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

Feng Zhao,Xin-Qiang Lan,Yan Du,Pei-YiChen,Jiao Zhao,Fang Zhao,4,Wen-HuiLee,Yun Zhang,＊

1 Key Laboratory ofSubtropicalMedicina lEdible Resources Deve lopm entand Utilization in Yunnan Province,Department ofBio logy and Chem istry,PuerUniversity,PuerYunnan 665000,China

2 Key Laboratory ofBioactive Peptides ofYunnan Province/Key Laboratory ofAnim alMode ls and Hum an Disease Mechanism s of Chinese Academy ofSciences,Kunm ing Institute of Zoo logy,Kunm ing Yunnan 650223,China

3 Departm entofClinica l Laboratories,First Affi liated HospitalofKunm ing Medica lUniversity,Kunm ing Yunnan 650032,China

4 Institute ofComparative Study ofTraditionalMateria Medica,Institute of Integrative Medicine ofFudan University,Shanghai200433,China

INTRODUCTION

The abundant use of traditional antibiotics has resulted in the emergence of many antibiotic-resistant isolates worldwide,causing threatening to human health and the rate of antibiotic discovery and production has been insufficient to respond to this crisis(Fischbach&Walsh,2009;Nathan,2004).Recent studies have shown that there are many components in the venom that possess antimicrobial activity. Snake venom,produced by specialized glands in the snake jawbone,is a particularly rich source of such antimicrobial com pounds(Tashima et al.,2012).Several venom com pounds have been used for antimicrobial effects,such as phospholipases A2,metalloproteinases,L-am ino acid oxidases and antimicrobial peptides(De Oliveira Junior et al.,2013).Cathelicidins have been identified as the main family of naturally-occurring antim icrobialpeptides from snake venom,which exhibit potential microbicidal properties against bacteria,fungiand some viruses(Zhang,2015).Currently,at least9 cathelicidin-type antimicrobial peptides have been identified from elapid snake venoms,most of which exhibit potential antim icrobial activities(Falcao et al.,2014;Zhao et al.,2008).Cationic antimicrobial peptides(AMPs)are considered as im portant candidate therapeutic agents com prising a diverse group of bactericidal molecules forwhich m icrobialorganism s show lower levels of resistance,for example pexiganan,an AMP developed for the treatment of diabetic foot infection(Ge et al.,1999).Although several peptide-antibiotics are well established clinically,such as polymixin B,AMPs alone without any modifications have not been widely used in clinical treatment(Hancock&Sahl,2006).As an unique family of AMPs,the cathelicidin peptides shared conserved N-terminal domains from a variety of species and exhibited effective antimicrobial activity and some cytotoxic activity towards eukaryotic cells(Johansson et al.,1998;Zanettietal.,1995).

We previously reported a novel cathelicidin-derived peptide from the king cobra. This reptile cathelicidin was termed OH-CATH30 and exhibited potential broad-spectrum antibacterial activityinvitroand relatively low toxicityinvivo(Li et al.,2012;Zhang et al.,2010).The aim of this study is to evaluate the efficacy of OH-CATH30 and its analog D-OH-CATH30(com prising allD-amino acids residues)against clinical isolates(collected from patients in hospital)compared with routinely utilized antibioticsinvitro. Meanwhile,the corresponding information is crucial for the cytotoxicity to eukaryotes and the immunogenicity of these peptides,which hemolysis assay and IL-6 assay were further investigated.Considering that most of AMPs have free radical scavenging ability,we also expanded nitric oxide assay and detected the release of nitric oxide(Figure 1A).

Figu re 1 Cytotoxicity o f OH-CATH30 and its analog

MATERIALS AND METHODS

Ethics statement

This study was approved by the Ethics Comm ittee of Puer University and the Biomedical Ethics Committee of the First Affiliated Hospital of Kunming Medical University.The data were analyzed anonymously,patient identity deduced or disclosed.

Clinical isolates,reference strains and materials

A total of 584 clinical isolates were collected between July 2013 and June 2016,which included 14 different species as follows:(i)Acinetobacterspp.(n=53);(ii)Citrobacterspp.(n=7);(iii)Enterobacterspp. (n=41);(iv)Escherichiaspp.(n=229);(v)Klebsiellaspp.(n=126);(vi)Proteusspp.(n=4);(vii)Pseudomonasspp.(n=76);(viii)Salmonellaspp.(n=16);(ix)Serratiaspp.(n=12);(x)Sphingobacteriumspiritivorum(n=1);(xi)Staphylococcusaureus(MSSA)(n=2)andS.aureus(MRSA)(n=2);(xii)Stenotrophomonasmaltophilia(n=9);(xiii)Streptococcuspneumoniae. (n=2);and(xiv)Yersiniaspp.(n=4). The species identification of each clinical isolates was confirmed with the Vitek 2 system(bioMérieux,France;part of the data shown in Supp lementary Figure S1).The antibiotic susceptibility of the clinical isolates was determ ined using the Kirby-Bauer disk diffusion method,in accordance with the Clinical and Laboratory Standards Institute(CLSI)2009 guidelines(Clinical and Laboratory Standards Institute,2009). Twelve control strains were also included:E.coliATCC 25922 and ATCC 35218,HaemophilusinfluenzaATCC 49766 and ATCC 49247,S.aureusATCC 25923 and ATCC 43300,KlebsiellapneumoniaeATCC 13883 and ATCC 700603,S.maltophiliaATCC 8090,EnterococcusfaecalisATCC 29212,EnterobactercloacaeATCC 13047 andPseudomonas aeruginosaATCC 27853.All strains were cultured in MHB Mueller-Hinton broth(MHB,pH 7.2)medium at37°C.Allother reagents were of analytical grade and were obtained from commercial sources.

An tibio tics

Ampicillin(Amp)and cefoperazone(CPZ)were acquired from the General Pharmaceutical Factory of Harbin Pharmaceutical Group(Harbin,China).Roxithrom ycin(ROX),chloram phenicol(CHL)and azithromycin(AZM)were from Sangon Biotech Co.,Ltd.(Shanghai,China).Amikacin(AMK),vancomycin(VAN),levofloxacin(LVX)and polymyxin(PB)were from Sigm a-Aldrich Co.LLC(Sigma,St.Louis,MO,USA).

Antimicrobial pep tides synthesis

Pexiganan(GIGKFLKKAKKFGKAFVKILKK-NH2),OH-CATH30(KFFKKLKNSVKKRAKKFFKKPRVIGVSIPF)and its analog D-OH-CATH30 (KFFKKLKNSVKKRAKKFFKKPRVIGVSIPF,italics indicate D-amino acids)were synthesized by solid-phase synthesis on an Applied Biosystems Model 433A peptide synthesizer according to the manufacturer’s standard protocols,as reported previously(Lietal.,2012).Then,the crude synthetic peptide was purified by reverse-phase high-performance lipid chromatography(RP-HPLC,Supplementary Figure S2).Mobile phase eluent Aconsisted of 0.1%TFA(aqueous)and mobile phase eluent B consisted of ACN/ddH2O/TFA 90/10/0.08%(v/v/v).The sample was added along with auto-sampler to a Thermo Scientific EASY loading column(Thermo Fisher Scientific,USA)(2 cm×100µm,5µm-C18)then to an analytic column(75 µm×100 mm,3 µm-C18)with a flow rate of 1.2 m L/m in. The presence of peptides was confirmed by an absorption peak at 215 nm. The purity of each synthetic peptidewas above 95%.The identity and purity ofeach peptide was further confirmed by matrix-assisted laser desorption ionization-time of flight(MALDI-TOF)mass spectrometry(Voyager;Supplementary Figure S2).The survey of the full scan MS spectra(m/z 300–1 800)was acquired in the Orbit rap with a 70 000-resolution(m/z 200).Dynam ic exclusion was set to 15s.The 10 m ost intense multiply charged ions(z≥2)were sequentially isolated and fragmented by higher-energy collisional dissociation(HCD)with a fixed injection time of 80 ms and 17 500-resolution(m/z 200).The conditions were as follows:spray voltage,2 kV;no sheath and auxiliary gas flow;heated capillary temperature,250°C;and norm alized HCD collision energy 27 eV;underfill ratio 0.1%.The MS/MS ion selection threshold was setat1×105counts.

Determination of MICs

MICs were determined by the broth microdilution method(Wiegand et al.,2008)according to the CLSI guidelines(Clinical and Laboratory Standards Institute,2009).In each well of a 96-well plate,100µL of MHB broth and bacteria(5×105colony-form ing unit(cfu)/m L)were incubated with various concentrations of antim icrobials for 16 h at37°C.The concentrations of the antimicrobials were the same for each of the tested bacterial strains in three independent experiments,and the MIC values were obtained without inter-experiment variations and expressed asµg/m L.For com pounds and antibiotics drugs,the interpretation criteria were set by using the CLSIpublished criteria.The concentration of antibiotics was initially set at 512 µg/m L,then diluted to 4 µg/m L.As for the(only two are listed)peptides used in this study(pexiganna,polymyxin B),the concentration was diluted from 256µg/m L to 2µg/m L.In each batch of susceptibility tests,the 12 control strains listed above were included as a quality control.

Hem o lytic assay and cytokine determ ination

Hemolytic assays were investigated using human red blood cells in liquid medium as previously reported(Yang et al.,2012).Serial dilutions of the samples were incubated with washed red blood cells(2%)at37°C for 30m in,the cells were then centrifuged,and the absorbance of the supernatant was measured at540 nm.Maximum hemolysis was determined by adding 1%Triton X-100 to the cellsamples(Liu et al.,2012).

An ELISA kit(Neobiosoence)was employed for IL-6 determination according to the manufacturer’s instructions.Nitric oxide was quantified using the Griess chemical method(Park etal.,1993).

IL-6 detectioninvitro:Prior to euthanization,mice were administered 3 mL of saline by in traperitoneal injection.The abdomen was kneaded for 2 m in before being opened up and the peritoneal fluid was collected.Afterexcision,them ice livers and kidneys were placed into physiological saline,cut(sliced)into pieces,homogenized with trypsin,ground into fluid,passed through a mesh sieve and centrifuged at2 000 r/m in for 10m in.The supernatant was discarded and the pellet was washed with PBS until the supernatant was clear. Then the cells were re-suspended in F12 medium and 190µL of cells(about 1×107)were added to each well of a 96-well plate before the adding 10µL of different concentrations of OH-CATH30 as the experimental group or F12 medium as the control.The plate was then incubated at36°C for 2 h,followed by centrifugation at 3 000 r/m in for 20 m in.The supernatant was retained for further determinations.

IL-6 detectioninvivo:Mice were intraperitoneally injected with 2m Lofsaline,then after5 hwere intraperitoneally injected with 5 mg/kg or 10 mg/kg of different types of OH-CATH30(Li et al.,2013).Mice were euthanized,then the abdomen was kneaded for 2 m in,after which it was opened and the peritoneal fluid was collected.After excision,the livers and kidneys were p laced into physiologicalsaline,sliced into pieces,homogenized with trypsin,ground into fluid,passed through a mesh sieve and centrifuged at 3 000 r/m in for 20 m in.The supernatant was discarded and the pellet was washed with PBS until the supernatantwas clear.

Statisticalanalysis

The data was analyzed using two-way ANOVA.The experimental data are expressed as MIC50and MIC90values.The level of statistical significance was setatP＜0.05.

RESULTS

Susceptibility test

Theinvitrosusceptibility of584 clinical isolates to three AMPs and nine antibiotics was tested,which was also represented by its cytotoxicity of prokaryotes cell(Figure 1A).Table 1 shows the MICs,with ranges of M IC50and M IC90values,of each antimicrobial agent tested against 14 different species.Overall,the efficacy of OH-CATH30 and D-OH-CATH30 was higher when com pared with 9 routine antibiotics,and slightly higher than pexiganan. Among the 584 clinical isolates tested,85%were susceptible to OH-CATH30 and its analog with a MIC≤64 mg/L(Figure 1B).ForEscherichiaspp.andKlebsiellaspp. (61%of all clinical isolates),OH-CATH30 was m ore active than the 9 antibiotics tested,and showed slightly higher activity than pexiganan. For 53 strains ofAcinetobacterspp.,includingmultidru g-resistantAcinetobacter baumannii(MRAB),the MIC90value of D-OH-CATH30 was doubled comparing with that of OH-CATH30,and polymixin B also had high rates of susceptibility(77%)toAcinetobacterspp.com pared with other antibiotics,except for pexiganan.For the 76 strains ofPseudomonasspp.,the activity of OH-CATH30 and its analog was higher than pexiganan,although these peptides are all showed higher activity than the 9 antibiotics,with the exception of levofloxacin.For the 16 strains ofSalmonellaspp.,all showed high susceptibility to the antimicrobial peptides and antibiotics,with the exception of ampicillin and cefoperazone.The rate of susceptibility(92%)to levofloxacin among these specieswas considerably higher than the other8 antibiotics.Among the 14 species of clinicalisolates tested,Serratiaspp.,for which there were 12 strains,had the lowest rate of susceptibility to antim icrobial peptides and the peptide-antibiotic(polymixin B).OH-CATH30 inhibited all of the species,com prising around 10 strains,ata concentration of≤64mg/L,with the exception ofProteusspp.andS.maltophilia.Of the four strains ofS.aureustested,including two MSSA and two MRSA,D-OH-CATH30 was found to be more effective at inhibiting MRSA than OH-CATH30,with a range of≤8mg/L.Overall,L-OH-CATH30 showed higher efficacy against two thirds of the tested clinical isolates than OH-CATH30.

Hemolysis assay

Generally,the peptide and its D-analog showed little difference in hemolytic activity to hum an red blood cells(Figure 1A).When the dose of L-OH-CATH30 or D-OH-CATH30 was lower than 125µg/m L,similar hemolysis rates of about 10%were observed,but as the dose increased to 250µg/m L,high hemolytic activities exceeding 70%for the peptide and 80%for its D-analog were detected(Figure 2).

Figu re 2 Hem o lytic activity o f the L-and D-OH-CATH30 pep tides

Nitric oxide release

The peptide and its D-analog displayed steady nitric oxide release when the concentration ranged from 500µg/m L to 6.25 µg/m L.The amount of nitric oxide released tripled than that in the vitam in C as controlgroup,suggesting thata low concentration of OHCATH30 stimulated relatively high nitric oxide release(Figure 3).

Figu re 3 Nitric oxide re lease induced by L-and D-OH-CATH30 pep tides in vitro

IL-6 immunogenicity response

L-OH-CATH30 is more effective in increasing the IL-6 concentration during an immunogenicity response bothin vitroandinvivocom pared with the peptide and its D-analog.Interestingly,OH-CATH30 showed the lowest IL-6 immunogenicity response in the kidneyinvitro,whereas L-OH-CATH30 showed clear induction of IL-6 in the kidneyin vivo.By com parison,the peptide and its D-analog promoted IL-6 potently in peritoneal fluidinvitrobuthad virtually no effect on the IL-6 immunogenicity responseinvivo(Figure 4,5).

DISCUSSION

Drug resistance in bacteria,in particular the rise ofmulti-drug resistance,poses a serious threat to hum an health that urgently requires new drugs with broad efficiency against clinically-encountered bacteria(Leid et al.,2012). AMPs,as novel therapeutic drugs,are being used increasingly to treat infections. In our previous study,OH-CATH30 was found to possess relative normal bactericidal activity and low toxicityinvivoandinvitro(Li et al.,2012),indicating that it is a com petitive candidate for the development of novel antimicrobialagent.

Here,we tested OH-CATH30 and its D-analog against 584 clinical isolates,including several of drug-resistant pathogens.The data showed that this peptide and its D-analog are broad ly active against important medical pathogens including MRSA,MRAB,S.pneumoniaeandPseudomonasspp.Importantly,this peptide showed high efficacy against Gram-positive bacteria equal to amikacin,and higher efficacy against almost all G ram-negative bacteria tested than that of the peptide-antibiotic polymixin B(shown in Table 1).This peptide also showed good antim icrobialactivity againstAcinetobacterspp.,which is primarily associated with nosocom ial infections in severely-ill patients in which them anagement of infections is difficult due to increasing resistance to multiple classes of antibacterial agents(Karageorgopoulos et al.,2008).Our results indicated that OH-CATH30 and its D-analog have higher efficacy against these strains com pared with routine antibiotics.

Usually,searching for new antibiotics,we particularly focus on the cytotoxicity and immunogenicity. Meanwhile,L-OH-CATH30 and D-OH-CATH30 showed low cytotoxicity to eukaryotic cells(red blood cell in our experiments),com pared to bacterial cells(their high bactericide activity).They had little difference in hem olytic activity and free radicalscavenging ability.IL-6 assay results show that both L-OH-CATH30 and D-OH-CATH30 promoted the release of IL-6 no m atterinvitroorinvivo,which varied in different tissue and treatment doses.Itindicated thatthis peptide could stimulate the immune system to protect against infectionsinvitroorinvivo.Furthermore,L-OH-CATH30 and D-OH-CATH30 also showed differences in bactericidal activity against a range of clinical isolates,suggesting that OH-CATH30 containing L-residues is more effective againstsom e pathogens than OH-CATH30 containing D-residues.

Figure 4 IL-6 induced by L- and D- OH-CATH30 peptides in vitro

Figu re 5 IL-6 induced by L-and D-OH-CATH30 pep tides in vivo

CONCLUSION

In summary,ourin vitrostudies demonstrated that OH-CATH30 and its D- analog are effective against a broad range of clinical isolates. This peptide exerts antimicrobial activity against a multitude of virulent bacteria from various human sources, and has the potential of treating a wide range of bacterial infections, OH-CATH30 is therefore a promising candidate for the development of a new broad-spectrum antimicrobial drug.

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHORS’CONTRIBUTIONS

F.Z.,W.H.L.and Y.Z.conceived the study.F.Z., X.Q.L., P.Y.C., J.Z. and F.Z. performed the experiments. Y.D. collected the clinical isolates. F.Z. wrote the paper. All authors read and approved the final version of the manuscript.

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