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Prevention of lethal murine candidiasis using 2-20, an antimicrobial peptide derived from the N-terminus of

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Prevention of lethal murine candidiasis using 2-20, an antimicrobial peptide derived from the N-terminus of
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  Peptides 24 (2003) 1807–1814 Prevention of lethal murine candidiasis using HP (2–20), anantimicrobial peptide derived from the N-terminusof   Helicobacter pylori  ribosomal protein L1 Patr´ıcia Damasceno Ribeiro, Enrique Medina-Acosta ∗  Laboratório de Biotecnologia, Centro de Biociˆ encias e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Avenida Alberto Lamego 2000, Parque Califórnia, Campos dos Goytacazes, CEP 28013-602, RJ, Brazil Received 4 April 2003; accepted 1 August 2003 Abstract Peptide HP (2–20),  A 2 KKVFKRLEKLFSKIQNDK  20 , is a cationic antimicrobial peptide derived from the N-terminus of   Helicobacter  pylori ribosomalprotein1,HpRpL1.NativepeptideHP(2–20)anditssyntheticderivativeshavebeenshowninvitrotoexhibitpotentkillingactivity against Gram-positive, Gram-negative and yeast cells, thus, making them promising candidates for treatment of polymicrobialinfections. However, the therapeutic potential of peptide HP (2–20) or its synthetic derivatives in any animal model of either bacterial orfungal diseases has not yet been investigated. In this study, we demonstrate that synthetic peptide amide HP (2–20), administered in sixdoses(300  geach;oneintraperitonealdoseatthetimeoftheinfection,followedbyfiveintravenousdosesat12hintervals)toCBA/Jmalemice experimentally infected with a lethal inoculum (1 × 10 9 CFU) of   Candida albicans , delayed the onset of disease, suppressed diseaseprogression, and greatly increased survival rate and time (16.6% by day 14), as compared with the untreated infected control mice (100%mortality by day 5). Further, using isotonic buffer systems differing in ionic strength, peptide HP (2–20) was shown in vitro to exhibitan ionic strength-dependent hemolytic activity, previously not detected. Repeated intravenous administration of uninfected control CBA/Jmale mice with peptide HP (2–20), however, caused neither morbidity nor mortality. These findings strongly evidence the therapeuticefficacy and safety values of peptide HP (2–20) as a lead drug for the treatment of acquired candidiasis.© 2003 Elsevier Inc. All rights reserved. Keywords:  Antibacterial peptide; Antifungal peptide; Antimicrobial peptide; Candidiasis 1. Introduction The worldwide increasing frequency of fungal infections,particularly, in immunocompromised patients, together withthe still limited clinical efficacies of triazole antifungalagents such as fluconazole and itraconazole to treat mycoses[20,22] and the emergence of azole-resistant fungal agents[19,21,27], which are refractory to the repeated treatmentwith fluconazole [19,21,27], demand a global effort for dis- covery of new classes of antimicrobial agents [30]. More  Abbreviations:  HP (2–20), peptide  A 2 KKVFKRLEKLFSKIQNDK  20 , de-rived from the N-terminal sequence of HpRpL1; HpRpL1,  Helicobac-ter pylori  ribosomal protein L1; CFU, colony forming units; RP-HPLC,reverse-phase high-pressure liquid chromatography; IC 50 , half-maximalinhibitory concentration; HC 50 , half-maximal hemolytic activity; S.D.,standard deviation; single letter abbreviations for the amino acid residuesare,  A , Ala;  K  , Lys;  V  , Val;  F  , Phe;  R , Arg;  E  , Glu;  L , Leu;  S  , Ser;  I  , Ile; Q , Gln;  N  , Asn;  D , Asp ∗ Corresponding author. Tel.: + 55-22-27261564; fax: + 55-22-27261661.  E-mail address:  enrique@medina-acosta.com (E. Medina-Acosta). troubling, the clinical use of amphotericin B that is activeagainst deep-seated mycoses, to which triazole antifungalagents are ineffective, is limited because of its severe toxi-city [7,27]. Thus, development of potent and safe antifun- gal agents against mycosis caused by antifungal-resistantstrains is urgently needed [3].Current interest in developing antimicrobial therapies,alternative to classical antibiotics, is focused on linear andcyclic antimicrobial peptides, which can be easily madein large scale, synthetically or by recombinant biology.Many known antimicrobial peptides occur widely in natureand constitute the front line of defence of many organ-isms against microbial infections [13]. The bulk of thedata indicate that antimicrobial peptides vary largely in: (a)amino acid composition and, therefore, net charge (mostare cationic, yet amphipathic) [8]; (b) length (8 to  < 100residues, some potentially bearing a membrane spanningdomain); (c) secondary structure (mostly are   -helical, butsome configure  -sheets); (d) target and mode of action: dis-tinctly from the mechanism of action of available antibiotic 0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved.doi:10.1016/j.peptides.2003.08.021  1808  P.D. Ribeiro, E. Medina-Acosta/Peptides 24 (2003) 1807–1814 agents, most antimicrobial peptides act on cell membranesby mainly altering their permeability and/or stability, andcreating membrane pores, rather than targeting a singlemolecule/enzyme. Some even interfere with the synthesisof the cell wall or the biosynthesis of essential cellularmolecules (i.e. glucans, chitins, etc.) [5]; (e) efficacy (nev- ertheless, most have been only tested in vitro) and (c) safety(most are not cytotoxic to hosts cells, i.e. non-hemolytic inhigh ionic strength buffer systems containing NaCl) [12].Resistance to classical antimicrobial therapies has thengreatly reduced the effectiveness of classical antibioticdrugs, leading to increased morbidity, mortality, and healthcare expenditure [30]. Possible drawbacks of the applicationof antimicrobial peptides are: (a) their exceedingly shorthalf-lives in the present of host serum [23]; (b) environmen-tal conditions, including high salt concentrations in bodyfluids, may reduce the antimicrobial activities [12], and (c)the potential emergence of resistant microbial strains, forexample by surface charge modifications or employment of microbial proteases that digest and inactivate antimicrobialpeptides [10]. In light of the similarities in mechanism(s) of action between therapeutic peptides and the diverse andwidely spread endogenous antimicrobial peptides, togetherwith the fact that endogenous antimicrobial peptides alsoregulate, in part, host innate immune responses [19], one can argue that microbial strains resistant to antimicrobialpeptides that alter membrane permeability/integrity are lesslikely to emerge. The emergence of such strains might alsohave consequences for the action of the endogenous an-timicrobial peptides. All things considered, peptide-basedantimicrobial therapies constitute a promising alternativeto, or in combination with, classical antibiotic drugs forprevention and suppression of pathology associated withmicrobial infections. Candida  species are often present as commensals on skinand mucosal surfaces occasionally causing opportunisticinfections [11,29]. With the increasing number of immuno-compromised hosts (i.e. patients presenting with HIV infec-tion or lymphoproliferative disorders, transplant recipientsundergoing immunosuppressive therapy, cancer patient un-dergoing chemotherapy), adequate treatment of   Candida infections is increasingly clinically relevant [6,28]. A large amount of work on the mechanisms of action of antimi-crobial peptides such as defensins, protegrins, histatins andlactoferrin-derived peptides has been performed in  Candidaalbicans , aiming at improving the treatment of candidiasis.In cases of polymicrobial infections [25], it ensues that combined therapeutic strategies are needed. Microbial in-terference, a naturally occurring phenomenon by which thepopulation density of a given microbial species is positivelyor negatively affected by coexisting species, thus, serves as arationale platform for the development of novel therapeuticstrategies. In this regard, it has been recently reported thatthe peptide  A 2 KKVFKRLEKLFSKIQNDK  20 , termed peptideHP (2–20), derived from the amino terminal of the  Heli-cobacter pylori  ribosomal protein L1 (HpRpL1) possessespotent antibacterial activity in vitro to which  H. pylori  isresistant (26). Furthermore, peptide HP (2–20) is endowedwith potent antifungal activity in vitro [17,18]. However, the therapeutic potential of peptide HP (2–20), as either anantibacterial or an antifungal agent, has not yet been testeddirectly in vivo, and thus, it is difficult to speculate on itstherapeutic value. In this study, we appraised the therapeu-tic efficacy and safety of synthetic peptide HP (2–20) usinga CBA/J mouse protection model of acquired lethal  C. albi-cans  infection by evaluating prolongation of survival. 2. Material and methods 2.1. Mice Male 4–6-week-old CBA/J mice (10–12g body weight),which are susceptible to  C. albicans  infection [2,14,24],were purchased from the University of São Paulo, Brazil,and maintained in the animal facility of the UniversidadeEstadual do Norte Fluminense Darcy Ribeiro, RJ, Brazil.They were housed five in a cage under standard conditionsof temperature and light and fed standard laboratory chowand water ad libitum. All procedures were performed in ac-cordance with the standards for humane handling, care andtreatment of research animals. 2.2. Cells The yeast strains used in this study were  C. albicans and  Candida tropicalis , both clinical isolates, and  Saccha-romyces cerevisiae  baker’s yeast, typed and kindly suppliedby Dr. Vˆania Maria Maciel Melo, Departamento de Micro-biologia, Universidade Federal do Ceará. Brazil. Yeast cellswere grown in Sabouraud dextrose agar (Difco Laboratories,Detroit, USA) plates for 48h at 28 ◦ C. 2.3. Growth inhibition curves/killing curves Colonies grown on Sabouraud dextrose agar plates werepick-lifted and resuspended in saline solution (150mMNaCl), shortly vortex, diluted in saline and counted un-der a microscope using an improved Newbauer bright-lineLevy double counting chamber (VWR Scientific, USA).Yeast cells (1  ×  10 4 CFU/ml) were cultured by triplicatein Sabouraud liquid medium in the presence of increas-ing concentration of peptide HP (2–20), as follows: 2.15,21.5, 107.5 and 215  M, in flat-bottom sterile 96-wellmicroplates, standing for 60h at 28 ◦ C. Growth was mon-itored reading  A 630nm  every 6h or 12h using a Dynatechmicroplate reader. 2.4. Peptide synthesis Peptide HP (2–20)  A 2 KKVFKRLEKLFSKIQNDK  20 , inits amide form was synthesised commercially (Genemed  P.D. Ribeiro, E. Medina-Acosta/Peptides 24 (2003) 1807–1814  1809 Synthesis Inc., CA, USA), essentially as described [9], using a Rink Amide AM Resin (Calbiochem-Novabiochem AG,Switzerland) and Fmoc-chemistry. Purification was carriedout by RP-HPLC using a Varian C18 column to achieved>95% grade. Mass spectrometry analysis indicated that pu-rified peptide HP (2–20) had a molecular weight of 2321.Purified peptide HP (2–20) was stored dry at  − 70 ◦ C, andreconstituted in sterile water at 8.6mM before use. 2.5. Hemolytic activity assay The hemolytic activity was determined as described by[12], with the following modifications. Type A + blood froma healthy individual was collected, after given informed con-sent, in vacuum tube containing EDTA as anticoagulant.The whole blood cells were harvested by centrifugation for10min at 2000 × g  at 20 ◦ C and washed three times in PBS(9mM sodium phosphate, pH 7.0 in 150mM NaCl). Cellswere reconstituted in PBS to 20% (v/v) packed cell vol-ume. This suspension was diluted 1:20 in either PBS (highionic strength) or in IGP (low ionic strength isotonic glu-cose phosphate buffer; 1mM potassium phosphate buffer,pH 7.0, supplemented with 287mM glucose as an osmo-protectant), and from this suspension 100  l were addedin triplicate to 100  l of the corresponding buffer contain-ing 2.15, 21.5, 107.5 or 215  M of peptide HP (2–20) in a96-well U-bottomed microtiter plate. No significant differ-ences were observed using either flat- or U-bottomed mi-crotiter plates (unpublished results). Blood samples werealso collected in the absence of anticoagulant and were de-fibrinated using sterile glass beads. Aliquots of 200  l of defibrinated blood were mixed with 2.15, 21.5, 107.5 or215  M of peptide HP (2–20). Complete control hemoly-sis was achieved by incubating processed blood sampleswith 1% Tween-20 in distilled water. All samples were in-cubated for 60min at 37 ◦ C and then centrifuged for 5minat 1500 × g  at 20 ◦ C. Aliquots of 100  l of the supernatantswere transferred to flat-bottomed microtiter plates, and theabsorbance was measured at 450nm. The hemolytic activitywas calculated as percentage, using the formula: % hemol-ysis = [(mean  A 450nm  value of triplicate peptide HP (2–20)treated sample  −  mean  A 450nm  value of triplicate buffertreated sample)/mean  A 450nm  value of triplicate Tween-20treated sample  −  mean  A 450nm  value of triplicate buffertreated sample)] × 100% [12]. 2.6. Determination of Candida albicans lethal inoculum Prior to the in vivo experiment,  C. albicans  were grown inSabouraud dextrose agar plates for 48h at 28 ◦ C. Colonieswere pick-lifted and resuspended in saline solution by vor-texing. Yeast cells were counted under a microscope usinga Newbauer chamber, sedimented by centrifugation and re-suspended in saline solution to yield 3 × 10 8 CFU/0.2ml or1 × 10 9 CFU/0.2ml. To ease interpretation of the potentialtherapeutic effects of peptide HP (2–20) in acquired lethalmurine candidiasis [1], the following experiment was con- ducted to determine the most adequate infection dose of  C. albicans  that causes a rapidly progressive disease with100% mortality in a short period of time (5 days). Fivemice per group were inoculated intraperitoneally with ei-ther 3 × 10 8 CFU (Group 1) or 1 × 10 9 CFU (Group 2) andmortality was monitored daily. 2.7. Experimental protocol For all in vivo experiments, the intraperitoneal route of infection was used, while the follow-up treatments were per-formed intravenously at the radial vein of the tail. To controlfor possible toxicity effects of the treatment of mice withpeptide HP (2–20) presented in saline as carrier, two groupsof two mice each were included: Group 3 mice were admin-istered intraperitoneally at day 1 with 100  g (ca. 8mg/kgbody weight) of peptide HP (2–20), followed by intravenousadministrations with 100  g of peptide HP (2–20) at 24hintervals during 4 days. Group 4 mice were administeredintraperitoneally at day 1 with 300  g (ca. 25mg/kg bodyweight) of peptide HP (2–20), followed by five intravenousadministrations with 300  g of peptide HP (2–20) at 12h in-tervals. For assessment of the therapeutic efficacy of peptideHP (2–20), two separate experiments were performed. In thefirst experiment, performed to monitored survival rates afterrepeated administration of low peptide doses spaced every24h, five mice (Group 5) were inoculated with 1 × 10 9 CFU C. albicans  mixed with 100  g of peptide HP (2–20) imme-diately before the procedure. The follow-up treatment con-sisted of four administrations with 100  g each peptide HP(2–20). In the second experiment, performed to evaluate thetherapeutic efficacy of higher doses of peptide HP (2–20)administered at closer intervals (12h), six mice (Group 6)were inoculated with 1 × 10 9 CFU  C. albicans  mixed with300  g of peptide HP (2–20) immediately before the pro-cedure. The follow-up treatment consisted of five adminis-trations with 300  g each. Lastly, 13 mice (Group 7) wereinoculated with 1 × 10 9 CFU  C. albicans  and these receivedno peptide HP (2–20) treatment at all. All groups of micewere examined for signs of toxicity or infection (lethargy,prostration, eye infection, mortality, survival time/rate). 3. Results 3.1. Antifungal activity of HP (2–20) tested in vitro Given that the objective of the present study was to eval-uate the therapeutic efficacy of the synthetic peptide amideHP (2–20) in a mouse protection model of lethal candidiasis,we reasoned that it was important to confirm in vitro, firstly,the antifungal activity and, secondly, the reported lack of hemolytic activity of the synthetic peptide HP (2–20). We,then, evaluated the killing effects of peptide HP (2–20),at increasing concentrations, on the in vitro growth curves  1810  P.D. Ribeiro, E. Medina-Acosta/Peptides 24 (2003) 1807–1814 Fig. 1. Time curves for the in vitro killing of (A)  Candida albicans , (B) Candida tropicalis , (C)  Saccharomyces cerevisiae  by peptide HP (2–20).Incubation mixtures contained a fixed number of yeast cells and theindicated increasing concentrations of peptide HP (2–20). of three fungi:  C. albicans ,  C. tropicalis  and  S. cerevisiae ,Fig. 1. Peptide HP (2–20) at 215  M inhibited (i.e. killed)100% growth of   C. albicans . Considering the standard de-viation (S.D.) values, no significant growth inhibition wasobserved at 107.5, 21.5 or 2.15  M, Fig. 1A.  C. tropicalis ,however, exhibited greater susceptibility to the antifungalpeptide HP (2–20), with 45, 90 and 100% inhibition of growth obtained at 21.5, 107.5 and 215  M, respectively.No detectable growth inhibition was observed at 2.15  M,Fig. 1B. Growth of   S. cerevisiae  was also greatly affectedby peptide HP (2–20), with 29, 85 and 100% killing ob-tained at 21.5, 107.5 and 215  M, respectively. As observedfor the other fungi, no inhibition of growth occurred at2.15  M, Fig. 1C. 3.2. Peptide HP (2–20) exhibits an ion strength- and dose-dependent hemolytic activity It is known that for peptide-based drugs, the lack of hemolytic activity distinguishes, for instance, an antimicro-bial peptide from a peptide-based venom. Moreover, be-cause the therapeutic index (i.e. the ratio HC 50  /IC 50 ) is of surmounting relevance for pharmacological studies, it is in-creasingly accepted that both hemolytic and killing assaysmust be performed in the same ionic strength buffer systems.Therefore, we re-appraised the issue of the reported lack of hemolytic activity of the synthetic peptide amide HP (2–20),particularly, since buffer systems differing in ionic strengthswere used in the previous studies [16–18]. Our experimen-tal design was based on the critical comparison study of thehemolytic and fungicidal activities of cationic antimicrobialpeptides published by Helmerhorst et al. [12], who pointed that since most cationic peptides are considerably hemolyticin low ionic strength buffers, at least two isotonic bufferssystems, differing in ionic strengths must be tested to cor-rectly infer on the therapeutic index. We, therefore, usedPBS (high ionic strength buffer system containing NaCl), aspreviously used by others for peptide HP (2–20) [16–18],and IGP (low ionic strength buffer system that contains noNaCl). To test the cytotoxicity of the synthetic peptide HP(2–20) against host cells, we assayed it for hemolytic activ-ity in vitro using human whole blood cells. As illustrated inFig. 2, in IGP, peptide HP (2–20) (net charge + 5, at neutralpH) exhibited a dose-dependent hemolytic activity (up to80% at 215  M). However, and more importantly, when as-sayed in PBS, the synthetic peptide HP (2–20), tested from2.15 to 215  M, exhibited < 20% hemolytic activity, as com-pared with control untreated samples. Furthermore, peptideHP (2–20), tested from 2.25 to 215  M, exhibited no de-tectable hemolytic activity using defibrinated human blood,a buffer system that closest reflects physiological conditions. 3.3. Therapeutic efficacy of peptide HP (2–20) To ease interpretation of the potential therapeutic effectsof peptide HP (2–20) in acquired lethal murine candidi-asis, it was important to determine the infective dose of  C. albicans  that causes a rapidly progressive disease with100% mortality in a short period of time (5 days) by theintraperitoneal route. We tested two doses: 3  ×  10 8 and1  ×  10 9 CFU, Fig. 3. Mice that were administered with 3 × 10 8 CFU slowly developed signs of disseminated can-didiasis with 20% survival rate at day 20 after inoculation.Mice that were administered with 1 × 10 9 CFU presentedwith a rapidly progressive and aggressive disease, with100% de mortality at day 5 after inoculation, Fig. 3. Thus,  P.D. Ribeiro, E. Medina-Acosta/Peptides 24 (2003) 1807–1814  1811Fig. 2. Hemolytic activity of peptide HP (2–20) in low and high ionic strength buffer systems tested on normal or defibrinated human blood. Each pointrepresents the mean value of triplicate samples ± S . D. Variation between triplicate experiments was  < 5%. for the in vivo protection experiments intended to evaluatethe therapeutic effect of peptide HP (2–20), an inoculumof 1 × 10 9 CFU administered intraperitoneally was used toestablish progressively lethal candidiasis.We next used a mouse protection model of induced lethalcandidiasis to evaluate the therapeutic potential of peptideHP (2–20) by directly testing its systemic antimycotic ac-tivity. Two experimental protocols were observed: a protec-tion/24h treatment protocol (Group 5 mice), consisting of daily intravenous administrations with 100  g (ca. 8mg/kgbody weight) of peptide HP (2–20), and a protection/12htreatment protocol (Group 6 mice), consisting of repeated(every 12h) intravenous administrations with 300  g (ca.25mg/kg body weight) of peptide HP (2–20).Mice that underwent the first experimental therapy pro-tocol (Group 5) presented with moderate increased survivalrate and time, as compared with those observed in the in-fected and untreated control mice (Group 7). At day 3 and4 after infection, survival rates were 40 and 20%, respec-tively, in the peptide HP (2–20) treated group, in contrast Fig. 3. Experimental determination of   Candida albicans  inoculum size that causes rapidly progressive lethal outcome of intraperitoneal infection in CBA/Jmale mice. to 7.5% at day 3 in the untreated control group, Fig. 4. Amore impressive preventive/suppressive effect of peptide HP(2–20) on the outcome of experimental lethal candidiasiswas observed applying the second therapy protocol (Group6), Fig. 4. At higher doses (300  g) of peptide HP (2–20),and using a more frequent scheme of systemic administra-tion (every 12h), treatment of infected mice (Group 6) re-sulted in, firstly, delayed in mortality onset (starting at day3, while 92.5% of the control infected and untreated micehad died by day 3); secondly, considerable prolongation insurvival time, thirdly, and most importantly, improved sur-vival rates (16.6% at day 14). 3.4. Safety of systemic repeated administration of micewith peptide HP (2–20) For any lead compound to be used safely in vivo, the for-mulation must not be toxic, in addition to exert no hemolyticactivity in physiological buffer systems. Given the potential
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