. We determined that the target of this peptide−Zn2+ complex is located within the cytoplasm, in addition to the previously reported membrane activity. Our data support a model in which the ClavA−Zn2+ complex uses its nucleolytic properties to kill bacteria.
Peptides were then purified via reverse phase high-performance liquid chromatography (HPLC) (Shimadzu LC-20AD) using a C18 semiprep column (Grace Davidson, Deerfield, IL), and masses were confirmed via electrospray ionization mass spectrometry in positive ion mode. The purity was determined using reverse phase HPLC with a C18 analytical column (Thermo). HPLC chromatograms and mass spectra are compiled in Figures S2−S5. Fractions containing the purified peptide were lyophilized overnight and dissolved in nanopure water. The peptides were then quantified via UV−vis spectrophotometry using molar extinction coefficients for the amino acid residues and the peptide bond as determined by Kuipers and Gruppen.54 All peptides were stored at 4 °C in solution and diluted before each experiment. Antimicrobial Assay. Antimicrobial activity was determined using the broth microdilution method described by Hancock.55 Three to five colonies of Escherichia coli (MG1655) or Bacillus subtilis (PS832) were added to Luria-Bertani broth (LB; Amresco, Solon, OH) containing 2-(N-morpholino)ethanesulfonic acid buffer (MES; Sigma-Aldrich) adjusted to pH 5.5 (LB 5.5) and incubated until midlogarithmic phase was achieved, at an OD600 of 0.4−0.6 (3−4 h for E. coli, 4−6 h for B. subtilis). Bacterial cultures containing 1 × 106 colony-forming units (cfu)/mL were then prepared in fresh medium for the determination of the minimum inhibitory concentration (MIC). A pH of 5.5 was chosen for these experiments because much of the previous research on ClavA showed that its activity is optimal at lower pH values.36,45,46 Additionally, this pH has been found to be biologically relevant for phagocytic hemocytes. For example, the pH values of hemocytes isolated from the tunicate A. ceratodes were found to be as low as 5.43 Peptide stock solutions were prepared at 2 times the maximal experimental concentration in 50 mM MES (pH 5.5) (MES 5.5), and 50 μL of 2-fold serial dilutions was made in a 96-well polypropylene plate (Greiner). To the serial dilutions was added 50 μL of a bacterial suspension of 1 × 106 cfu/mL, for a final concentration of bacteria of 5 × 105 cfu/mL. Plates were then incubated at 37 °C for 18−20 h, and the MIC was observed as the lowest concentration that visually inhibited bacterial growth. The MIC values reported represent the mode of three trials. For experiments using metal ions, the 2-fold peptide dilutions were incubated with solutions containing the metal ions used in the experiments at 2 times the peptide concentration for ∼30 min prior to the addition of the bacterial suspension to allow adequate time for binding. The peptide/metal solutions were then used in the same manner stated above. All of the metal ions used showed MICs greater than 256 μM, the maximal metal concentration used in any experiment. The minimum bactericidal concentration (MBC) was determined after the antimicrobial assay had been performed by spotting 10 μL of the MIC mixture and the next two higher peptide concentrations onto an agar plate and incubating the plate overnight. The MBC values reported are the peptide concentrations that resulted in agar plates without bacterial growth. Varied Zn2+ Concentration Assay. To determine the optimal Zn2+ concentration for running experiments as well as how much Zn2+ is required to potentiate the activity of ClavA, one concentration of ClavA was incubated with several different concentrations of Zn2+ and bacterial growth was monitored. E. coli cultures of 1 × 106 cfu/mL were prepared using the same
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MATERIALS AND METHODS Solid Phase Peptide Synthesis, Purification, and Identification. ClavA and all substitution and fluorescent mutants were synthesized, purified, and quantified using methods previously described by our lab.26 Briefly, peptides were synthesized manually via standard solid phase peptide synthesis using fluorenylmethyloxycarbonyl (Fmoc) chemistry on Rink amide resin (Matrix Innovation, Quebec City, QC). Peptides used for fluorescence microscopy were labeled with 5(6)-carboxytetramethylrhodamine (TMR; Sigma) on a lysine residue. The phenylalanine residue in the fourth position of the peptide sequence was replaced with a lysine protected with a 4methyltrityl group, which can be selectively deprotected at low acid concentrations. The lysine amine group was then used to attach to the carboxylic acid of the TMR. A scheme of this synthesis can be found in Figure S1. 1404
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were considered cleared when 100 μL of culture plated directly produced no colonies, indicating fewer than 10 cfu/mL. DiSC3(5) Depolarization Assay. B. subtilis cultures were grown at 37 °C in LB 5.5 to midlogarithmic phase, at an OD600 of 0.4−0.6, washed three times with MES 5.5, and diluted to OD600 of 0.01 in MES 5.5 containing 20 mM glucose (Fisher). The bacteria were then incubated for 1 h with 0.4 μM 3,3′dipropylthiadicarbocyanine iodide [DiSC3(5); Chemodex, St. Gallen, Switzerland], a membrane potential active fluorophore. The 1 h incubation allows time for the fluorophore to be drawn to polarized membranes and self-quench, as evidenced by the >90% decrease in fluorescence. The bacterial solutions were then mixed with serial dilutions of peptide treatments, 50% Triton X-100 (T-X100; Fisher), or MES 5.5. T-X100 was used as a positive depolarization control, and MES 5.5 was the negative control. Fluorescence was monitored every 2 min for 1 h at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. Values represent the percent fluorescence recovery averaged over three trials ± the standard error. β-Galactosidase Leakage Assay. The amount of βgalactosidase leakage caused by ClavA or ClavA-Zn2+ was determined as described previously.26,58,59 E. coli transformed with a gene for cytoplasmic β-galactosidase production was grown in LB 5.5 at 37 °C to an OD600 of 0.4−0.6, after which overexpression of β-galactosidase was induced for 1 h via the addition of a 1 mM final concentration of isopropyl β-D-1thiogalactopyranoside (IPTG; Fisher). Cells were then washed three times in MES buffer and suspended in fresh LB 5.5. Aliquots of the culture were then mixed with either ClavA, ClavA-Zn2+, or 50% T-X100. Mixtures were incubated for 1 h at 37 °C and then centrifuged at 4400 rpm and 4 °C for 10 min, and the supernatant, containing any leaked β-galactosidase, was recovered. o-Nitrophenyl-β-galactoside (ONPG; Thermo), which β-galactosidase cleaves into the yellow product onitrophenol (ONP),60 was then added to the supernatant at a final concentration of 0.8 mg/mL, and the absorbance was monitored at 405 nm every 5 min for 1 h. Values reported are an average of the 1 h points for three trials; error bars represent the standard error. Potassium Efflux. B. subtilis was grown to midlogarithmic phase (OD600 = 0.4−0.6) in LB 5.5, washed, and resuspended at a concentration of 1 × 108 cfu/mL in MES 5.5. Cells were then mixed with ClavA or ClavA-Zn2+ at twice their respective MBCs to yield solutions containing 5 × 108 cfu/mL B. subtilis and peptides at the MBC. The potassium concentration was then monitored every 10 min for 1 h using a potassium sensitive electrode and reference electrode (MI-442 and MI402, respectively; Microelectrodes, Bedford, NH) connected to a pH meter (Orion Star A111; Thermo) measuring potential in millivolts. Cells were then lysed using a combination of freeze− thaw cycles and sonication, and the potential of the resulting solution was used as an internal positive control. Percentages were calculated using a blank bacterial sample with no peptide as a negative control. Confocal Fluorescence Microscopy. E. coli cultures were grown to an OD600 of ∼1.0 (1 × 109 cfu/ml) in LB 5.5, washed, and suspended in MES 5.5 containing 20 mM glucose. Aliquots were incubated with 8 μM ClavA-TMR or ClavA-TMR-Zn2+ for 1 h at room temperature. This concentration was used for both treatments because higher ClavA-TMR concentrations would cause oversaturation of the image. Mixtures were then spun down and resuspended in fresh MES 5.5 with 20 mM
procedure that was used for the antimicrobial assay. These cultures were incubated with solutions containing ClavA at a final concentration of 8 μM and either 0, 2, 4, 8, or 16 μM Zn2+. The mixtures were then incubated at 37 °C for 4 h, after which dilutions were plated on LB agar and allowed to grow overnight at 37 °C. The next morning colonies were counted and percentages calculated on the basis of a control culture containing neither ClavA nor Zn2+ as 100%. Tunicate Collection and Hemolymph Harvest. To determine biologically relevant Zn2+ concentrations, S. clava tunicate specimens were collected from shallow water dock lines at Avery Point, CT, in January 2016 (41° 18.975 N, 72° 3.647 W). Tunicates were rinsed in sterile deionized water and stored in 10% (w/v) menthol before being dissected at the lab. The hemolymph was harvested from the tunicates via dissection and heart puncture. The hemolymph was then filtered through a 70 μm mesh nylon filter (Carolina Biological Supply, Burlington, NC) and stored at −20 °C until it was ready to use. Atomic Absorption Spectroscopy. The Zn2+ concentration was measured using atomic absorption spectroscopy (AAS). Digestion of the samples was required prior to AAS analysis. Hemolymph samples were first dried via heating at 120 °C in an aluminum block heater for ∼2 days in glass test tubes with Teflon screw caps removed. Next, 5 mL of 70% nitric acid (Sigma-Aldrich) was added to each tube and allowed to rest at room temperature for 1 h. Tubes were then capped and heated in an aluminum heating block for 3 h at 90 °C. Samples were then cooled to room temperature, diluted to 50 mL, and analyzed by AAS along with standards prepared via a similar process. Circular Dichroism (CD) Spectroscopy. CD spectra for the purpose of both determining the relative α-helical content of ClavA and its mutants and determining the effect of Zn2+ on ClavA were recorded using a Jasco J-710 spectropolarimeter. All samples contained 50 μM ClavA. The experiments performed on ClavA and the mutants were performed in a 50:50 solution of H2O and 2,2,2-trifluoroethanol (TFA). The Zn2+ titration experiments were performed in MES 5.5 buffer. Molar ellipticity Θ was calculated from the raw data using the following equation: Θ=θ
MRW lc
where θ is the measured ellipticity, MRW is the mean residue molecular weight of the peptide, calculated as the molar mass divided by the number of residues, l is the path length in centimeters, and c is the molar concentration.56 The percent αhelix reported was calculated using the equation % α‐helix = −100
Θ222 − 3000 33000
where Θ222 is the molar ellipticity at 222 nm.57 Time Kill Kinetics. E. coli cultures of 1 × 106 cfu/mL were prepared using the same procedure that was used for the antimicrobial assay. Aliquots of the diluted culture were then mixed with the indicated treatments in equal volumes and then incubated at 37 °C for the indicated periods of time. At each time point, aliquots of 10 μL were removed, serially diluted to 1:1000, plated on LB agar, and grown for 16−18 h. Colonies were counted and plotted versus time. In instances in which it was necessary to do so, dilutions were adjusted up or down to obtain plates with an appropriate number of colonies. Cultures 1405
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activity.27 Circular dichroism shows that the three His to Ala mutants have a percentage of helicity higher than that of ClavA (Figure S6), but ClavA-H21A has the smallest increase in helicity. Because ClavA-H17A and ClavA-H17A-H21A are less active than ClavA-H21A, the helicity increase is not the reason the antimicrobial activity in ClavA-H21A is better than those in the other two mutants. Notably, marked differences were observed when the peptides were combined with zinc ions. Table 2 summarizes these results as well as the activities of the
glucose prior to imaging to remove any unbound peptide. Cells were imaged on a Nikon A1R spectral confocal microscope using a 60× oil immersion lens. In Vitro DNA Cleavage. Samples were prepared by mixing 10 μM base pair pUC19 with 1 μM ClavA-Zn2+, 1 μM ClavA with 20 μM diethylenetriaminepentaacetic acid (DTPA), or 2 μM Zn2+ in MES 5.5. Mixtures were allowed to stand at room temperature for the indicated periods of time, and reactions were stopped using 3× loading dye containing 1 mM EDTA prior to loading on the gel. After all samples at various time points were prepared, the samples were loaded on a 1% agarose gel containing ethidium bromide, and the gel was run at 100 V for 90 min. Gels were imaged using a Bio-Rad GelDoc XR+ Imager and quantified using Image Lab version 5.0. A correction factor of 1.47 was applied to the data for supercoiled DNA to account for the difficulty of staining with ethidium bromide.61
Table 2. Minimum Inhibitory Concentrations for ClavA, ClavA Mutants, ClavC, and Magainin 2 without and with Zn2+a
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RESULTS AND ANALYSIS The Antimicrobial Activity of ClavA Is Potentiated by the Addition of Zn2+. To examine whether Zn2+ ions affect the activity of ClavA, we determined its MIC against E. coli in the presence of Zn2+ and various biologically relevant metal ions using the broth microdilution method.55 Magainin 2 (GIGKFLHSAKKFGKAFVGEIMNS-NH2) and clavanin C (ClavC, VFHLLGKIIHHVGNFVYGFSHVF-NH2) were used for comparison as well as positive controls. Table 1 summarizes
peptide with with with with with
Ca2+ Fe2+ Ni2+ Cu2+ Zn2+
MIC without Zn2+ (μM)
MIC with Zn2+ (μM)
ClavA ClavA-H17A ClavA-H21A ClavA-H17A-H21A ClavC magainin 2
64 >128 64 >128 4 8
4 >128 32 >128 4 8
a
Zinc ion concentrations are 2 times that of the peptide. The MIC of Zn2+ has been found to be >256 μM, the highest concentration employed in this study.
other peptides used in this study in the presence and absence of Zn2+. ClavA-H17A and ClavA-H17A-H21A remained inactive, whereas ClavA-H21A showed a modest 2-fold increase in activity in the presence of additional Zn2+. These results suggest that His17 is more important for the formation of a putative ClavA-Zn2+ complex (this is not necessarily a 1:1 complex as the nomenclature might suggest; however, we prefer to use this notation until we learn more about the nature of this complex). We also determined MBC values. ClavA had an MBC equal to its MIC, which means that the growth inhibition seen at that concentration was due to the peptide bringing about cell death. In the presence of Zn2+, however, the MBC was 8 μM, twice the MIC, meaning that at MICs of ClavA-Zn2+ there were still living E. coli cells that were in some way inhibited from growing by the peptide. The 8-fold decrease in MBC between ClavA and ClavA-Zn2+ indicates an improvement in the overall bactericidal activity of ClavA upon addition of Zn2+ ions. ClavA Shows Improved Activity Even at Substoichiometric Zn 2+ Concentrations. To examine how the concentration of Zn2+ affects the activity of ClavA, we incubated bacteria with 8 μM ClavA and varied the concentration of Zn2+. Figure 1 summarizes these results. The percent viability was determined by plating dilutions of each sample, counting colonies, and comparing the result to an untreated control. All of the concentrations of Zn2+ used in the study, from 16 to 2 μM, resulted in clearance of a vast majority of the E. coli, and growth was present only in samples that contained no additional Zn2+ because the concentration of ClavA used was below the MBC for the peptide alone. These results indicate that even at very low levels of Zn2+, ClavA activity is greatly improved. This could indicate that one Zn2+ ion interacts with several ClavA units, though more study is required for definitive proof of this. S. clava Hemolymph Contains a Sufficient Concentration of Zn2+ for Biological Relevance. We next evaluated whether the Zn2+ concentration that was observed to increase the activity of ClavA in the antimicrobial assays was biologically relevant. For this purpose, we harvested live S. clava and
Table 1. Minimum Inhibitory Concentrations for ClavA with Various Metalsa ClavA ClavA ClavA ClavA ClavA ClavA
peptide
MIC (μM) 64 >128 64 64 32 4
a
All metal ion concentrations are 2 times that of the peptide. The MICs of the metals used have all been found to be >256 μM, the highest concentration employed in this study.
the MIC values for ClavA and ClavA incubated with the various biologically relevant metals. The activity of ClavA is maintained upon addition of Fe2+ and Ni2+ ions, abolished with Ca2+, and improved after adding Cu2+ and Zn2+ ions. As we hypothesized, ClavA in the presence of Zn2+ showed the greatest increase in activity, with a 16-fold increase, effectively making ClavA-Zn2+ as active as ClavC, the most potent peptide in the clavanin family.62 ClavC, on the other hand, was found to remain unaffected by the addition of any of the metal ions reported in this study. We hypothesized that the increase in activity for ClavA when combined with Zn2+ ions was the result of the presence of His17 and His21 in its sequence. To explore this, mutants of ClavA containing substitutions to one or both of these potentially important histidines were synthesized. The H17A mutant, ClavA-H17A, and the double mutant, ClavA-H17AH21A, had lower activity in the absence of additional Zn2+ ions, whereas ClavA-H21A remained as active as the wild type. Depending on the position of histidine to alanine substitutions, an increase in the helical content of peptides can be observed,63 which in turn can result in an increase in antimicrobial 1406
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ClavA Exerts Its Bactericidal Activity Faster in the Presence of Zn2+. We next sought to determine the rate at which ClavA and ClavA-Zn2+ killed bacteria. To do this, we performed a time kill kinetic study by counting viable colonies from a bacterial suspension in a solution of ClavA or ClavAZn2+. Figure 3 shows that when both ClavA and ClavA-Zn2+
Figure 1. Effect of varied Zn2+ concentrations on the antimicrobial activity of 8 μM ClavA. These data are plotted on a logarithmic scale on the y-axis. Values represent the average of three trials ± the standard error of the mean (*P ≤ 0.05; N.S., not significant).
determined the concentration of Zn2+ ions in the hemolymph, a fluid found in invertebrates that serves as a blood analogue, using atomic absorption spectroscopy. The concentration of Zn2+ ions was determined to be 26.7 ± 7.1 μM, indicating that enough Zn2+ ions are found in the hemolymph to potentiate the activity of ClavA. Some percentage of these ions is likely to be tightly bound to proteins in the hemolymph or hemocytes; however, considering the fact that the viability of E. coli was 128 64 2
>128 64 2
observed across all of the time points (Figure 8B). The nicked form can be observed to increase at first, but after 30 min, it starts to decrease; at the same time, the amount of linearized DNA increases. To prove that ClavA and Zn2+ together are required for DNA cleavage to occur, several controls were run. Panels C and D of Figure 8 show that, upon exposure to ClavA in the absence of Zn2+, the relative concentrations of each of the forms of DNA remained constant. The chelator DTPA is necessary for this system because Zn2+ has a ubiquitous presence in laboratory settings.76 This same unchanging phenotype can also be observed for Zn2+ and DNA (Figure 8E,F), as well as DNA in the absence of treatment (Figure 8G,H). The level of DNA damage was also found to be dependent on the concentration of ClavA-Zn2+ present in the sample, with lower ClavA-Zn2+ concentrations resulting in lower relative nuclease activity (not shown). These results imply that DNA damage could be a part of the mechanism of ClavA, specifically when bound to Zn2+.
a
JW5831, JW1848, and JW1847 are deletion mutants of znuA, znuB, and znuC, respectively. Each column represents the MIC against each mutant strain of bacteria.
peptide that does not utilize Zn2+, remains unchanged. For the ΔznuB and ΔznuC strains (JW1848 and JW1847, respectively), the MIC for ClavA alone increases beyond the scope of the experiment, whereas the MIC for ClavA-Zn2+ increases 16-fold. For these strains, the activity of ClavC improves slightly, but this variation could be due to a side effect of the mutation process. The remaining activity of ClavA in the mutants is most likely due to the fact that some zinc is likely to diffuse into ZnuB without the aid of ZnuA. Overall, it appears that cytoplasmic zinc is important to the activity of ClavA, especially when the peptide is not pre-exposed to zinc. To prove that the change in activity is due to the decreased internal Zn2+ concentration from the deletion of the zinc transport system, and not just some feature of the deletion of a membrane protein, we measured the activity of ClavA, ClavAZn2+, and ClavC against three E. coli strains with deletions to the nickel transport system, NikABC. Overall, although the MIC values are not the same, as expected for different E. coli strains, the pattern observed for the wild type remains (i.e., ClavA shows improved activity in the presence of additional Zn2+ ions). These results confirm that the deletion of Zn2+ transporters, and the subsequent decrease in intracellular Zn2+ concentration, is what causes the increase in MIC observed above (Table 4).
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DISCUSSION In these studies, ClavA has been shown to have increased antimicrobial activity in the presence of Zn2+ not only through a 16-fold reduction in MIC but also through an increased rate of cell killing. The work presented above not only provides insight into the activity of ClavA in the presence of Zn2+ ions but also sheds light on how ClavA interacts with bacteria. The results of the membrane depolarization, β-galactosidase leakage, and potassium leakage experiments combine to paint a picture of a peptide that is membrane active and forms small pores. This behavior has been previously described by van Kan and co-workers, who established the important role played by phenylalanine and glycine residues in the interaction of ClavA with the bacterial membrane.46,67 The question that arises from our results is how Zn2+ ions potentiate the activity of ClavA. It is conceivable that like DCD-1L, a peptide that oligomerizes into a trimer of dimers,77,78 ClavA uses Zn2+ ions to selfassemble into a higher oligomeric state to destabilize the bacterial membrane. Our ESI-MS data (Figure S8) indicate the presence of ClavA2-Zn2+ (a dimer of ClavA bound through a Zn2+ ion) in the gas phase; however, such a complex might not exist in solution. On the other hand, higher oligomers, ClavAxZn2+, could also exist in solution but were not detected under the ionization conditions used in the ESI-MS experiment. Interestingly, binding of zinc to His17 and His21 can result in a nucleation site for α-helix folding once the peptide interacts with the bacterial membrane,79 because the CD measurements determined in buffer indicated that upon binding of Zn2+ the αhelicity of ClavA increases from 4.2 to 9.6%. Alternatively, the interaction of Zn2+ with the phospholipid membrane is known to result in less hydrophilic phosphate groups,80 which could favor the binding and internalization of ClavA without the need for an oligomer to form. Both scenarios need to be tested before any conclusions can be reached. It is clearer that a large component of the synergy between ClavA and zinc ions occurs in the cytoplasmic space. As evidenced by the microscopy images, ClavA and/or a ClavAZn2+ complex is internalized before cell killing occurs. Furthermore, E. coli mutants with deleted zinc transporters are less susceptible to the antimicrobial activity of ClavA. It is unlikely that a putative ClavA-Zn2+ complex will use the ABC transporter designed for Zn2+ given the large difference in size between the metal ion and any ClavA-Zn2+ complex. Within the cytoplasmic space, the ClavA-Zn2+ complex can target different
Table 4. Antimicrobial Activity of ClavA, ClavA-Zn2+, and ClavC against Three Mutant E. coli Strains with Deleted Nickel Ion Transportersa peptide
MIC (JW3441) (μM)
MIC (JW3442) (μM)
MIC (JW3444) (μM)
ClavA ClavA-Zn2+ ClavC
32 8 4
32 8 2
32 8 2
a JW3441, JW3442, and JW3444 are deletion mutants of nikA, nikB, and nikD, respectively. Each column represents the MIC against each mutant strain of bacteria.
ClavA-Zn2+ Cleaves Supercoiled DNA in Vitro. Because cytoplasmic zinc appears to be important to the function of ClavA and microscopy shows ClavA present in the cytoplasm, it is likely that ClavA-Zn2+ is forming in the cytoplasm and damaging an internal target. While most AMPs are active against bacterial membranes, a small subset has been shown to be internalized and bind to DNA.72−75 Therefore, ClavA-Zn2+ was tested for its in vitro DNA cleavage ability. As shown in Figure 8A, DNA cleavage by ClavA preincubated with Zn2+ ions can be visibly observed after 60 min at room temperature, with the intensity of the supercoiled and nicked forms decreasing, along with a concomitant increase in the linearized form. A steady decrease in the level of supercoiled DNA was 1410
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Biochemistry
Figure 8. In vitro DNA cleavage gels and quantification. (A) Gel of ClavA-Zn2+ cleaving DNA as can be observed by the decrease in nicked and supercoiled DNA concentrations, especially visible at 60 and 120 min. (B) Quantification of the concentration of the DNA forms after cleavage with ClavA-Zn2+. The increase in the linearized concentration, along with the decrease in the supercoiled DNA concentration, is evidence of DNA cleavage. (C) Gel of ClavA with the Zn2+ chelator DTPA. The chelator was necessary because of the ubiquitous nature of Zn2+. The gel for ClavA alone can be found in Figure S7. (D) Quantification of the concentration of the DNA forms after incubation with ClavA and the chelator DTPA. No cleavage is reported. (E) Gel of DNA incubated with Zn2+. No cleavage occurs. (F) Quantification of the concentration of the DNA forms after incubation with Zn2+. No change in concentration is observed. (G) Gel showing DNA incubated in buffer for various times. No cleavage occurs in buffer alone. (H) Quantification of the DNA incubated in buffer. The concentrations of the three forms remain constant over time. Legend: S, supercoiled DNA; N, nicked DNA; L, linear DNA.
biomolecules. Herein, we propose that like other antimicrobial peptides, the ClavA-Zn2+ complex targets DNA.22,26,74,81−83 The proposed nuclease activity of ClavA in combination with Zn2+ ions is a novel mechanism for natural AMPs that bind zinc. Histatin 5, a human salivary peptide, binds to copper and zinc ions and has nuclease activity;84,85 however, when the copper-binding site is removed, the DNA cleavage activity is abolished despite the presence of Zn2+.85 Other host defense peptides and/or proteins that interact with Zn2+ ions have been previously reported; however, no DNA cleavage activity has been observed in those cases. For example, human protein S100A12, a protein expressed and released by neutrophils,86 acts by binding and sequestering Zn2+ ions, depriving bacteria of such an essential metal ion.87 The aforementioned DCD-1L, a peptide derived from dermcidin, adopts an α-helical conformation in which the binding of the Zn2+ ions occurs via residues in an i, i + 4 motif, but no DNA cleavage activity has been reported.77 Kappacin, an AMP produced from κcasein found in bovine milk, enhances its membranolytic effect in the presence of Zn2+ ions.88 Similarly, an antibacterial hexapeptide isolated from human amniotic fluid containing one lysine, two glycines, and three glutamic acids potentiates its activity by Zn2+ via an unknown mechanism.89 Interestingly, Cu2+ and Mg2+ cations do not have the same effect on this hexapeptide.89 Brogden et al. have also reported that ovine pulmonary surfactant-associated anionic AMPs require zinc as a cofactor.90 However, not all anionic AMPs increase their activity in the presence of zinc ions. For instance, maximin H5, an anionic AMP isolated from the toad Bombina maxima, does not increase its activity in the presence of up to 1 mM zinc ions. Finally, we can also add to this collection of examples mutants of the Cardin and Weintraub motif peptides containing histidine residues instead of the amino acids Lys and Arg. These peptides become more active in the presence of Zn2+ ions; however, no cytoplasmic activity has been reported. For the mutants of the Cardin and Weintraub motif peptides, it was hypothesized that the metal ion increases the affinity of the peptides for heparin.91
Overall, we hypothesize that the already recognized membrane activity of ClavA combines with its nuclease activity to create a putative “one−two punch” of antimicrobial activity. Additional studies will be necessary to elucidate the structure of the ClavA complex with Zn2+. A better understanding of the role of Zn2+ ions in the chemistry of ClavA and other tunicate AMPs is key to improving our understanding of zinc trafficking and its role in the immune system of tunicates. In addition, the unique strategy reported here for ClavA will be useful for the development of novel AMP-based strategies for the treatment of infections. This is particularly important because ClavA is being targeted for the development of new peptide-based antimicrobial strategies because the peptide and its nanoformulations have been shown to be effective for the clearing of sepsis infections in mice.44,92
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b01046. Additional experimental details (PDF) Movie 1 (AVI) Movie 2 (AVI) Movie 3 (AVI) Movie 4 (AVI)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Alfredo M. Angeles-Boza: 0000-0002-5560-4405 Funding
This work was supported by a grant from the Lyme Disease Association and generous start-up funds from the University of Connecticut. 1411
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Biochemistry Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful to Prof. Peter Setlow and Prof. Ashis Basu for their generous gifts of B. subtilis and E. coli, respectively. S.A.J. thanks Daben Libardo for his review and comments.
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ABBREVIATIONS ClavA, clavanin A; ClavA-Zn2+, clavanin A incubated with Zn2+; AMPs, antimicrobial peptides; HDPs, host defense peptides; Fmoc, fluorenylmethyloxycarbonyl; TMR, 5(6)-carboxytetramethylrhodamine; LB, Luria-Bertani broth; MES, 2-(Nmorpholino)ethanesulfonic acid buffer; LB 5.5, Luria-Bertani broth adjusted with 2-(N-morpholino)ethanesulfonic acid buffer to pH 5.5; MES 5.5, 2-(N-morpholino)ethanesulfonic acid buffer adjusted to pH 5.5; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; AAS, atomic absorption spectroscopy; DiSC3(5), 3,3′-dipropylthiadicarbocyanine iodide; T-X100, Triton X-100; IPTG, isopropyl β-D-1-thiogalactopyranoside; ONPG, o-nitrophenylβ-galactoside; ClavC, clavanin C; ClavA-TMR, clavanin A labeled with 5(6)-carboxytetramethylrhodamine; ClavA-TMRZn2+, clavanin A labeled with 5(6)-carboxytetramethylrhodamine incubated with Zn2+.
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DOI: 10.1021/acs.biochem.6b01046 Biochemistry 2017, 56, 1403−1414
