Using Chemical Synthesis to Probe Structure–Activity Relationships of

6 days ago - Glycocin F, a bacteriocin produced by Lactobacillus plantarum KW30, is glycosylated with two N-acetyl-d-glucosamine sugars, and has been ...
1 downloads 3 Views 2MB Size
Download PDF
Articles Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX

Using Chemical Synthesis to Probe Structure−Activity Relationships of the Glycoactive Bacteriocin Glycocin F Sean W. Bisset,†,‡ Sung-Hyun Yang,§ Zaid Amso,§ Paul W. R. Harris,§,‡ Mark L. Patchett,† Margaret A. Brimble,§,‡ and Gillian E. Norris*,†,‡ †

Institute of Fundamental Sciences, Massey University, Colombo Rd, Palmerston North 4442, New Zealand Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, New Zealand § School of Chemical Sciences, The University of Auckland, 23 Symonds St, Auckland 1142, New Zealand ‡

S Supporting Information *

ABSTRACT: Glycocin F, a bacteriocin produced by Lactobacillus plantarum KW30, is glycosylated with two Nacetyl-D-glucosamine sugars, and has been shown to exhibit a rapid and reversible bacteriostasis on susceptible cells. The roles of certain structural features of glycocin F have not been studied to date. We report here the synthesis of various glycocin F analogues through solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL), allowing us to probe the roles of different structural features of this peptide. Our results indicate that the bacteriostatic activity of glycocin F is controlled by the glycosylated interhelical loop, while the glycosylated flexible tail appears to be involved in localizing the peptide to its cellular target.

B

been produced in vitro.20 Additionally, the chemical synthesis of sublancin 168 has been reported.21,22 While all six glycocins contain variations of the (C−Xn−C)2 motif consistent with nested disulfide bonds, a folding motif found in a number of unmodified eukaryote-produced toxins,17 disulfide bonds are apparently not required for enterocin 96 activity.23 The sugar moieties in glycocins are linked to the peptide scaffold through specific cysteine, serine, or threonine residues, with only glucose and N-acetyl-D-glucosamine (GlcNAc) moieties having been identified in the natural products.13,17 The mechanisms of action of these glycocins are unknown, although sublancin 168 is thought to target the large mechanosensitive channel and/or the glucose phosphoenolpyruvate phosphotransferase system (PTS) of susceptible cells.24 Glycocin F (GccF) is a 43 residue peptide produced by Lactobacillus plantarum KW30 and contains two nested disulfide bonds and two GlcNAc moieties, one β-O-linked to Ser18, and the other β-S-linked to Cys43.15,25 Glycosylated cysteine residues are relatively rare in prokaryotes and have only been confirmed in three other bacteriocins, sublancin 168, ASM1 and thurandacin.17,20,26 In contrast to sublancin, which is bactericidal,24 purified GccF has been found to exhibit a potent and reversible bacteriostatic effect on target cells. Interestingly, recovery from stasis occurs when free GlcNAc is added.15 It possesses a moderate spectrum of activity, inhibiting certain strains of some Lactobacillus and Enterococcus species at low

acteriocins are ribosomally synthesized antimicrobial peptides, produced by nearly all species of bacteria, and usually exhibit narrow-range bactericidal or bacteriostatic modes of action.1 They inhibit the growth of susceptible cells via five main mechanisms: formation of lethal membrane pores by targeting cell membrane molecules,2,3 inhibition of DNA replication,4 inhibition of transcription,5 interference with septum formation, and interference with cell wall synthesis.6 Bacteriocins from lactic acid bacteria (LAB) have received considerable attention for many years due to their use and potential use in the food industry. They are generally regarded as safe (GRAS), are easily digested by human gastrointestinal proteases, and are stable over wide pH and temperature ranges. They also have antimicrobial activity against various pathogens and most importantly show no cross-resistance with antibiotics.7,8 Bacteriocins that induce bacteriostasis in target cells include a number of peptides produced by Lactobacilli, although no mechanisms have been elucidated to date.9−11 Bacteriocins produced by Gram positive bacteria are structurally diverse, and many classification schemes have been developed. In 2013, Cotter and co-workers7 developed a simplified classification scheme that divided low molecular weight bacteriocins into those that are modified (bearing posttranslational modifications which typically contribute to their stability and/or activity12,13) and those that are unmodified. One distinctive group of post-translationally modified bacteriocins is the glycocins, which have one or two amino acid side chains bearing covalently linked mono- or disaccharide moieties.12 Five glycocins (glycocin F, ASM1, sublancin, enterocin F4-9, and enterocin 9614−19) have been purified from bacterial cultures, whereas thurandacin has only © XXXX American Chemical Society

Received: January 17, 2018 Accepted: April 12, 2018

A

DOI: 10.1021/acschembio.8b00055 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology nanomolar concentrations, and one strain of Escherichia coli, JM109(DE3), is susceptible to micromolar concentrations (Supplementary Figure S49). GccF has been biochemically confirmed to be glycoactive, as enzymatic27 removal of the loop-bound, O-linked GlcNAc abolished activity.15 The only other bacteriocins that are unequivocally glycoactive are enterocin F4-9, which requires glycosylation on serines 46 and 38 for activity20 and enterocin 96.23 The NMR structure of GccF was determined in 2011,25 followed by that of sublancin 168 in 2014,26 confirming that these two glycocins share a similar core peptide scaffold, despite their different modes of action. Genetic analysis of seven Lb. plantarum mutants selected for resistance to GccF suggested a GlcNAc-specific phosphoenolpyruvate phosphotransferase system (PTS) transporter may be acting as a receptor for GccF.28 Although a subsequent knockout of the PTS transporter gene and complementation tests confirmed its involvement in GccF activity, just how the formation of a GccF:transporter complex induces bacteriostasis remains unknown. PTS transporter transmembrane (EIIC) domains typically form homodimers (a notable exception being the heterodimeric mannose PTS transporter), with the other domains/subunits (EIIA and EIIB) located in the cytoplasm.29 Import of sugars via dimeric PTS components is believed to occur through an alternating elevator-style shuttling mechanism.30 As both GccF GlcNAcs are necessary for full activity, it is possible that one or both GlcNAcs bind to the sugar-binding sites of EIIC domains. Using I-TASSER, we created a model of the Lb. plantarum GlcNAc transporter based on the structure of the EIIC domain of the diacetyl chitobiose PTS transporter from Bacillus cereus31 and “in silico” docked the two GccF GlcNAcs into the monosaccharide binding sites,17 which showed that the length of the C-terminal tail could be critical for this putative mode of GccF-PTS interaction. In order to better understand the contribution of the various structural features of GccF to its inhibitory activity, we wanted to alter different parts of the molecule and test these modified analogues for activity, but despite much effort production of recombinant GccF was unsuccessful. With the development of an efficient in vitro total chemical synthetic protocol for GccF,32 these experiments became possible. Previous work had shown that the nested disulfide bonds are absolutely required for GccF activity.15 However, whether both disulfide bonds are necessary or just one, and if so, which one, is not known. As the glycosylated loop is bounded by the inner disulfide bond and is relatively constrained, loop size and conformation could also play a role in GccF activity. Furthermore, selective proteolysis of the molecule had already shown that removal of the Cterminal His-Cys(β-GlcNAc) decreased activity ∼75 fold, while removing the entire C-terminal “tail” reduced activity ∼200 fold.15 Clearly, the identity of the tail residues, and possibly the length of the tail, contribute to activity.

replaced by Lys (GccFSer38Lys) which gave an overall positive charge, which we predicted would decrease antimicrobial activity. The tail was also reduced in length by three amino acids, through removal of residues Ser38, Ser39, and Ser40 to produce GccFΔSer38−40. We also aimed to investigate the effect on GccF activity of replacing the S-linked GlcNAc with either glucose (GccFCys43‑Glc) or mannose (GccFCys43‑Man; Figure 1).

Figure 1. Modifications present in each glycocin F analogue are highlighted. Modifications in blue represent changes to the interhelical loop. Modifications in red represent changes to the flexible tail, and the purple and green ovals signify deletions of the respective disulfide bonds. Dotted circles represent sites of amino acid deletions, while colored-in circles represent amino acids that have been substituted. GccFΔGlyI is missing Gly13. GccFΔGlyII is missing Gly13 and Gly15, and GccFΔGlyIII is missing Gly13, Gly15, and Gly19. The scissors indicate the enzymatic cleavage site of trypsin.

We had previously shown that the two disulfides present in GccF are necessary for activity and that their reduction to cysteine abolished all activity. What is unknown is how each individual disulfide, and the ring size they span, contribute to the three-dimensional structure and the activity of the molecule. The role of each disulfide linkage was probed by synthesizing capped cysteine variants en route to the synthesis of the full-length molecule. This was accomplished by irreversible reaction with the alkylation reagent iodoacetamide to produce the monodisulfide analogues GccFCys5/28 and GccFCys12/21 (Figure 1). We also examined the consequences of contracting the 8-residue loop between the Cys12−Cys21 disulfide bond which contains the important O-linked GlcNAc. This was performed by removal of residues Gly13, Gly15, and/ or Gly19 to give a seven, six or five amino acid loop, respectively, to produce the analogues GccFΔGlyI, GccFΔGlyII, and GccFΔGlyIII. The synthetic strategy devised to prepare ‘native’ GccF and four tail-modified analogues (GccFSer38Lys, GccFΔSer38−40, GccFCys43−Man, and GccFCys43−Glc) was analogous to the most

■

RESULTS AND DISCUSSION Chemical Synthesis of Modified Glycocin F Analogues. To unravel the molecular architecture responsible for the bioactivity of glycocin F, we aimed to chemically modify key regions in the native structure. We first focused on determining whether the charge and length of the C-terminal tail affect activity. It was previously found that synthetic glycocin F-NH2 was only half as active as the wild-type molecule when the only chemical difference between the two molecules was an amidated C-terminus.32 Thus, Ser38 was B

DOI: 10.1021/acschembio.8b00055 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology

Figure 2. (a) The original scheme for glycocin F chemical synthesis, as described in Brimble et al.,32 involved synthesizing the peptide in three fragments, which were then ligated between Met11 and Cys12 and between His27 and Cys28. The latter ligation, however, resulted in epimerization of His27 (green). (b) The current scheme for glycocin F chemical synthesis only contains one ligation step, between Met11 and Cys12.

recently described synthesis of native GccF.33 The original protocol involved creating three polypeptide fragments [Lys1Met11-COSCH2CH2CO-(Lys)5, Thz12-His27-COSBn (Bn = benzyl; Thz = thiazolidine), and Cys28-Cys43] through solid phase peptide synthesis, followed by native chemical ligation and oxidative folding to produce a full-length, properly folded peptide with both disulfides intact.32 However, epimerization at

one of the linkage points (between His27 and Cys28) resulted in two populations of mature peptides bearing either D-His27 or L-His27 (Figure 2a). An alternative strategy was therefore developed to reduce the epimerization of His27 and used successfully for the preparation of the above tail-modified analogues. The new strategy involved synthesis of the sidechain protected, middle peptide fragment Thz12-His27-COOH C

DOI: 10.1021/acschembio.8b00055 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology Table 1. Activity of Synthetic and Enzymatically Prepared GccF Derivatives Modification Native C-terminal tail

Interhelical loop

Disulfide bonds a

Analogue

Description

IC50 nM

GccFNative GccFSyn GccFCys43SerΔGlcNAc GccFΔSer38−40 GccFCys43−Man GccFCys43−Glc GccFSer38Lys GccF1−32ΔGlcNAca GccF1−41ΔGlcNAca GccFΔGlyI GccFΔGlyII GccFΔGlyIII GccFCys12/21 GccFCys5/28

Native peptide purified from Lb. plantarum KW30 Chemically synthesized, native GccF Cys43−GlcNAc replaced with unmodified Ser Ser38, Ser39, and Ser40 removed Cys43 GlcNAc replaced with Mannose Cys43 GlcNAc replaced with Glucose Ser38 replaced with Lys Residues 33−43 enzymatically removed with trypsin Residues 42−43 enzymatically removed with chymotrypsin Gly13 removed from loop Gly13 and Gly15 removed from loop Gly13, Gly15, and Gly19 removed from loop Disulfide bridge between Cys12 and Cys21 disrupted Disulfide bridge between Cys5 and Cys28 disrupted

2 ± 0.20 1.13 ± 0.20 107 ± 21 50 ± 8.4 1015 ± 203 661 ± 132 1.71 ± 0.19 ∼400 ∼150 57.6 ± 6.8 2480 ± 540 2710 ± 370 No activity 8560 ± 1710

Enzymatically prepared GccF analogues.

out using slightly different methodology on an amino-capped C-terminus analogue consistently increased the IC50 from 0.9 nM for WT GccF to 1.6 nM.32 In this case, the positive charge on His42 would not be neutralized, giving the C-terminal region a positive charge, which also appears to be detrimental to activity. These results imply that the environment of the cysteine-linked GlcNAc is important for the function of the molecule, and that it functions better in a neutral environment. The Role of Residues 33−43, the “Flexible Tail”, in Activity. GccFΔSer38−40 possesses both GlcNAc moieties linked as in the wild-type bacteriocin but has Ser38, -39, and -40 removed, making the tail considerably shorter (Figure 1). Surprisingly, GccFΔSer38−40 with an IC50 of approximately 50 nM was more active than both GccFCys43SerΔGlcNAc and GccF1−41ΔGlcNAc (Table 1). We expected that the removal of three neutral residues from the tail would have a significant effect if, as predicted, the C-terminal sugar interacts with the PTS cytosolic-facing binding site. We therefore expected to see a reduction in activity similar to that observed for GccFCys43SerΔGlcNAc. The fact that it is more active than constructs without the sugar suggests that both sugars do not need to bind simultaneously to the PTS transporter, leading to the hypothesis that the function of the PTS may be to position the GccF molecule in the cell membrane near to its true target. In contrast, enzymatic removal of 10 C-terminal residues plus the C-terminal GlcNAc to produce GccF1−32ΔGlcNAc increased the IC50 to 400 nM, a 200-fold decrease in activity showing that residues 33−37 must have a role in GccF activity. Whether this contribution is due to the properties of residues involved (two histidines, two serines, and a glycine) or whether a tail of a certain length is physically necessary for full function is still being explored. Because the string (residues 35−40) of neutral relatively small residues in the tail is likely to contribute to its flexibility25 (which may be important for function) and previous analogues with an amidated C-terminus exhibited slightly reduced activity,32 we replaced serine 38 with a lysine to produce GccFSer38Lys. Lysine carries a positive charge at acidic and neutral pH and is a bulky side chain that may restrict flexibility, hence it was placed at the edge of the group of serines while avoiding proximity to Tyr41. This change had a similar effect to amidation of the C-terminal cysteine, raising the IC50 from 1.13 to 1.71 nM.

via 9-fluoroenylmethoxycarbonyl (Fmoc)-solid phase peptide synthesis on 2-chlorotrityl chloride-based (2-ClTrtCl) resin, followed by low-temperature, solution-phase thioesterification of the C-terminal carboxylic acid of His27 and global deprotection.33 For the synthesis of the disulfide-disrupted, decreased loop and GccFCys43SerΔGlcNAc analogues, another strategy was developed using only two fragments (Lys1−Met11 and Cys12−Cys43 for GccFCys5/28 and GccFCys12/21; Lys1−Met11 and Cys12−Cys42 for GccFΔGlyI; Lys1−Met11 and Cys12− Cys41 for GccFΔGlyII; Lys1−Met11 and Cys12−Cys40 for GccFΔGlyIII; Lys1−Met11 and Cys12−Ser43 for GccFCys43SerΔGlcNAc). This simpler synthetic route resulted in the successful prevention of His epimerization and reduction of the total number of synthetic steps from eight to five (Figure 2b). All prepared GccF analogues had the expected monoisotopic masses, showing the correct modifications had been incorporated, and had intact disulfide bonds as designed (Supporting Information Table S7). Far UV circular dichroism (CD) spectra showed that analogues with changes in the tail residues and the monosaccharide linked to the peptide were similar to that of GccFSyn, showing that the secondary structure of the molecule had not been disrupted (Supporting Information Figure S47b). The spectra of the analogues in which the size of the loop was decreased (Supporting Information Figure S47a) showed what appeared to be reduced helical content, while the far UV spectra of GccFCys12/21 and GccFCys5/28 showed that these two analogues had lost alpha-helicity (Supporting Information Figure S47c). The C-Terminal Tail Sugar Is Not Essential for Activity. GccFCys43SerΔGlcNAc contains an non-glycosylated Ser at position 43, resulting in a monoglycosylated analogue (Figure 1). The IC50 of GccFCys43SerΔGlcNAc is 107 nM, making it approximately 95-fold less active than di-glycosylated, GccFSyn (Table 1). Previous findings had shown that when residues 42 and 43 plus the C-terminal GlcNAc were removed to produce GccF1−41ΔGlcNAc, the IC50 increased to 150 nM.15 While these results implicate the C-terminal GlcNAc in GccF activity, evidence indicates that His42 could also be involved. At pH 5.6, the pH of the media used in these experiments, His42 will carry a positive charge. As such, it will act to neutralize the negative charge on the C-terminal carboxylate, which may be important for GlcNAc-receptor recognition. Interestingly, assays carried D

DOI: 10.1021/acschembio.8b00055 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology

Figure 3. GccFSyn, along with GccF analogues containing different sugars at the C-terminal Cys43 position, were tested for bacteriostatic activity against Lb. plantarum ATCC 8014. Analogues were tested at 10× their IC50 concentrations. (a) GccFSyn was tested with and without 1 mM GlcNAc, which can be seen to protect the cells for the duration of the experiment. (b) GccFCys43−Man was tested with and without 1 mM mannose, which can be seen to not provide any protective effect. (c) GccFCys43−Glc was tested with and without 1 mM glucose, which, as in the case of b, provided no protection. (d) Trypsin-digested GccF (GccF1−32ΔGlcNAc) was tested with and without 1 mM GlcNAc, which can be seen to protect the cells from stasis. For both GccFCys43−Man and GccFCys43−Glc, GlcNAc provided a protective effect.

The Identity of the C-Terminal Sugar Is Important. In order to test the role of the C-terminal GlcNAc in targeting GccF to a specific membrane receptor, a mannose (GccFCys43−Man) and a glucose (GccFCys43−Glc) were substituted for the GlcNAc linked to Cys43 (Figure 1). The rationale behind this is the relatively high abundance of mannose and glucose PTS transporters in the cell membranes of Firmicutes, proteins that could possibly act as receptors for these monosaccharide-modified GccFs. As the essential Ser18-linked GlcNAc remained unchanged, we expected that if the Cterminal monosaccharide was not recognized by the GlcNAc specific PTS transporter, both new analogues would have similar activities to GccFCys43SerΔGlcNAc. Unexpectedly, both analogues displayed IC50 values that were significantly higher (less active) than GccFCys43SerΔGlcNAc, with GccFCys43−Man exhibiting an IC50 of ∼1000 nM, and GccFCys43−Glc exhibiting an IC50 of ∼660 nM (Table 1). The IC50 values of GccF with these modifications were higher than those so far discussed, adding further credence to the hypothesis that the function of the cysteine-linked GlcNAc is to localize the bacteriocin adjacent to its primary cellular target. Both mannose and glucose (each of which have PTS transporters) linked to Cys43 appear to severely attenuate activity, probably by preventing GccF from interacting with its as yet unidentified primary target. This hypothesis was further supported by experiments designed to test the ability of GlcNAc to rescue cells in the presence of either GccFCys43−Man or GccFCys43−Glc . Lb. plantarum ATCC 8014 cells were grown in an excess of each construct (10 μM GccFCys43−Man and 6 μM GccFCys43−Glc, 10

times the IC50 values) supplemented with either 1 mM GlcNAc or 1 mM of the specific Cys43-linked sugar. Additionally, Lb. plantarum ATCC 8014 cells were also grown with 10 times the IC50 concentration of trypsin-digested GccF (GccF1−32), with and without supplementation of 1 mM GlcNAc (Figure 3). Cells were protected from the bacteriostatic effects of all three analogues for at least 15 h (900 min) by the addition of 1 mM GlcNAc. However, 1 mM mannose and 1 mM glucose had no effect on the inhibition of cell growth by GccFCys43−Man or GccFCys43−Glc, respectively, which is further evidence that the Cterminal sugar is not directly contributing to the bacteriostasis. The Interhelical Loop Region Is Essential for Glycocin F Activity. Previous experiments had shown that the GlcNAc attached to Ser18 is vital for activity.15 However, the role of the loop in correctly presenting the monosaccharide to its receptor is not known. Three analogues designed to test the effect of decreasing the size of the interhelical loop were synthesized. Glycines 13, 15, and 19 were sequentially removed to form GccFΔGlyI, GccFΔGlyII, and GccFΔGlyIII (Figure 1). The removal of one glycine from the loop decreased activity approximately 50-fold (IC50 of GccFΔGlyI ∼ 57 nM; Table 1), which is similar to the effect seen when the length of the C-terminal tail was reduced by removing three serine residues. When either two or three glycines were removed from the interhelical loop (GccFΔGlyII and GccFΔGlyIII, respectively), activity was substantially reduced, with a greater than 2000-fold decrease in the IC50 for both modifications (Table 1). CD spectra of these analogues showed a significant reduction in α-helical structure (Supporting Information Figure S47). As both nested disulfide bonds remain intact in these analogues, reducing the size of the E

DOI: 10.1021/acschembio.8b00055 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology

GccF has provided a new avenue with which to study targeted modifications of GccF without the need for recombinant modification.32 Using this approach, we have been able to study modifications to the flexible C-terminal tail, interhelical loop, and C-terminal sugar of GccF, as well as separately disrupting each of the two nested disulfide bonds. Typically, targeted mutagenesis is used to study functionality in bacteriocins using minor modifications.35 While some of the analogues tested here could conceivably have been generated using recombinant technologies, other modifications might not have been attainable. For example, directing the addition of two different sugars to the loop and tail positions of glycocin F is currently impossible to encode genetically. Any change in the loop between residues 12 and 21 significantly reduces the bacteriostatic activity and appears to decrease the α-helical content of GccF (Supporting Information Figure S47). The results suggest that the receptor not only recognizes the O-linked GlcNAc but interacts with a surface that may comprise a significant portion of the molecule. Space filling models of the loop region of GccF show that a large cavity is formed between the underside of the loop and the ‘stem’ of the molecule (formed by the helices17). Although there are no hydrogen bonds between the GlcNAc and the polypeptide chain, the sugar appears to be nestled in a groove formed by the top surface of the loop. Looking down on this surface, it is easy to visualize how both the peptide and GlcNAc could be involved in recognition by a receptor. It is also clear that any disruption to the loop will change the way the GlcNAc is presented on the polypeptide chain to a potential receptor, and may change the architecture of the (C−(X)n−C)2 scaffold. That the disulfide bonds appear to also be essential for the activity of GccF is not surprising. The (C−Xn−C)2 peptide scaffold is common to many unmodified toxins.36−43 For all of these toxins, and the glycocins GccF and sublancin, both disulfide bonds must be intact for activity, presumably through maintaining the integrity and stability of the loop and/or holding the two α-helices in a specific position. Combining this result with that of decreasing the interhelical loop size, it appears that the Ser18-linked GlcNAc, interhelical loop, and αhelices are all required for GccF to successfully bind to its primary target. The role of the glycosylated C-terminal tail seems to be primarily to localize the bacteriocin to the correct cell-surface receptor, as removal of the tail reduces the IC50 value but does not abolish bacteriostatic activity (Table 1). Substitution of the C-terminal sugar with glucose or mannose further decreases the IC50 values (∼660 nM for GccFCys43−Glc and ∼1000 nM for GccFCys43−Man, compared to 400 nM for GccF1−32ΔGlcNAc), suggesting the C-terminal tail sugar binds first, tethering the bacteriocin to the cell, facilitating the efficient interaction of the Ser-linked GlcNAc with its primary target. The receptors being targeted by the tail sugars of GccFCys43−Glc and GccFCys43−Man are presumably localized to regions of the cell surface distant from the primary GccF target, hence the decrease in activity. Interestingly, the inability of supplemented mannose or glucose to rescue the cells from stasis indicates that the tail interaction itself does not initiate bacteriostasis (Figure 3). The results of this work suggest an order of events with regard to GccF-induced bacteriostasis and a model for GccF activity (Figure 5). First, as GccF diffuses through the thick peptidoglycan layer of Gram-positive cells, the C-terminal GlcNAc samples its environment to find extracellular targets (i.e., the GlcNAc-specific PTS EIIC). Binding to this receptor

interhelical loop is possibly placing strain on the structure, causing at least one of the helices to unwind. Whether the change in structure of the loop is alone responsible for the loss of activity or whether it is due to a more global structural change has yet to be determined. On the basis of the effects of these changes, it appears GccF-receptor interaction involves structures of the peptide loop bearing the O-linked GlcNAc and possibly residues of the flanking helical regions. Disulfide Bonds Contribute to the Stability of the Loop. Previous work has shown that reduction of GccF completely abolished activity, indicating that the disulfide bonds are vital for GccF’s functionality.15 However, what is not known is whether both disulfides are important, and the effect each one has on activity. To test this, two analogues were synthesized with the cysteines of each disulfide capped to prevent formation of the disulfide between Cys5 and Cys28 (GccFCys5/28) or Cys12 and Cys21 (GccFCys12/21). GccFCys12/21 was the only analogue tested that lacked any detectable activity, even on an agar indicator plate (Figure 4).

Figure 4. An indicator plate containing Lb. plantarum ATCC 8014 was titrated with 5 μL drops of either 45 μM GccF (top left), 500 μM GccFCys5/28 (top right), or 500 μM of GccFCys12/21 (bottom). Activity of GccFCys5/28 was substantially reduced compared to native GccF, while GccFCys12/21 was completely inactive.

The loss of the disulfide bond between Cys12 and Cys21 will increase the size of the glycosylated loop from 8 to 22 residues and possibly result in some loss of helical structure. GccFCys5/28, though active, was substantially less active than GccFSyn, with an IC50 of ∼8.6 μM (Table 1). While the loss of the disulfide bond between Cys5 and Cys28 should not affect the loop, it could conceivably promote flexibility in the region due to the axis of rotation around the Cys12/Cys21 disulfide. Its loss also significantly reduced the α-helical content of the peptide (Supporting Information Figure S47c), suggesting that the conformation of the peptide (C-Xn−C)2 scaffold is important for function. Glycocin F Possibly Works through a Two-Step Binding Mechanism. Glycocin F is one of the few bacteriocins with verified bacteriostatic activity. It is somewhat remarkable because under optimized culture conditions, the most susceptible cells (Lb. plantarum ATCC 14917) are inhibited within 15 min of exposure to picomolar concentrations of GccF, which equates to approximately 24 molecules per cell (Supporting Information Figure S48). However, despite its discovery over two decades ago,34 the mechanism of action has remained elusive. The recent chemical synthesis of F

DOI: 10.1021/acschembio.8b00055 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology

bicarbonate (AmBic), 60% acetonitrile (MeCN), and 100 mL fractions collected. Fractions were tested for activity using an indicator plate assay,15 and those with activity (eluted by AmBic+ 60% MeCN) were pooled then concentrated by lyophilization. The lyophilized powder was dissolved in distilled water before being further purified by HPLC (2 mL injection volume; Jupiter C18 10 × 250 mm; linear gradient: 0.1% trifluoroacetic acid (TFA), 20% MeCN to 0.08% TFA, 40% MeCN over 30 min at a flow rate of 4 mL min−1). Peak fractions were collected manually and tested for activity using indicator plates. Active fractions were then combined and lyophilized for storage at −20 °C. Purified bacteriocin was quantified using absorbance at 280 and 205 nm. The yield of purified GccF was 0.5−1 mg L−1 of culture. Antimicrobial Activity Assays. Bacteriocin analogues to be tested were serially diluted in MRS media (Merck) and added to the wells of a flat-bottomed 96-well multititer plate. An equal amount of log phase Lb. plantarum ATCC 8014 cells was added to each well to a final volume of 300 μL and growth monitored by measuring the optical density at 600 nm (OD600) in a plate reader (MultiSkan GO) over 15 h at 30 °C. Growth of bacteriocin-treated cells was compared directly to an untreated control, and plots of inhibition over time were generated. All growth curves were done in triplicate. IC50 values were determined by plotting the maximum inhibition achieved by each concentration and interpolating the concentration that would be expected to provide 50% growth inhibition.

Figure 5. Models of GccF−receptor interaction. (a) Both GlcNAc moieties of GccF bind to the same receptor, the GlcNAc-specific PTS EIIC domain (green). (b) The tail-bound GlcNAc binds to the GlcNAc-specific PTS EIIC domain, while the loop bound GlcNAc binds to a nearby membrane-bound primary target (red). The cell membrane is shown in gray. In both models, only binding by the loopbound GlcNAc results in bacteriostasis.

effectively ‘tethers’ the bacteriocin to the cell. The GlcNAcbearing interhelical loop is then positioned to interact with a nearby target that may or may not be the PTS, setting up a rapid signaling response that results in bacteriostasis. There are many things about this hypothetical mechanism that need to be tested, including the identity of this unknown receptor and the mechanism used to transmit the ‘stop signal’ to the cell. Work is ongoing in our laboratory to find the missing pieces of the puzzle and to see if they can be applied to develop a new targeted antimicrobial therapy.

■

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.8b00055. Description of chemical synthesis, chemical and physical characterization of proteins, and supporting figures (DOCX)

METHODS

Synthesis of Glycocin F Analogues. Peptide syntheses were accomplished on either 2-chlorotrityl chloride (2-ClTrtCl)-based resin via 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis (FmocSPPS) or aminomethyl polystyrene resin via tert-butyloxycarbonyl (Boc)-SPPS. The purified peptides were then ligated under native chemical ligation (NCL) conditions in the presence of 100 mM 4mercaptophenylacetic acid (MPAA) and 20 mM tris(2-carboxyethyl)hydrochloride (TCEP·HCl). Following NCL, the formyl protecting groups of Trp residues and the acetate groups of GlcNAc moieties were removed by treatment with a mixture of hydrazine/2mercaptoethanol (v/v; 25:37.5) in N-methyl-2-pyrrolidone (NMP)/ guanidine hydrochloride (Gn·HCl)/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; v/v/v; 22.5:5.6:9.4). Following solid phase extraction, the glycopeptides were individually subjected to oxidative folding by treatment with 2 mM cysteine, 0.25 mM cystine, and 0.1 mM (ethylenedinitrilo)tetra-acetic acid (EDTA) in 1.5 M Gn· HCl (pH 8.2) at 4 °C. Pleasingly, all glycocin F analogues were produced as single folded species after 16 h, as determined by reversed phase-high performance liquid chromatography (RP-HPLC). Details of the synthesis of each of the GccF analogues are provided in the Supporting Information. RP-HPLC purification afforded glycocin F analogues in >95% purity. The presence of disulfide bonds in all analogues was confirmed by ESI-MS, and the masses were consistent with the calculated mass of the desired glycopeptides (Supporting Information Table S7). Production and Purification of Native Glycocin F. Native glycocin F was purified from the supernatant of a 3-day-old Lb. plantarum KW30 culture as follows. Briefly, 50 mL of an overnight culture of Lb. plantarum KW30 in MRS media was used to inoculate 4 L of MRS, which was incubated without shaking or aeration for 3 days at 25 °C. Cells were removed from the media by centrifugation at 6000g for 30 min, after which the pH was reduced, if necessary, to approximately pH 4.5 using formic acid. A total of 100 mL of SPSephadex (GE Healthcare) pre-equilibrated in 50 mM sodium formate at pH 4.0 was mixed with the 4 L of clarified supernatant and stirred overnight at RT using an overhead stirrer. The unbound fraction was decanted until the resin formed a slurry that was packed into an XK30 (GE-Healthcare) column. The column was then washed with 1 L of equilibration buffer, and bound protein was eluted with 1 L of 50 mM MOPS (pH 7.2), followed by 300 mL of 50 mM ammonium

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Sean W. Bisset: 0000-0003-1974-1047 Margaret A. Brimble: 0000-0002-7086-4096 Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS S.W.B. was supported by a doctoral scholarship from the Maurice Wilkins Centre for Molecular Biodiscovery. REFERENCES

(1) Cavera, V. L., Arthur, T. D., Kashtanov, D., and Chikindas, M. L. (2015) Bacteriocins and their position in the next wave of conventional antibiotics. Int. J. Antimicrob. Agents 46, 494−501. (2) Kjos, M., Salehian, Z., Nes, I. F., and Diep, D. B. (2010) An Extracellular Loop of the Mannose Phosphotransferase System Component IIC Is Responsible for Specific Targeting by Class IIa Bacteriocins. J. Bacteriol. 192, 5906−5913. (3) Willey, J. M., and van der Donk, W. A. (2007) Lantibiotics: Peptides of Diverse Structure and Function. Annu. Rev. Microbiol. 61, 477−501. (4) Heddle, J. G., Blance, S. J., Zamble, D. B., Hollfelder, F., Miller, D. A., Wentzell, L. M., Walsh, C. T., and Maxwell, A. (2001) The antibiotic microcin B17 is a DNA gyrase poison: Characterisation of the mode of inhibition. J. Mol. Biol. 307, 1223−1234. (5) Kuznedelov, K., Semenova, E., Knappe, T. A., Mukhamedyarov, D., Srivastava, A., Chatterjee, S., Ebright, R. H., Marahiel, M. A., and Severinov, K. (2011) The Antibacterial Threaded-lasso Peptide

G

DOI: 10.1021/acschembio.8b00055 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology Capistruin Inhibits Bacterial RNA Polymerase. J. Mol. Biol. 412, 842− 848. (6) Hasper, H. E., Kramer, N. E., Smith, J. L., Hillman, J. D., Zachariah, C., Kuipers, O. P., de Kruijff, B., and Breukink, E. (2006) An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II. Science 313, 1636−1637. (7) Cotter, P. D., Ross, R. P., and Hill, C. (2013) Bacteriocins - a viable alternative to antibiotics? Nat. Rev. Microbiol. 11, 95−105. (8) Cleveland, J., Montville, T. J., Nes, I. F., and Chikindas, M. L. (2001) Bacteriocins: safe, natural antimicrobials for food preservation. Int. J. Food Microbiol. 71, 1−20. (9) Atrih, A., Rekhif, N., Moir, A. J. G., Lebrihi, A., and Lefebvre, G. (2001) Mode of action, purification and amino acid sequence of plantaricin C19, an anti-Listeria bacteriocin produced by Lactobacillus plantarum C19. Int. J. Food Microbiol. 68, 93−104. (10) Hernandez, D., Cardell, E., and Zarate, V. (2005) Antimicrobial activity of lactic acid bacteria isolated from Tenerife cheese: initial characterization of plantaricin TF711, a bacteriocin-like substance produced by Lactobacillus plantarum TF711. J. Appl. Microbiol. 99, 77−84. (11) Barbosa, M., Todorov, S., Ivanova, I., Belguesmia, Y., Choiset, Y., Rabesona, H., Chobert, J.-M., Haertlé, T., and Franco, B. (2016) Characterization of a two-peptide plantaricin produced by Lactobacillus plantarum MBSa4 isolated from Brazilian salami. Food Control 60, 103−112. (12) Alvarez-Sieiro, P., Montalban-Lopez, M., Mu, D. D., and Kuipers, O. P. (2016) Bacteriocins of lactic acid bacteria: extending the family. Appl. Microbiol. Biotechnol. 100, 2939−2951. (13) Arnison, P. G., et al. (2013) Ribosomally synthesized and posttranslationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat. Prod. Rep. 30, 108−160. (14) Oman, T. J., Boettcher, J. M., Wang, H. A., Okalibe, X. N., and van der Donk, W. A. (2011) Sublancin is not a lantibiotic but an Slinked glycopeptide. Nat. Chem. Biol. 7, 78−80. (15) Stepper, J., Shastri, S., Loo, T. S., Preston, J. C., Novak, P., Man, P., Moore, C. H., Havlíček, V., Patchett, M. L., and Norris, G. E. (2011) Cysteine S-glycosylation, a new post-translational modification found in glycopeptide bacteriocins. FEBS Lett. 585, 645−650. (16) Maky, M. A., Ishibashi, N., Zendo, T., Perez, R. H., Doud, J. R., Karmi, M., and Sonomoto, K. (2015) Enterocin F4-9, a Novel OLinked Glycosylated Bacteriocin. Appl. Environ. Microbiol. 81, 4819− 4826. (17) Norris, G. E., and Patchett, M. L. (2016) The glycocins: in a class of their own. Curr. Opin. Struct. Biol. 40, 112−119. (18) Izquierdo, E., Wagner, C., Marchioni, E., Aoude-Werner, D., and Ennahar, S. (2009) Enterocin 96, a Novel Class II Bacteriocin Produced by Enterococcus faecalis WHE 96, Isolated from Munster Cheese. Appl. Environ. Microbiol. 75, 4273−4276. (19) Hata, T., Tanaka, R., and Ohmomo, S. (2010) Isolation and characterization of plantaricin ASM1: A new bacteriocin produced by Lactobacillus plantarum A-1. Int. J. Food Microbiol. 137, 94−99. (20) Wang, H., Oman, T. J., Zhang, R., Garcia de Gonzalo, C. V., Zhang, Q., and van der Donk, W. A. (2014) The Glycosyltransferase Involved in Thurandacin Biosynthesis Catalyzes Both O- and SGlycosylation. J. Am. Chem. Soc. 136, 84−87. (21) Hsieh, Y. S. Y., Wilkinson, B. L., O’Connell, M. R., Mackay, J. P., Matthews, J. M., and Payne, R. J. (2012) Synthesis of the Bacteriocin Glycopeptide Sublancin 168 and S-Glycosylated Variants. Org. Lett. 14, 1910−1913. (22) Katayama, H., Asahina, Y., and Hojo, H. (2011) Chemical synthesis of the S-linked glycopeptide, sublancin. J. Pept. Sci. 17, 818− 821. (23) Nagar, R., and Rao, A. (2017) An iterative glycosyltransferase EntS catalyzes transfer and extension of O- and S-linked monosaccharide in enterocin 96. Glycobiology 27, 766−776. (24) Garcia de Gonzalo, C. V., Denham, E. L., Mars, R. A. T., Stülke, J., van der Donk, W. A., and van Dijl, J. M. (2015) The Phosphoenolpyruvate:Sugar Phosphotransferase System Is Involved

in Sensitivity to the Glucosylated Bacteriocin Sublancin. Antimicrob. Agents Chemother. 59, 6844−6854. (25) Venugopal, H., Edwards, P. J. B., Schwalbe, M., Claridge, J. K., Libich, D. S., Stepper, J., Loo, T., Patchett, M. L., Norris, G. E., and Pascal, S. M. (2011) Structural, Dynamic, and Chemical Characterization of a Novel S-Glycosylated Bacteriocin. Biochemistry 50, 2748− 2755. (26) Garcia de Gonzalo, C. V., Zhu, L., Oman, T. J., and van der Donk, W. A. (2014) NMR structure of the S-linked glycopeptide sublancin 168. ACS Chem. Biol. 9, 796−801. (27) Harty, D. W. S., Chen, Y. J., Simpson, C. L., Berg, T., Cook, S. L., Mayo, J. A., Hunter, N., and Jacques, N. A. (2004) Characterisation of a novel homodimeric N-acetyl-beta-D-glucosaminidase from Streptococcus gordonii. Biochem. Biophys. Res. Commun. 319, 439− 447. (28) Drower, K. R. Bacteriostatic diglycocylated bacteriocin glycocin F targets a sugar-specific transporter. Master’s Thesis, Massey University, Manawatu, New Zealand, 2014. (29) Deutscher, J., Ake, F. M. D., Derkaoui, M., Zebre, A. C., Cao, T. N., Bouraoui, H., Kentache, T., Mokhtari, A., Milohanic, E., and Joyet, P. (2014) The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions. Microbiol. Mol. Biol. Rev. 78, 231−256. (30) McCoy, J. G., Ren, Z. N., Stanevich, V., Lee, J., Mitra, S., Levin, E. J., Poget, S., Quick, M., Im, W., and Zhou, M. (2016) The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport. Structure 24, 956−964. (31) Cao, Y., Jin, X. S., Levin, E. J., Huang, H., Zong, Y. N., Quick, M., Weng, J., Pan, Y. P., Love, J., Punta, M., Rost, B., Hendrickson, W. A., Javitch, J. A., Rajashankar, K. R., and Zhou, M. (2011) Crystal structure of a phosphorylation-coupled saccharide transporter. Nature 473, 50−U58. (32) Brimble, M. A., Edwards, P. J., Harris, P. W. R., Norris, G. E., Patchett, M. L., Wright, T. H., Yang, S.-H., and Carley, S. E. (2015) Synthesis of the Antimicrobial S-Linked Glycopeptide, Glycocin F. Chem. - Eur. J. 21, 3556−3561. (33) Amso, Z., Bisset, S. W., Yang, S.-H., Harris, P. W. R., Wright, T. H., Navo, C. D., Patchett, M. L., Norris, G. E., and Brimble, M. A. (2018) Total chemical synthesis of glycocin F and analogues: Sglycosylation confers improved antimicrobial activity. Chem. Sci. 9, 1686−1691. (34) Kelly, W. J., Asmundson, R. V., and Huang, C. M. (1996) Characterization of plantaricin KW30, a bacteriocin produced by Lactobacillus plantarum. J. Appl. Bacteriol. 81, 657−662. (35) Etayash, H., Azmi, S., Dangeti, R., and Kaur, K. (2015) Peptide Bacteriocins - Structure Activity Relationships. Curr. Top. Med. Chem. 16, 220−241. (36) Chagot, B., Dai, L., Pil, J., Tytgat, J., Nakajima, T., Corzo, G., Darbon, H., Ferrat, G., and Pimentel, C. (2005) An unusual fold for potassium channel blockers: NMR structure of three toxins from the scorpion Opisthacanthus madagascariensis. Biochem. J. 388, 263−271. (37) Srinivasan, K. N., Sivaraja, V., Huys, I., Sasaki, T., Cheng, B., Kumar, T. K. S., Sato, K., Tytgat, J., Yu, C., San, B. C. C., Ranganathan, S., Bowie, H. J., Kini, R. M., and Gopalakrishnakone, P. (2002) kappahefutoxin1, a novel toxin from the scorpion Heterometrus fulvipes with unique structure and function - Importance of the functional diad in potassium channel selectivity. J. Biol. Chem. 277, 30040−30047. (38) Saucedo, A. L., Flores-Solis, D., Rodriguez de la Vega, R. C., Ramirez-Cordero, B., Hernandez-Lopez, R., Cano-Sanchez, P., Navarro, R. N., Garcia-Valdes, J., Coronas-Valderrama, F., de Roodt, A., Brieba, L. G., Possani, L. D., and del Rio-Portilla, F. (2012) New Tricks of an Old Pattern Structural Versatility Of Scorpion Toxins With Common Cysteine Spacing. J. Biol. Chem. 287, 12321−12330. (39) Nolde, S. B., Vassilevski, A. A., Rogozhin, E. A., Barinov, N. A., Balashova, T. A., Samsonova, O. V., Baranov, Y. V., Feofanov, A. V., Egorov, T. A., Arseniev, A. S., and Grishin, E. V. (2011) Disulfidestabilized Helical Hairpin Structure and Activity of a Novel Antifungal H

DOI: 10.1021/acschembio.8b00055 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Articles

ACS Chemical Biology Peptide EcAMP1 from Seeds of Barnyard Grass (Echinochloa crusgalli). J. Biol. Chem. 286, 25145−25153. (40) Nakamura, T., Furunaka, H., Miyata, T., Tokunaga, F., Muta, T., Iwanaga, S., Niwa, M., Takao, T., and Shimonishi, Y. (1988) Tachyplesin, A Class Of Antimicrobial Peptide From The Hemocytes Of The Horseshoe-Crab (Tachypleus-Tridentatus) - Isolation And Chemical-Structure. J. Biol. Chem. 263, 16709−16713. (41) Silva, P. I., Daffre, S., and Bulet, P. (2000) Isolation and characterization of gomesin, an 18-residue cysteine-rich defense peptide from the spider Acanthoscurria gomesiana hemocytes with sequence similarities to horseshoe crab antimicrobial peptides of the tachyplesin family. J. Biol. Chem. 275, 33464−33470. (42) Mandard, N., Bulet, P., Caille, A., Daffre, S., and Vovelle, F. (2002) The solution structure of gomesin, an antimicrobial cysteinerich peptide from the spider. Eur. J. Biochem. 269, 1190−1198. (43) Sukumar, M., Rizo, J., Wall, M., Dreyfus, L. A., Kupersztoch, Y. M., and Gierasch, L. M. (1995) The Structure Of Escherichia-Coli Heat-Stable Enterotoxin-B By Nuclear-Magnetic-Resonance And Circular-Dichroism. Protein Sci. 4, 1718−1729.

I

DOI: 10.1021/acschembio.8b00055 ACS Chem. Biol. XXXX, XXX, XXX−XXX