King cobra peptide OH-CATH30 as a potential candidate drug through clinic drug-resistant isolates

2018·3.5 MB·

by  Zhao, Feng

ZOOLOGICAL RESEARCH
King cobra peptide OH-CATH30 as a potential
candidate drug through clinic drug-resistant isolates
FengZhao1,2,4,*,Xin-QiangLan2,YanDu3,Pei-YiChen1,JiaoZhao1,FangZhao1,4,Wen-HuiLee2,YunZhang2,*
1KeyLaboratoryofSubtropicalMedicinalEdibleResourcesDevelopmentandUtilizationinYunnanProvince,DepartmentofBiologyand
Chemistry,PuerUniversity,PuerYunnan665000,China
2KeyLaboratoryofBioactivePeptidesofYunnanProvince/KeyLaboratoryofAnimalModelsandHumanDiseaseMechanismsofChinese
AcademyofSciences,KunmingInstituteofZoology,KunmingYunnan650223,China
3DepartmentofClinicalLaboratories,FirstAffiliatedHospitalofKunmingMedicalUniversity,KunmingYunnan650032,China
4InstituteofComparativeStudyofTraditionalMateriaMedica,InstituteofIntegrativeMedicineofFudanUniversity,Shanghai200433,China
ABSTRACT Keywords: Cathelicidin;Antibacterialagent;Clinical
Cationicantimicrobialpeptides(AMPs)areconsidered isolates;OH-CATH30
asimportantcandidatetherapeuticagents,whichexert
INTRODUCTION
potentmicrobicidalpropertiesagainstbacteria,fungi
The abundant use of traditional antibiotics has resulted in
and some viruses. Based on our previous findings
theemergenceofmanyantibiotic-resistantisolatesworldwide,
king cobra cathelicidin (OH-CATH) is a 34-amino
causingthreateningtohumanhealthandtherateofantibiotic
acid peptide that exerts strong antibacterial and discovery and production has been insufficient to respond to
weak hemolytic activity. The aim of this research this crisis (Fischbach & Walsh, 2009; Nathan, 2004). Recent
is to evaluate the efficacy of both OH-CATH30 and studies have shown that there are many components in the
venom that possess antimicrobial activity. Snake venom,
its analog D-OH-CATH30 against clinical isolates
produced by specialized glands in the snake jawbone, is
comparing with routinely utilized antibiotics in vitro.
a particularly rich source of such antimicrobial compounds
In this study, 584 clinical isolates were tested (Tashimaetal.,2012). Severalvenomcompoundshavebeen
(spanning 2013–2016) and the efficacy of the used for antimicrobial effects, such as phospholipases A2,
candidate peptides and antibiotics were determined metalloproteinases, L-amino acid oxidases and antimicrobial
peptides (De Oliveira Junior et al., 2013). Cathelicidins
by a broth microdilution method according to
have been identified as the main family of naturally-occurring
the CLSI guidelines. Among the 584 clinical
antimicrobialpeptidesfromsnakevenom,whichexhibitpotential
isolates, 85% were susceptible to OH-CATH30
microbicidalpropertiesagainstbacteria,fungiandsomeviruses
and its analogs. Both L- and D-OH-CATH30 (Zhang,2015).Currently,atleast9 cathelicidin-typeantimicrobial
showedhigherefficacyagainst(toward)Gram-positive
bacteria and stronger antibacterial activity against
nearly all Gram-negative bacteria tested compare
Received:01December2017;Accepted:08February2018
with antibiotics. The highest bactericidal activity
Foundation items: This work was supported by grants from the
was detected against Acinetobacter spp., including
NationalNaturalSciencesFoundationofChina(31572268,31560596),
multi-drug-resistantAcinetobacterbaumannii (MRAB)
the Key Research Program of the Chinese Academy of Sciences
and methicillin-resistant Staphylococcus aureus
(KJZD-EW-L03),"YunnanScholar"Program,theYunnanAppliedBasic
(MRSA). The overall efficacy of OH-CATH30 and its
Research Projects (2016FD076), the National Training Program of
analogswashigherthanthatofthe9routinelyused
InnovationandEntrepreneurshipforUndergraduates(201510685001,
antibiotics. OH-CATH30 is a promising candidate
201610685001),andPuerUniversity(RCXM003&CXTD011)
drug for the treatment of a wide variety of bacterial
*Corresponding authors, E-mail: [email protected]; zhangy@
infectionswhichareresistanttomanyroutinelyused
mail.kiz.ac.cn
antimicrobialagents.
DOI:10.24272/j.issn.2095-8137.2018.025
SciencePress ZoologicalResearch39(2):87-96,2018 87
peptideshavebeenidentifiedfromelapidsnakevenoms,most Wepreviouslyreportedanovelcathelicidin-derivedpeptide
of which exhibit potential antimicrobial activities (Falcao et from the king cobra. This reptile cathelicidin was
al., 2014; Zhao et al., 2008). Cationic antimicrobial peptides termed OH-CATH30 and exhibited potential broad-spectrum
(AMPs) are considered as important candidate therapeutic antibacterial activity in vitro and relatively low toxicity in vivo
agents comprising a diverse group of bactericidal molecules (Li et al., 2012; Zhang et al., 2010). The aim of this study
forwhichmicrobialorganismsshowlowerlevelsofresistance, is to evaluate the efficacy of OH-CATH30 and its analog
for example pexiganan, an AMP developed for the treatment D-OH-CATH30(comprisingallD-aminoacidsresidues)against
of diabetic foot infection (Ge et al., 1999). Although several clinicalisolates(collectedfrompatientsinhospital)compared
peptide-antibiotics are well established clinically, such as with routinely utilized antibiotics in vitro. Meanwhile, the
polymixin B, AMPs alone without any modifications have not corresponding information is crucial for the cytotoxicity to
beenwidelyusedinclinicaltreatment(Hancock&Sahl,2006). eukaryotes and the immunogenicity of these peptides, which
AsanuniquefamilyofAMPs,thecathelicidinpeptidesshared hemolysis assay and IL-6 assay were further investigated.
conserved N-terminal domains from a variety of species and Considering that most of AMPs have free radical scavenging
exhibited effective antimicrobial activity and some cytotoxic ability, we also expanded nitric oxide assay and detected the
activity towards eukaryotic cells (Johansson et al., 1998; releaseofnitricoxide(Figure1A).
Zanettietal.,1995).
A
in vitro OH-CATH 30 (L- / D-) in vivo
IL-6
NO IL-6
NO
Low cytotoxity
High bactericidal activity
B
100
OH-CATH30
D-OH-CATH30
80
%) 60
y (
c
n
e
u 40
q
e
Fr
20
0
≤2 4 8 16 32 64 128 >128
MICs of OH-CATH30 and D-OH-CATH30 (μg/mL)
Figure1CytotoxicityofOH-CATH30anditsanalog
A:TheworkflowofcytotoxicitydeterminationtoL-OH-CATH30andD-OH-CATH30;B:DistributionofL-OH-CATH30andD-OH-CATH30MICsfor584bacterialisolates.
88 www.zoores.ac.cn
MATERIALSANDMETHODS (v/v/v). The sample was added along with auto-sampler to
a Thermo Scientific EASY loading column (Thermo Fisher
Ethicsstatement
Scientific,USA)(2cm×100µm,5µm-C18)thentoananalytic
This study was approved by the Ethics Committee of Puer
column (75 µm×100 mm, 3 µm-C18) with a flow rate of 1.2
University and the Biomedical Ethics Committee of the First
mL/min. The presence of peptides was confirmed by an
AffiliatedHospitalofKunmingMedicalUniversity.Thedatawere
absorption peak at 215 nm. The purity of each synthetic
analyzedanonymously,patientidentitydeducedordisclosed.
peptidewasabove95%.Theidentityandpurityofeachpeptide
Clinicalisolates,referencestrainsandmaterials was further confirmed by matrix-assisted laser desorption
A total of 584 clinical isolates were collected between July ionization-time of flight (MALDI-TOF) mass spectrometry
2013 and June 2016, which included 14 different species as (Voyager; Supplementary Figure S2). The survey of the full
follows: (i) Acinetobacter spp. (n=53); (ii) Citrobacter spp. scan MS spectra (m/z 300–1 800) was acquired in the Orbit
(n=7); (iii) Enterobacter spp. (n=41); (iv) Escherichia spp. rapwitha70000-resolution(m/z200).Dynamicexclusionwas
(n=229); (v) Klebsiella spp. (n=126); (vi) Proteus spp. (n=4); set to 15s. The 10 most intense multiply charged ions (z≥2)
(vii)Pseudomonas spp. (n=76); (viii)Salmonellaspp. (n=16); were sequentially isolated and fragmented by higher-energy
(ix) Serratia spp. (n=12); (x) Sphingobacterium spiritivorum collisional dissociation (HCD) with a fixed injection time of 80
(n=1);(xi)Staphylococcusaureus(MSSA)(n=2)andS.aureus ms and 17 500-resolution (m/z 200). The conditions were as
(MRSA)(n=2); (xii)Stenotrophomonasmaltophilia(n=9); (xiii) follows: sprayvoltage,2kV;nosheathandauxiliarygasflow;
Streptococcus pneumoniae. (n=2); and (xiv) Yersinia spp. heated capillary temperature, 250 °C; and normalized HCD
(n=4). The species identification of each clinical isolates collision energy 27 eV; underfill ratio 0.1%. The MS/MS ion
was confirmed with the Vitek 2 system (bioMérieux, France; selectionthresholdwassetat1×105counts.
part of the data shown in Supplementary Figure S1). The
DeterminationofMICs
antibiotic susceptibility of the clinical isolates was determined
MICs were determined by the broth microdilution method
using the Kirby-Bauer disk diffusion method, in accordance
(Wiegand et al., 2008) according to the CLSI guidelines
with the Clinical and Laboratory Standards Institute (CLSI)
(Clinical and Laboratory Standards Institute, 2009). In each
2009 guidelines (Clinical and Laboratory Standards Institute,
well of a 96-well plate, 100 µL of MHB broth and bacteria
2009). Twelve control strains were also included: E. coli
(5×105 colony-forming unit (cfu)/mL) were incubated with
ATCC25922andATCC35218, HaemophilusinfluenzaATCC
variousconcentrationsofantimicrobialsfor16hat37°C.The
49766 and ATCC 49247, S. aureus ATCC 25923 and ATCC
concentrationsoftheantimicrobialswerethesameforeachof
43300,KlebsiellapneumoniaeATCC13883andATCC700603,
the tested bacterial strains in three independent experiments,
S. maltophilia ATCC 8090, Enterococcus faecalis ATCC
and the MIC values were obtained without inter-experiment
29212,EnterobactercloacaeATCC13047andPseudomonas
variations and expressed as µg/mL. For compounds and
aeruginosa ATCC 27853. All strains were cultured in MHB
antibiotics drugs, the interpretation criteria were set by using
Mueller-Hintonbroth(MHB,pH7.2)mediumat37°C.Allother
the CLSI published criteria. The concentration of antibiotics
reagents were of analytical grade and were obtained from
was initially set at 512 µg/mL, then diluted to 4 µg/mL. As for
commercialsources.
the(onlytwoarelisted)peptidesusedinthisstudy(pexiganna,
Antibiotics polymyxinB),theconcentrationwasdilutedfrom256µg/mLto
Ampicillin(Amp)andcefoperazone(CPZ)wereacquiredfrom 2 µg/mL. In each batch of susceptibility tests, the 12 control
theGeneralPharmaceuticalFactoryofHarbinPharmaceutical strainslistedabovewereincludedasaqualitycontrol.
Group(Harbin,China).Roxithromycin(ROX),chloramphenicol
Hemolyticassayandcytokinedetermination
(CHL)andazithromycin(AZM)werefromSangonBiotechCo.,
Hemolytic assays were investigated using human red blood
Ltd. (Shanghai, China). Amikacin (AMK), vancomycin (VAN),
cells in liquid medium as previously reported (Yang et al.,
levofloxacin(LVX)andpolymyxin(PB)werefromSigma-Aldrich
2012). Serial dilutions of the samples were incubated with
Co.LLC(Sigma,St.Louis,MO,USA).
washedredbloodcells(2%)at37°Cfor30min,thecellswere
Antimicrobialpeptidessynthesis thencentrifuged, andtheabsorbanceofthesupernatantwas
Pexiganan(GIGKFLKKAKKFGKAFVKILKK-NH2),OH-CATH30 measuredat540nm. Maximumhemolysiswasdeterminedby
(KFFKKLKNSVKKRAKKFFKKPRVIGVSIPF) and its analog adding1%TritonX-100tothecellsamples(Liuetal.,2012).
D-OH-CATH30 (KFFKKLKNSVKKRAKKFFKKPRVIGVSIPF, An ELISA kit (Neobiosoence) was employed for IL-6
italicsindicateD-aminoacids)weresynthesizedbysolid-phase determination according to the manufacturer’s instructions.
synthesis on an Applied Biosystems Model 433A peptide Nitric oxide was quantified using the Griess chemical method
synthesizer according to the manufacturer’s standard protocols, (Parketal.,1993).
asreportedpreviously(Lietal.,2012). Then,thecrudesynthetic IL-6 detection in vitro: Prior to euthanization, mice were
peptide was purified by reverse-phase high-performance lipid administered 3 mL of saline by intraperitoneal injection. The
chromatography (RP-HPLC, Supplementary Figure S2). Mobile abdomenwaskneadedfor2minbeforebeingopenedupand
phase eluent A consisted of 0.1% TFA (aqueous) and mobile theperitonealfluidwascollected.Afterexcision,themicelivers
phase eluent B consisted of ACN/ddH2O/TFA 90/10/0.08% and kidneys were placed into physiological saline, cut(sliced)
ZoologicalResearch39(2):87-96,2018 89
intopieces,homogenizedwithtrypsin,groundintofluid,passed tested, 85% were susceptible to OH-CATH30 and its analog
throughameshsieveandcentrifugedat2000r/minfor10min. with a MIC≤64 mg/L (Figure 1B). For Escherichia spp. and
The supernatant was discarded and the pellet was washed Klebsiella spp. (61% of all clinical isolates), OH-CATH30
with PBS until the supernatant was clear. Then the cells was more active than the 9 antibiotics tested, and showed
werere-suspendedinF12mediumand190µLofcells(about slightly higher activity than pexiganan. For 53 strains of
1×107) were added to each well of a 96-well plate before the Acinetobacterspp.,includingmultidrug-resistantAcinetobacter
adding10µLofdifferentconcentrationsofOH-CATH30asthe baumannii (MRAB), the MIC value of D-OH-CATH30 was
90
experimental group or F12 medium as the control. The plate doubled comparing with that of OH-CATH30, and polymixin
wasthenincubatedat36°Cfor2h,followedbycentrifugation B also had high rates of susceptibility (77%) to Acinetobacter
at 3 000 r/min for 20 min. The supernatant was retained for spp. compared with other antibiotics, except for pexiganan.
furtherdeterminations. For the 76 strains of Pseudomonas spp., the activity of
IL-6 detection in vivo: Mice were intraperitoneally injected OH-CATH30 and its analog was higher than pexiganan,
with2mLofsaline,thenafter5hwereintraperitoneallyinjected although these peptides are all showed higher activity than
with 5 mg/kg or 10 mg/kg of different types of OH-CATH30 the 9 antibiotics, with the exception of levofloxacin. For the
(Li et al., 2013). Mice were euthanized, then the abdomen 16strainsofSalmonellaspp.,allshowedhighsusceptibilityto
was kneaded for 2 min, after which it was opened and the theantimicrobialpeptidesandantibiotics,withtheexceptionof
peritoneal fluid was collected. After excision, the livers and ampicillinandcefoperazone.Therateofsusceptibility(92%)to
kidneyswereplacedintophysiologicalsaline,slicedintopieces, levofloxacinamongthesespecieswasconsiderablyhigherthan
homogenizedwithtrypsin,groundintofluid,passedthrougha theother8antibiotics.Amongthe14speciesofclinicalisolates
mesh sieve and centrifuged at 3 000 r/min for 20 min. The tested, Serratiaspp., forwhichtherewere12strains, hadthe
supernatant was discarded and the pellet was washed with lowest rate of susceptibility to antimicrobial peptides and the
PBSuntilthesupernatantwasclear. peptide-antibiotic(polymixinB).OH-CATH30inhibitedallofthe
species,comprisingaround10strains,ataconcentrationof≤
Statisticalanalysis
64mg/L,withtheexceptionofProteusspp. andS.maltophilia.
The data was analyzed using two-way ANOVA. The
Of the four strains of S. aureus tested, including two MSSA
experimental data are expressed as MIC and MIC values.
50 90 andtwoMRSA,D-OH-CATH30wasfoundtobemoreeffective
ThelevelofstatisticalsignificancewassetatP<0.05.
atinhibitingMRSAthanOH-CATH30,witharangeof≤8mg/L.
RESULTS Overall, L- OH-CATH30 showed higher efficacy against two
thirdsofthetestedclinicalisolatesthanOH-CATH30.
Susceptibilitytest
Theinvitrosusceptibilityof584clinicalisolatestothreeAMPs Hemolysisassay
and nine antibiotics was tested, which was also represented Generally,thepeptideanditsD-analogshowedlittledifference
by its cytotoxicity of prokaryotes cell (Figure 1A). Table 1 in hemolytic activity to human red blood cells (Figure 1A).
shows the MICs, with ranges of MIC and MIC values, of WhenthedoseofL-OH-CATH30orD-OH-CATH30waslower
50 90
each antimicrobial agent tested against 14 different species. than 125 µg/mL, similar hemolysis rates of about 10% were
Overall, the efficacy of OH-CATH30 and D-OH-CATH30 was observed, but as the dose increased to 250 µg/mL, high
higher when compared with 9 routine antibiotics, and slightly hemolyticactivitiesexceeding70%forthepeptideand80%for
higher than pexiganan. Among the 584 clinical isolates itsD-analogweredetected(Figure2).
100
D-OH-CATH30
80 L-OH-CATH30
%) Saline
sis ( 60 Triton X-100
y
ol
m
ae 40
H
20
0
0 100 200 300
Concentration (μg/mL)
Figure2HemolyticactivityoftheL-andD-OH-CATH30peptides
Peptidesatvariousconcentrationswereincubatedwithhumanredbloodcellsfor4hat37°C.
90 www.zoores.ac.cn
Table1MICsofthreeantimicrobialpeptidesandnineantibioticsdeterminedfor584clinicisolates,andtheMIC andMIC values
50 90
Percentage Percentage
ofisolates ofisolates
OH-CATH30MIC(mg/L) D-OH-CATH30MIC(mg/L) AmpbMIC(mg/L) CPZbMIC(mg/L)
Isolates No. susceptibletoc susceptibletoc
(%) (%)
MIC50a MIC90a Range MIC50a MIC90a Range MIC50a,*** MIC90a,*** Range MIC50a,*** MIC90a,*** Range
Acinetobactersp. 53 16 32 8–>128 8 16 2–>128 >256 >256 8–>256 0 >256 >256 4–>256 2
Citrobactersp. 7 8 16 4–>128 8 >128 4–>128 >256 >256 8–>256 20 >256 >256 4–>256 40
Enterobactersp. 41 8 32 4–>128 8 >128 4–>128 >256 >256 64–>256 0 >256 >256 4–>256 33
Escherichiasp. 229 8 64 2–>128 8 >128 2–>128 >256 >256 8–>256 5 >256 >256 4–>256 25
Klebsiellasp. 126 8 64 4–>128 16 128 2–>128 >256 >256 16–>256 0 >256 >256 4–>256 29
Proteussp. 4 >128 >128 8–>128 8 128 8–>128 >256 >256 16–>256 0 256 256 4–256 33
Pseudomonassp. 76 8 128 4–>128 16 >128 4–>128 >256 >256 16–>256 5 256 >256 4–>256 23
Salmonellasp. 16 8 8 4–>128 4 >128 4–>128 64 >256 4–>256 54 32 >256 4–>256 54
Serratiasp. 12 >128 >128 4–>128 8 64 8–>128 >256 >256 128–>256 0 256 >256 4–>256 36
Sphingobacteriumspiritivorum 1 8 8 8 16 16 16 >256 >256 >256 0 >256 >256 >256 0
Staphylococcusaureus(MSSA)d 2 16 16 16 32 64 32–64 >256 >256 >256 0 >256 >256 >256 0
Staphylococcusaureus(MRSA)e 2 16 32 16–32 8 8 8 >256 >256 >256 0 >256 >256 >256 0
Stenotrophomonasmaltophilia 9 8 >128 4–>128 8 32 4–32 >256 >256 128–>256 0 256 >256 4–>256 13
Streptococcuspneumoniae 2 8 8 8 8 >128 8–>128 >256 >256 >256 0 >256 >256 >256 0
Yersiniasp. 4 8 >128 8–>128 8 >128 8–>128 >256 >256 >256 0 >256 >256 >256 0
ZoologicalResearch39(2):87-96,2018 91
Continued
Percentage Percentage Percentage Percentage
ofisolates ofisolates ofisolates ofisolatese
ROXbMIC(mg/L) AZMbMIC(mg/L) AMKbMIC(mg/L) PBbMIC(mg/L)
Isolates No. susceptible susceptible susceptible susceptible
toc(%) toc(%) toc(%) toc(%)
MIC50a,*** MIC90a,*** Range MIC50a MIC90a,*** Range MIC50a MIC90a,* Range MIC50a MIC90a Range
Acinetobactersp. 53 >256 >256 8–>256 N/A >256 >256 4–>256 N/A >256 >256 4–>256 28 <2 32 2–>128 77
Citrobactersp. 7 >256 >256 32–>256 N/A 16 >256 4–>256 N/A 8 >256 4–>256 60 <2 >128 2–>128 100
Enterobactersp. 41 >256 >256 128–.256 N/A 8 >256 4–>256 N/A <4 >256 4–>256 67 <2 128 2–>128 69
Escherichiasp. 229 >256 >256 16–>256 N/A 8 128 4–>256 N/A <4 >256 4–>256 66 <2 128 2–>128 78
Klebsiellasp. 126 256 >256 32–>256 N/A 8 >256 4–>256 N/A <4 >256 4–>256 58 <2 >128 2–>128 74
Proteussp. 4 >256 >256 >256 N/A 32 128 32–128 N/A <4 4 4 100 <2 >128 2–>128 67
Pseudomonassp. 76 128 >256 4–>256 N/A 64 >256 4–>256 N/A 8 >256 4–>256 54 <2 >128 2–>128 67
Salmonellasp. 16 128 256 128–>256 N/A <4 64 4–64 N/A <4 32 4–>256 69 <2 64 2–64 77
Serratiasp. 12 >256 >256 32–>256 N/A 16 >256 4–>256 N/A <4 64 4–>256 73 >128 >128 2–>128 9
Sphingobacteriumspiritivorum 1 >256 >256 >256 N/A 16 16 16 N/A <4 <4 <4 100 <2 <2 <2 100
Staphylococcusaureus(MSSA)d 2 256 >256 256–>256 0 <4 16 <4–16 50 <4 <4 <4 100 4 >128 4–>128 0
Staphylococcusaureus(MRSA)e 2 >256 >256 >256 0 8 >256 8–>256 0 <4 <4 <4 100 8 8 8 0
Stenotrophomonasmaltophilia 9 128 >256 8–>256 N/A 64 >256 4–>256 N/A 32 >256 4–>256 50 <2 64 2–64 88
Streptococcuspneumoniae 2 >256 >256 >256 0 8 >256 8–>256 0 <4 >256 <4–>256 100 <2 >128 <2–>128 50
Yersiniasp. 4 32 >256 32–>256 N/A 16 256 16–256 N/A <4 64 4–64 100 <2 >128 2–>128 100
92 www.zoores.ac.cn
Continued
Percentage Percentage Percentage Percentage
ofisolates ofisolates ofisolates ofisolatese
CHLbMIC(mg/L) PEXbMIC(mg/L) LVXbMIC(mg/L) VANbMIC(mg/L)
Isolates No. susceptible susceptible susceptible susceptible
toc(%) toc(%) toc(%) toc(%)
MIC50a MIC90a,*** Range MIC50a MIC90a Range MIC50a,* MIC90a,*** Range MIC50a MIC90a,*** Range
Acinetobactersp. 53 128 256 8–>256 5 8 16 4–128 56 128 >256 16–>256 N/A 128 256 8–>256 5
Citrobactersp. 7 8 32 4–32 80 <4 32 4–32 60 256 >256 4–>256 N/A 8 32 4–32 80
Enterobactersp. 41 16 >256 4–>256 53 <4 32 4–128 72 256 >256 64–>256 N/A 16 >256 4–>256 53
Escherichiasp. 229 <4 128 4–>256 58 <4 128 4–256 55 128 >256 8–>256 N/A <4 128 4–>256 58
Klebsiellasp. 126 16 256 4–>256 46 >256 32 4–128 48 8 >256 8–>256 N/A 16 256 4–>256 46
Proteussp. 4 8 256 8–256 67 8 16 4–128 33 128 256 128–256 N/A 8 256 8–256 67
Pseudomonassp. 76 <4 >256 4–>256 68 <4 64 4–128 63 >256 >256 64–>256 N/A <4 >256 4–>256 68
Salmonellasp. 16 <4 256 4–>256 84 <4 4 4–128 92 >256 >256 4–>256 N/A <4 256 4–>256 84
Serratiasp. 12 64 >256 4–>256 0 <4 16 4–32 55 >256 >256 64–>256 N/A 64 >256 4–>256 0
Sphingobacteriumspiritivorum 1 <4 <4 <4 100 <4 <4 <4 100 256 256 256 N/A <4 <4 <4 100
Staphylococcusaureus(MSSA)d 2 <4 32 <4–32 50 8 8 8 0 <4 8 <4–8 50 <4 32 <4–32 50
Staphylococcusaureus(MRSA)e 2 <4 <4 <4 100 16 32 16–32 0 <4 >256 <4–>256 50 <4 <4 <4 100
Stenotrophomonasmaltophilia 9 32 >256 4–>256 50 <4 256 4–256 63 128 >256 128–>256 N/A 32 >256 4–>256 50
Streptococcuspneumoniae 2 <4 <4 <4 100 16 32 16–32 0 <4 <4 <4 50 <4 <4 <4 100
Yersiniasp. 4 <4 <4 <4 100 <4 4 4 100 256 >256 4–>256 N/A <4 <4 <4 100
a:MIC andMIC ,MICsatwhich50%,or90%ofisolatesareinhibited,respectively;b:Amp,Ampicillin;CPZ,Cefoperazone;ROX,Roxithromycin;CHL,Chloramphenicol;AZM,Azithromycin;
50 90
AMK,Amikacin;VAN,Vancomycin;LVX,Levofloxacin;PB,PolymyxinB;PEX,Pexiganan;c: InterpretivecriteriabasedonCLSIrecommendations(2009);d: MSSA,Methicillin-sensitive
Staphylococcusaureus;e:MRSA,Methicillin-resistantStaphylococcusaureus.N/A:Notapplicable.*:P<0.05,OH-CATH30vs.thetreateddrugsorcompounds.
ZoologicalResearch39(2):87-96,2018 93
Nitricoxiderelease Theamountofnitricoxidereleasedtripledthanthatinthevitamin
ThepeptideanditsD-analogdisplayedsteadynitricoxiderelease Cascontrolgroup,suggestingthatalowconcentrationofOH-
when the concentration ranged from 500µg/mL to 6.25µg/mL. CATH30stimulatedrelativelyhighnitricoxiderelease(Figure3).
D-OH-CATH30
L-OH-CATH30
Vitamin c
0.5
0.4
0
4
5 0.3
D
O
0.2
0.1
0.0
500 250 125 100 50 25 12.5 6.25
Concentration (μg/mL)
Figure3NitricoxidereleaseinducedbyL-andD-OH-CATH30peptidesinvitro
IL-6immunogenicityresponse this peptide showed high efficacy against Gram- positive
L-OH-CATH30 is more effective in increasing the IL-6 bacteria equal to amikacin, and higher efficacy against
concentration during an immunogenicity response both in almost all Gram-negative bacteria tested than that of the
vitro and in vivo compared with the peptide and its D- peptide-antibioticpolymixinB(showninTable1). Thispeptide
analog. Interestingly, OH-CATH30 showed the lowest IL-6 alsoshowedgoodantimicrobialactivityagainstAcinetobacter
immunogenicity response in the kidney in vitro, whereas spp., which is primarily associated with nosocomial infections
L-OH-CATH30 showedclear induction ofIL-6 in the kidneyin inseverely-ill patientsinwhichthemanagement ofinfections
vivo. Bycomparison, thepeptideanditsD-analogpromoted is difficult due to increasing resistance to multiple classes
IL-6potentlyinperitonealfluidinvitrobuthadvirtuallynoeffect of antibacterial agents (Karageorgopoulos et al., 2008). Our
ontheIL-6immunogenicityresponseinvivo(Figure4,5). results indicated that OH-CATH30 and its D- analog have
higher efficacy against these strains compared with routine
DISCUSSION antibiotics.
Usually, searching for new antibiotics, we particularly
Drugresistanceinbacteria,inparticulartheriseofmulti-drug focus on the cytotoxicity and immunogenicity. Meanwhile,
resistance, poses a serious threat to human health that L-OH-CATH30 and D-OH-CATH30 showed low cytotoxicity to
urgently requires new drugs with broad efficiency against eukaryoticcells(redbloodcellinourexperiments),compared
clinically-encountered bacteria (Leid et al., 2012). AMPs, to bacterial cells (their high bactericide activity). They had
as novel therapeutic drugs, are being used increasingly to littledifferenceinhemolyticactivityandfreeradicalscavenging
treat infections. In our previous study, OH-CATH30 was ability. IL-6 assay results show that both L-OH-CATH30 and
found to possess relative normal bactericidal activity and low D-OH-CATH30promotedthereleaseofIL-6nomatterinvitro
toxicity in vivo and in vitro (Li et al., 2012), indicating that orinvivo,whichvariedindifferenttissueandtreatmentdoses.
it is a competitive candidate for the development of novel Itindicatedthatthispeptidecouldstimulatetheimmunesystem
antimicrobialagent. to protect against infections in vitro or in vivo. Furthermore,
Here,wetestedOH-CATH30anditsD-analogagainst584 L-OH-CATH30 and D-OH-CATH30 also showed differences
clinicalisolates, includingseveralofdrug-resistantpathogens. in bactericidal activity against a range of clinical isolates,
ThedatashowedthatthispeptideanditsD-analogarebroadly suggesting that OH-CATH30 containing L- residues is more
active against important medical pathogens including MRSA, effectiveagainstsomepathogensthanOH-CATH30containing
MRAB, S. pneumoniae and Pseudomonas spp. Importantly, D-residues.
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Figure 4 IL-6 induced by L- and D- OH-CATH30 peptides in vitro
ELISA was used for IL-6 determination. *: P<0.05).

Figure 4 IL-6 induced by L- and D- OH-CATH30 peptides in vitro
ELISA was used for IL-6 determination. *: P<0.05).

Figure4IL-6indFuigcuered 5b ILy-6L i-naduncdedD b-y OL-H a-nCd ADT- OHH3-0CpATeHp3ti0d peesptiidnesv iint rvoivo
Mouse was intraperitoneal injected 2 mL saline 5 h after they were intraperitoneal injected 10 mg/kg and 5 mg/kg the above pe ptides. ***:
ELISFAigwuarse u5s eILd-fo6rP i<InL0d-.60u0dc1ee).td e rbmyin Lat-i oann.d*: DP-< O0.0H5-.CATH30 peptides in vivo
Mouse was intraperitoneal injected 2 mL saline 5 h after they were intraperitoneal injected 10 mg/kg and 5 mg/kg the above peptides. ***:
P<0.001).

Figure5IL-6inducedbyL-andD-OH-CATH30peptidesinvivo
Mousewasintraperitonealinjected2mLsaline5haftertheywereintraperitonealinjected10mg/kgand5mg/kgtheabovepeptides.***:P<0.001.
CONCLUSION AUTHORS’CONTRIBUTIONS
Insummary,ourinvitrostudiesdemonstratedthatOH-CATH30
F.Z.,W.H.L.andY.Z.conceivedthestudy. F.Z.,X.Q.L.,P.Y.C.,J.Z.andF.Z.
anditsD-analogareeffectiveagainstabroadrangeofclinical
performedtheexperiments.Y.D.collectedtheclinicalisolates.F.Z.wrotethe
isolates. This peptide exerts antimicrobial activity against a
paper.Allauthorsreadandapprovedthefinalversionofthemanuscript.
multitude of virulent bacteria from various human sources, 19 / 19
and has the po tential of treating a wide range of bacterial REFERENCES
infections, OH-CATH30 is therefore a promising candidate for
thedevelopmentofanewbroad-spectrumantimicrobialdrug. Clinical and Laboratory Standards Institute. 2009. M07-A8 Methods
for dilution antimicrobial susceptibility tests for bacteria that grow
COMPETINGINTERESTS
aerobically-eighth edition. Wayne, PA, USA: Clinical and Laboratory
Theauthorsdeclarethattheyhavenocompetinginterests. StandardsInstitute.
19 / 19 ZoologicalResearch39(2):87-96,2018 95
De Oliveira Junior NG, E Silva Cardoso MH, Franco OL. 2013. Snake antimicrobialpeptideOH-CATH30selectivelyregulatestheinnateimmune
venoms: attractiveantimicrobialproteinaceouscompoundsfortherapeutic responsetoprotectagainstsepsis.JournalofMedicinalChemistry,56(22):
purposes.CellularandMolecularLifeSciences,70(24):4645–4658. 9136–9145.
Falcao CB, De La Torre BG, Pérez-Peinado C, Barron AE, Andreu D, LiuZC,ZhangR,ZhaoF,ChenZM,LiuHW,WangYJ,JiangP,ZhangY,Wu
Rádis-BaptistaG.2014.Vipericidins: anovelfamilyofcathelicidin-related Y,DingJP,LeeWH,ZhangY.2012.Venomicandtranscriptomicanalysisof
peptidesfromthevenomglandofSouthAmericanpitvipers.AminoAcids, centipedeScolopendrasubspinipesdehaani.JournalofProteomeResearch,
46(11):2561–2571. 11(12):6197–6212.
FischbachMA,WalshCT.2009.Antibioticsforemergingpathogens.Science, NathanC.2004.Antibioticsatthecrossroads.Nature,431(7011):899–902.
325(5944):1089–1093. ParkE,QuinnMR,WrightCE,Schuller-LevisG.1993.Taurinechloramine
GeYG,MacDonaldDL,HolroydKJ,ThornsberryC,WexlerH,ZasloffM. inhibitsthesynthesisofnitricoxideandthereleaseoftumornecrosisfactor
1999.Invitroantibacterialpropertiesofpexiganan,ananalogofmagainin. inactivatedRAW264.7cells.JournalofLeukocyteBiology,54(2):119–124.
AntimicrobialAgentsandChemotherapy,43(4):782–788. TashimaAK,ZelanisA,KitanoES,IanzerD,MeloRL,RioliV,Sant’anna
Hancock REW, Sahl HG. 2006. Antimicrobial and host-defense peptides SS,SchenbergACG,CamargoACM,SerranoSMT.2012.Peptidomicsof
as new anti-infective therapeutic strategies. Nature Biotechnology, 24(12): three Bothrops snake venoms: insights into the molecular diversification
1551–1557. of proteomes and peptidomes. Molecular & cellular proteomics, 11(11):
Johansson J, Gudmundsson GH, Rottenberg ME, Berndt KD, Agerberth 1245–1262.
B. 1998. Conformation-dependent antibacterial activity of the naturally WiegandI,HilpertK,HancockREW.2008.Agarandbrothdilutionmethods
occurring human peptide LL-37. Journal of Biological Chemistry, 273(6): to determine the minimal inhibitory concentration (MIC) of antimicrobial
3718–3724. substances.NatureProtocols,3(2):163–175.
Karageorgopoulos DE, Kelesidis T, Kelesidis I, Falagas ME. 2008. Yang XW, Lee WH, Zhang Y. 2012. Extremely abundant antimicrobial
Tigecycline for the treatment of multidrug-resistant (including peptidesexistedintheskinsofninekindsofChineseodorousfrogs.Journal
carbapenem-resistant) Acinetobacter infections: a review of the scientific ofProteomeResearch,11(1):306–319.
evidence.JournalofAntimicrobialChemotherapy,62(1):45–55. ZanettiM,GennaroR,RomeoD.1995.Cathelicidins:anovelproteinfamily
LeidJG,DittoAJ,KnappA,ShahPN,WrightBD,BlustR,ChristensenL, withacommonproregionandavariableC-terminalantimicrobialdomain.
Clemons CB, Wilber JP, Young GW, Kang AG, Panzner MJ, Cannon CL, FEBSLetters,374(1):1–5.
YunYH,YoungsWJ,SeckingerNM,CopeEK.2012.Invitroantimicrobial Zhang Y, Zhao H, Yu GY, Liu XD, Shen JH, Lee WH, Zhang Y. 2010.
studies of silver carbene complexes: activity of free and nanoparticle Structure-function relationship of king cobra cathelicidin. Peptides, 31(8):
carbeneformulationsagainstclinicalisolatesofpathogenicbacteria.Journal 1488–1493.
ofAntimicrobialChemotherapy,67(1):138–148. ZhangY.2015.Whydowestudyanimaltoxins?ZoologicalResearch,36(4):
Li SA, Lee WH, Zhang Y. 2012. Efficacy of OH-CATH30 and its analogs 183–222.
againstdrug-resistantbacteriainvitroandinmousemodels.Antimicrobial Zhao H, Gan TX, Liu XD, Jin Y, Lee WH, Shen JH, Zhang Y. 2008.
AgentsandChemotherapy,56(6):3309–3317. Identificationandcharacterizationofnovelreptilecathelicidinsfromelapid
LiSA,XiangY,WangYJ,LiuJ,LeeWH,ZhangY.2013.Naturallyoccurring snakes.Peptides,29(10):1685–1691.
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