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Correlations of Cationic Charges with Salt Sensitivity and Microbial Specificity of Cystine-stabilized β-Strand Antimicrobial Peptides*

  • James P. Tam
    Correspondence
    To whom correspondence should be addressed: Vanderbilt University, Dept. of Microbiology and Immunology, A-5119 MCN, Nashville, TN 37232-2363. Tel.: 615-343-1465; Fax: 615-343-1467
    Affiliations
    Department of Microbiology and Immunology, Vanderbilt University, A5119 MCN, Nashville, Tennessee 37232-2363
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  • Yi-An Lu
    Affiliations
    Department of Microbiology and Immunology, Vanderbilt University, A5119 MCN, Nashville, Tennessee 37232-2363
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  • Jin-Long Yang
    Affiliations
    Department of Microbiology and Immunology, Vanderbilt University, A5119 MCN, Nashville, Tennessee 37232-2363
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grant AI46164 from the United States Public Health Service.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:October 23, 2002DOI:https://doi.org/10.1074/jbc.M208429200
      The electrostatic interaction of the charge cluster of an amphipathic peptide antibiotic with microbial membranes is a salt-sensitive step that often determines organism specificity. We have examined the correlation between charge clusters and salt insensitivity and microbial specificity in linear, cyclic, and retro-isomeric cystine-stabilized β-strand (CSβ) tachyplesin (TP) in a panel of 10 test organisms. Cyclic tachyplesins consisting of 14 and 18 amino acids are constrained by an end-to-end peptide backbone and two or three disulfide bonds to cross-brace the anti-parallel β-strand that approximates a “β-tile” structure. Circular dichroism measurements of β-tile TPs showed that they displayed ordered structures. Control peptides containing the same number of basic amino acids as TP but lacking disulfide constraints were highly salt sensitive. Cyclic TP analogues with six cationic charges were more broadly active and salt-insensitive than those with fewer cationic charges. Reducing their proximity or number of cationic charges, particularly those with three or fewer basic amino acids, led to a significant decrease in potency and salt insensitivity, but an increased selectivity to certain Gram-positive bacteria. An end-group effect of the dibasic N-terminal Lys of TP in the open-chain TP and its retroisomer was observed in certain Gram-negative bacteria under high-salt conditions, an effect that was not found in the cyclic analogs. These results suggest that a stable folded structure together with three or more basic amino acids closely packed in a charged region in CSβ peptides is important for salt insensitivity and organism specificity.
      CSβ
      cystine-stabilized β-strand
      Acm
      acetamidomethyl
      DCC
      N,N-dicyclohexylcarbodiimide
      DCM
      dichloromethane
      DIC
      N,N-diisopropylcarbodiimide
      DIEA
      N,N-diisopropylethylamine
      DMF
      dimethylformamide
      Me2SO
      dimethylsulfoxide
      HOBt
      N-hydroxybenzotriazole
      MALDI/MS
      matrix-assisted laser desorption ionization mass spectrometry
      MBHA resin methylbenzhydrylamine resin
      MeBzl, methylbenzyl
      MIC
      minimal inhibition concentration
      PG-1
      protegrin-1
      RP-HPLC
      reverse phase-high performance liquid chromatography
      Rt
      retention time
      RTD-1
      rhesus theta defensin
      TCEP
      tris(carboxyethyl)phosphine
      TFE
      trifluoroethanol
      TP-1
      tachyplesin-1
      TSB
      trypticase soy broth
      Boc
      t-butyloxycarbonyl
      LPS
      lipopolysaccharide
      A common feature shared among the 500 antimicrobial peptides found in nature is their tendency to form amphipathic structures that cluster basic and hydrophobic amino acids into distinctive regions (
      • Zasloff M.
      ). This secondary structure is important for productive interactions of antimicrobial peptides with the microbial surfaces in models proposed independently by Shai (
      • Shai Y.
      ), Matsuzaki (
      • Matsuzaki K.
      ), and Huang and co-workers (
      • Yang L.
      • Weiss T.M.
      • Lehrer R.I.
      • Huang H.W.
      ) to explain the microbial killing mechanisms. In these models, the charged region provides the initial interaction with the negatively charged headgroups of the microbial surfaces whereas the hydrophobic region facilitates the displacement of lipids and entry of the peptide into the cell interior.
      Cystine-stabilized β-strand (CSβ)1 peptides belong to a major structural family of antimicrobial peptides that are characterized by one or more cystine bonds in their β-strand scaffoldings. This family is richly represented by defensins, protegrins, and tachyplesins that generally possess a cluster of three or more basic amino acids in their charged regions. These CSβ peptides exhibit various degrees of salt sensitivity. The protegrins and tachyplesins are salt insensitive whereas the α- and β-defensins are rapidly inactivated at physiological salt concentration of about 100 mm (high salt condition). Furthermore, the salt-dependent inactivation of β-defensin-1, an antimicrobial peptide found in lung epithelia, may play an important role in innate immunity in cystic fibrosis (
      • Goldman M.J.
      • Anderson G.M.
      • Stolzenberg E.D.
      • Kari U.P.
      • Zasloff M.
      • Wilson J.M.
      ). The charged region may be useful in designing membrane-active transportants for intracellular delivery of functionally active cargoes that include peptides and proteins to inhibit protein-protein interactions (
      • Futak S.
      • Suzuki T.
      • Ohashi W.
      • Yagami T.
      • Tanaka S.
      • Ueda K.
      • Sugiura Y.
      ). Recent findings have shown that synthetic peptides containing six or more basic amino acids in continuous sequences are cell permeable and can act as transportants to eukaryotic cells (
      • Wender P.A.
      • Mitchell D.J.
      • Pattabiraman K.
      • Pelkey E.T.
      • Steinman l.
      • Rothbard J.B.
      ). However, the importance of basic amino acids present in a charge cluster of an amphipathic antimicrobial peptides under physiological conditions is poorly understood. To this end, we have used tachylesin-1 as a model because it is one of the most potent antimicrobial peptides known today (
      • Nakamura T.
      • Furanaka H.
      • Miyata T.
      • Tokunaga F.
      • Muta T.
      • Iwanaga S.
      • Niwa M
      • Taokao T.
      • Shimonishi Y.
      ,
      • Miyata T.
      • Tokunaga F.
      • Yoneya T.
      • Yoshikawa K.
      • Iwanaga S.
      • Niwa M.
      • Takao T.
      • Shimonishi Y.
      ). TP-1 exhibits a broad-spectrum activity against bacteria and fungi with minimal inhibition concentration (MIC) values in the submicromolar range under high salt conditions that may render some antimicrobial peptides inactive. Furthermore, TP-1 has also been shown to play a role in the proinflammatory response because it forms complexes with bacterial lipopolysaccharides that neutralize the factor C-activating activity of LPS in a manner similar to that of anti-LPS factor (
      • Nakamura T.
      • Furanaka H.
      • Miyata T.
      • Tokunaga F.
      • Muta T.
      • Iwanaga S.
      • Niwa M
      • Taokao T.
      • Shimonishi Y.
      ).
      Tachyplesins isolated from horseshoe crabs consisting of 17–19 amino acids contain two cross-bracing disulfides stabilizing an antiparallel β-strand connected by a reverse turn as determined by two-dimensional NMR (
      • Kawano K.
      • Yoneya T.
      • Miyata T.
      • Yoshikawa K.
      • Tokunaga F.
      • Terada Y.
      • lwanage S.
      ). They are similar in structure and activity profile to protegrins (
      • Kokryakov V.N.
      • Harwig S.S.
      • Panyutich E.A.
      • Shevchenko A.A.
      • Aleshina G.M.
      • Shamova O.V.
      • Korneva H.A.
      • Lehrer R.J.
      ), a family of peptides found in pig intestines. Because of their potency and relatively small sizes, these peptides are attractive targets for structure-activity studies that may lead to therapeutics to treat infections.
      Cyclic CSβ antimicrobial peptides known as cyclotides have also been discovered in plants and animals (
      • Gustafson K.R.
      • Sowder II R.C.
      • Henderson L.E.
      • Parsons I.C.
      • Kashman Y.
      • Cardellina II J.H.
      • McMahon J.B.
      • Buckheit Jr., R.W.
      • Pannell L.K.
      • Boyd M.R.
      ,
      • Witherup K.M.
      • Bogusky M.J.
      • Anderson P.S.
      • Ramjit H.
      • Ransom R.W.
      • Wood T.
      • Sardana M.
      ,
      • Saether O.
      • Craik D.J.
      • Campbell I.D.
      • Sletten K.
      • Juul J.
      • Norman D.G.
      ,
      • Claeson P.
      • Goransson U.
      • Johansson S.
      • Luijendijk T.
      • Bohlin L.
      ,
      • Hallock Y.F.
      • Sowder II R.C.
      • Pannell L.K.
      • Hughes C.B.
      • Johnson D.G.
      • Gulakowsk R.
      • Cardellina II J.H.
      • Boyd M.R.
      ). These end-to-end constrained peptides include the Rhesus theta defensin (RTD-1) that has been identified from Rhesus monkey leukocytes (
      • Tang Y.-Q.
      • Yuan J.
      • Ösapay G.
      • Ösapay K.
      • Tran D.
      • Miller C.J.
      • Ouellette A.J.
      • Selsted M.E.
      ). RTD-1, an 18-amino acid and salt-insensitive cyclic peptide, contains a β tile-like structure with a two β-strand framework constrained by three evenly spaced cross-bracing disulfide bonds that partition RTD-1 into a four ring-like structure (Fig. 1). Because the structures and TP-1 are relatively similar, the stable β-tile template of RTD-1 can be exploited for structure-activity study (
      • Tam J.P., Lu, Y.-A.
      • Yang J.-L.
      ,
      • Tam J.P., Wu, C.
      • Yang J.-L.
      ). Previously, we have replaced the hydrophobic amino acids with glycine in β-tile tachyplesin analogues and found that they retain most of their antimicrobial activity and salt insensitivity (
      • Tam J.P., Lu, Y.-A.
      • Yang J.-L.
      ), suggesting that the constraint elements of the β-tile design that provide the conformational stability are sufficient to exert activity in high salt conditions.
      Figure thumbnail gr1
      Figure 1Amino acid sequences and topology of charged amino acid in RTD-1 , PG-1 , TP-18, cTP-18, and their retropeptides.
      The topology of charge cluster in tachyplesins and the CSβ antimicrobial peptides is due to their folded structure. Their electrostatic interactions with the microbial surfaces can be weakened under high salt conditions of 100 mm NaCl similar to physiological ionic conditions. In tachyplesins, most of the charged residues are clustered on one side of the β-tile structure. By maintaining a β-tile structure that provides a stable structure, we reasoned that it may be possible to correlate the number of basic amino acids in tachyplesin analogues to salt sensitivity. In cyclic peptides, there appears to be a correlation between the number of charges and salt sensitivity. Cyclic peptides having about 30 residues such as plant cyclotides that include kalata and circulin A and B as well as cyclopsychotride (
      • Gustafson K.R.
      • Sowder II R.C.
      • Henderson L.E.
      • Parsons I.C.
      • Kashman Y.
      • Cardellina II J.H.
      • McMahon J.B.
      • Buckheit Jr., R.W.
      • Pannell L.K.
      • Boyd M.R.
      ,
      • Witherup K.M.
      • Bogusky M.J.
      • Anderson P.S.
      • Ramjit H.
      • Ransom R.W.
      • Wood T.
      • Sardana M.
      ,
      • Saether O.
      • Craik D.J.
      • Campbell I.D.
      • Sletten K.
      • Juul J.
      • Norman D.G.
      ,
      • Claeson P.
      • Goransson U.
      • Johansson S.
      • Luijendijk T.
      • Bohlin L.
      ) with a cystine-knot motif (
      • Pallaghy P.K.
      • Nielsen K.J.
      • Craik D.J.
      • Norton R.S.
      ,
      • McDonald N.Q.
      • Hendrickson W.A.
      ,
      • Narasimhan L.
      • Singh J.
      • Humblet C.
      • Guruprasad K.
      • Blundell T.
      ) are salt sensitive and are far less basic than the salt-insensitive tachyplesins, protegrins, and RTD-1 that contain six cationic charges.
      The charge cluster in an amphipathic peptide may also be a result of the side chain and the N-terminal amines of a dibasic amino acid. The open-chain CSβ structures in tachyplesins, protegrins, and bactenecin-1 contain an invariable basic amino acid, Lys or Arg, at their N terminus and a carboxamide at their C terminus. The end group effect in providing localized cationic charges to confer salt insensitivity can be conveniently examined by comparing TP-1 with its retroisomer that maintains similar amino acid compositions as TP-1 but contains transposed N-terminal Lys and C-terminal Gly in its sequence. Furthermore, the end-group effect of the open-chain TP-1 can be compared with cyclic TP-1, which does not have end groups.
      In this report, we describe the correlation of cationic charges with activity spectra and salt sensitivity using linear, cyclic, and retroisomeric tachyplesin-1 analogs. In particular, we have exploited the β-tile design to constrain 14- and 18-residue tachyplesin analogs with an up-and-down arrangement of side chains (Fig. 1). Our results show that the number, proximity, and topology of charged amino acids are important contributions to organism specificity and salt sensitivity.

      DISCUSSION

      We have exploited the β-tile template based on the cyclic cystine-stabilized β-strand structure of RTD-1 to study the importance of charge clusters in the full-length and truncated tachyplesin to salt sensitivity and organism specificity. The β-tile has an advantage to maintain a stable structure due to its multiple intramolecularly constraints. Our previous study has shown that the 18-residue β-tile peptides display stable β-strand structures (
      • Tam J.P., Lu, Y.-A.
      • Yang J.-L.
      ,
      • Tam J.P., Wu, C.
      • Yang J.-L.
      ,
      • Tam J.P., Lu, Y.-A.
      • Yang J.-L.
      ). Conformational stability in a folded structure appears to be an important factor contributing to salt insensitivity. These β-tile peptides with four or more cationic charges are generally broadly active and salt insensitive whereas the linear or cyclic control peptides with six cationic charges as TP-18 but without disulfide constraint are found to be considerably less active and more salt sensitive. These results are in agreement with previous structure-activity studies that focus on the role of the disulfide bonds of tachyplesins. Deletion of two disulfide bridges has caused a significant decrease in all activities (
      • Matsuzaki K.
      • Nakayama M.
      • Fukui M.
      • Otaka A.
      • Funakoshi S.
      • Fujii N.
      • Bessho K.
      • Miyajima K.
      ,
      • Matsuzaki K.
      • Yoneyama S.
      • Fujii N.
      • Miyajima K.
      • Yamada K.
      • Kirino Y.
      • Anzai K.
      ). Although Rao (
      • Rao A.G.
      ) has found that antimicrobial activity can be retained when all four cysteines are simultaneously substituted with other amino acids suggesting that disulfide-bonded β-strand structure may not be absolutely essential for antimicrobial activity, it is likely that the study by Rao is performed under low salt conditions in which linear tachyplesins retain significant antimicrobial activity as observed in our control peptides.
      The topological clusters of cationic charges in CSβ-stabilized antimicrobial peptides can be roughly classified into two categories, mono-cluster and bi-cluster. Tachyplesins belong to the mono-cluster with four or more of the cationic charges clustered on one side of the amphipathic surface. The mono-cluster arrangement is also found in the one-disulfide bactenecin-1 with four cationic charges clustered at one end of a CSβ structure. RTD-1 is also mono-clustered with all its cationic charges distributed fairly uniformly on the top face of the β-strands. In contrast, protegrins are bi-clustered with three cationic charges distributed on each end of the anti-parallel strands separated a hydrophobic region. Similar observation can be made for α-helical peptides that are known to be salt-insensitive. Both cercopins from silk moth and magainins from frogs contain monoclusters of cationic residues contributed by four or more basic amino acids. Taken together, they suggest the cationic charge region in a monocluster amphipathic structure of an antimicrobial peptide may require four or more basic amino acids to be broadly active and salt insensitive.
      The minimal number of basic amino acids in a charge cluster of the β-tile tachyplesin analogs appears to be four to afford broad organism specificity and salt insensitivity. TP-18, cTP-18, and their retroisomers containing a cluster of six basic amino acids are broadly active against four Gram-negative and three Gram-positive bacteria as well as three fungi, exhibiting MIC values in a narrow range, and generally, under 1 μm in low and high salt conditions. Truncating the 18- to 14-member ring and retaining a cluster of six basic amino acids in cTP-14a and cTP-14c does not appear to be critical in affecting their broad spectrum activity spectra, but generally lead to an increase in salt sensitivity. A possible explanation is that the ring truncation deletes hydrophobic amino acids and alters the hydrophobicity of these analogs that also contribute to salt sensitivity and organism specificity. For example, both cTP-14a and its retroisomer rcTP-14b are broadly active. Under low salt conditions, cTP-14a missing the four hydrophobic residues in the D ring of cTP-18 (Cys7,12, Tyr8, and Ile11) but retaining six cationic amino acids shows comparable antimicrobial activity (within 2-fold difference) of cTP-18 in eight of ten test organisms with E. coli and E. faecalis being exceptions. Under high salt conditions and again comparing to cTP-18, cTP-14a shows a selective decrease of 3–9-fold in potency against three of the four test Gram-negative bacteria but only one of the three Gram-positive bacteria (Table III). The activity profile of rcTP-14b, the retro analog of cTP-14a, also shows increased salt sensitivity similar to cTP-14a. Interestingly, the activity profiles of cTP-14a and cTP-14b are fairly similar to RTD-1, which is generally more active in low than in high salt conditions.
      The separation of basic amino acids in a cluster also contributes to organism specificity and salt sensitivity. An advantage of the β-tile design is that their proximity can be readily adjusted by shortening the ring size. The β-tile ccTP-18 differs from cTP-18 and contains a cluster of four basic amino acids and an additional constraint, a cystine replacing two Arg in cTP-18. Consequently, it is less cationic but more hydrophobic than cTP-18, and also significantly less potent than cTP-18 and TP-18. Thus, the ccTP-14 peptides designed to increase the proximity of the charge cluster and to restore the charge/hydrophobic balance as TP-18 are generally more active than ccTP-18. The antimicrobial activity ccTP-14a lacking the D ring of ccTP-18 increases 3-fold in low salt and almost 5 fold in high salt conditions. The importance of increasing the proximity of charge clusters is evident in ccTP-14c containing only three charged amino acids that are 2-to-3-fold more active than ccTP-18 in both low and high conditions. Furthermore, the decrease of cationic charges also leads organism specificity. They are generally more active against Gram-positive than Gram-negative bacterial and fungi. The ccTP-14d containing two cationic charges increases its selectivity to Gram-positive bacteria with MICs 1–3.8 μm and is considerably less active against Gram-negative bacteria and fungi with MICs of 7–112 μm. It loses significant activity againstP. aeruginosa and is nearly inactive against C. kefyr.
      Another effect of increasing proximity of charge clustering is placing a dibasic amino acid as a part of a cationic cluster at the N terminus of an open-chain peptide. Dibasic amino acids such as Arg or Lys are commonly found at the N termini of many antimicrobial peptides. TP-18 has a dibasic Lys at the amino terminus and the Gly at the C terminus. Thus, TP-18 contains both α- and ε-amine at amino position not found in the retroisomer rTP-18. We show that the presence of an N-terminal Arg or N-terminal Lys in a cationic cluster may also contribute to salt insensitivity. When TP-18 and rTP-18 are compared under high salt conditions, TP-18 is found to be 6-, 4-, and 2-fold more potent than rTP-18 in three of the four tested Gram-negative bacteria, E. coli, P. aeruginosa, and K. oxytoca, respectively. The end-group effect also has an adverse effect on M. luteus, one of three Gram-positive bacteria being tested. Under high-salt conditions and comparing with TP-18, the MIC values of rTP-18 decrease 10-fold from 0.1 to 1 μm. Because of the close-chain arrangement, cyclic peptides do not have the end-group problem seen in the open-chain peptides. Consequently, the end-group effect on the Gram-negative bacteria is not observed in the corresponding set of cyclic peptides cTP-18 and rTP-18. Our results are consistent with the antimicrobial effects of known retroisomers that contain open-chain peptides with helical or β-strand structures. Merrifield et al. (
      • Merrifield R.B.
      • Juvvadi P.
      • Andreu D.
      • Ubach J.
      • Boman A.
      • Boman H.
      ,
      • Juvvadi P.
      • Vunnam S.
      • Merrifield R.B.
      ) have reported that retroisomers of the α-helical cecropin-melittin hybrids are as active as the parent peptide against five test bacterial strains, only one bacterial strain is resistant to the retroisomer.
      Although the retroisomers of TP-18, cTP-18, or cTP-14 and their parents have the same calculated hydrophobicity values, their experimental values as determined by RP-HPLC show small variations (Table I). In each pair, the retention time of a retroisomer is found to elute 0.5–2.5 min longer than its parent peptide. These results suggest that the direction of amide backbone may also play a subtle role in their interactions with microbial surfaces of interactions.
      Based on the models on the mechanism of actions of antimicrobial peptides (
      • Shai Y.
      ,
      • Matsuzaki K.
      ,
      • Yang L.
      • Weiss T.M.
      • Lehrer R.I.
      • Huang H.W.
      ), their initial interactions with microbial membranes heavily populated by negatively charged phospholipids are believed to be electrostatic. Thus, increasing ionic strength under the high salt conditions will likely weaken the electrostatic charge interactions and the activity of the antimicrobial peptides. Our results suggest that the topology and quantity of a charge cluster may provide low affinity molecular recognition to microbial surfaces and confer organism specificity and salt insensitivity. The principle of clustering cationic and hydrophobic regions of antimicrobial peptides has inspired the design of novel antimicrobials with β- and γ-peptide backbones (
      • Liu D.
      • DeGrado W.F
      ) as well as dendrimeric peptides with δ- and ε-peptide backbones (
      • Karig G.
      • Fuchs A.
      • Busing A.
      • Brandstetter T.
      • Scherer S.
      • Bats J.W.
      • Eschenmoser A.
      • Quinkert G.
      ). Studies in the requirements of cationic clusters in the amphipathic design may further the understanding and lead to the development of antimicrobial peptides with high specificity and potency that are useful as therapeutic agents.

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