World J Microbiol Biotechnol (2013) 29:1217–1224 DOI 10.1007/s11274-013-1284-6
ORIGINAL PAPER
A heterodimer comprised of two bovine lactoferrin antimicrobial peptides exhibits powerful bactericidal activity against Burkholderia pseudomallei Aekkalak Puknun • Jan G. M. Bolscher • Kamran Nazmi • Enno C. I. Veerman • Sumalee Tungpradabkul • Surasakdi Wongratanacheewin • Sakawrat Kanthawong Suwimol Taweechaisupapong
•
Received: 20 November 2012 / Accepted: 6 February 2013 / Published online: 13 February 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Melioidosis is a severe infectious disease that is endemic in Southeast Asia and Northern Australia. Burkholderia pseudomallei, the causative agent of this disease, has developed resistance to an increasing list of antibiotics, demanding a search for novel agents. Lactoferricin and lactoferrampin are two antimicrobial domains of lactoferrin with a broad spectrum of antimicrobial activity. A hybrid peptide (LFchimera) containing lactoferrampin (LFampin265–284) and a part of lactoferricin (LFcin17–30) has strikingly higher antimicrobial activities compared to the individual peptides. In this study, the antimicrobial activities of this chimeric construct (LFchimera1), as well as of another one containing LFcin17–30 and LFampin268–284, a shorter fragment of LFampin265–284 (LFchimera2), and the constituent peptides were tested against 7 isolates of A. Puknun S. Wongratanacheewin S. Kanthawong Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand A. Puknun S. Wongratanacheewin S. Kanthawong S. Taweechaisupapong Melioidosis Research Center, Khon Kaen University, Khon Kaen 40002, Thailand J. G. M. Bolscher K. Nazmi E. C. I. Veerman Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU Universiteit Amsterdam, 1081 LA Amsterdam, The Netherlands S. Tungpradabkul Faculty of Science, Department of Biochemistry, Mahidol University, Bangkok 10400, Thailand S. Taweechaisupapong (&) Faculty of Dentistry, Department of Oral Diagnosis, Biofilm Research Group, Khon Kaen University, Khon Kaen 40002, Thailand e-mail:
[email protected]
B. pseudomallei and compared to the preferential antibiotic ceftazidime (CAZ). All isolates including B. pseudomallei 979b shown to be resistant to CAZ, at a density of 105 CFU/ml, could be killed by 5–10 lM of LFchimera1 within 2 h, while the other peptides as well as the antibiotic CAZ only inhibited the B. pseudomallei strains resulting in an overgrowth in 24 h. These data indicate that LFchimera1 could be considered for development of therapeutic agents against B. pseudomallei. Keywords Antimicrobial activity Antimicrobial peptide Burkholderia pseudomallei Lactoferrin LFchimera
Introduction Burkholderia pseudomallei is classified by the Centers for Disease Control and Prevention as a category B warfare agent (Rotz et al. 2002) and is the causative agent of melioidosis, a severe infectious disease with a wide range of clinical presentations. Melioidosis is highly endemic in Thailand and northern Australia; recently, however, melioidosis has been increasingly recognized in Central and South America (Inglis et al. 2006). With the increase in global travel by humans and global transport of animals, melioidosis is considered an emerging infectious disease. B. pseudomallei is intrinsically resistant to many of the currently marketed antibiotics, which limits their therapeutic use during patient treatment (Chaowagul 2000). The current standard treatment with agents to which B. pseudomallei is susceptible requires 2–4 weeks of parenteral therapy e.g. with ceftazidime (CAZ) as initial intensive therapy, followed by 3–6 months of oral eradication therapy e.g. with trimethoprim/sulfamethoxazole, doxycycline, chloramphenicol or a combination therapy. Despite this vigorous
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intervention, relapse occurs in 13–23 % of the cases (Cheng and Currie 2005) and the mortality rate in treated patients with severe melioidosis is[40 % (White 2003). In addition, reports have indicated the emergence of B. pseudomallei strains that are resistant to CAZ and trimethoprim (Dance et al. 1989; Wuthiekanun et al. 2005). Therefore, the search for novel anti-infective agents and short treatment regimens effective at clearing B. pseudomallei infections remains a vital area of investigation. Lactoferrin (LF) is a multifunctional iron-binding protein, which has broad-spectrum antimicrobial activity against bacteria, fungi, protozoa and viruses (Farnaud and Evans 2003). Most of LF’s activity has been attributed to the highly cationic N-terminal region of the protein (Gifford et al. 2005). Lactoferricin (LFcin) and lactoferrampin (LFampin) which are bioactive peptides that can be isolated from the N-terminal region of LF by proteolytic digestion display higher antimicrobial activity than the native protein (Bellamy et al. 1992; Yamauchi et al. 1993; Van Der Kraan et al. 2004; Bolscher et al. 2006). Recently, a chimeric construct (LFchimera) was designed by linking LFcin17–30 and LFampin265–284 peptides into one molecule displaying strikingly stronger antimicrobial activities against several microorganisms, including Gram-positive and Gram-negative bacteria, Candida albicans and parasites than its constituent peptides LFcin17–30 and LFampin265–284 (Bolscher et al. 2009; Leon-Sicairos et al. 2009; FloresVillasenor et al. 2010; Bolscher et al. 2012; Silva et al. 2012). In addition, the other LFchimera with chimerical structure containing LFcin17–30 and a shorter LFampin peptide, (LFampin268–284 instead of LFampin265–284) was reported to display bactericidal activity against Pseudomonas aeruginosa, down-regulated the extracellular virulence factors and biofilm formation of P. aeruginosa, and was significantly more effective than its constituent peptides LFcin17–30 and LFampin268–284 as well as the native LF (Xu et al. 2010). By screening for the susceptibility of B. pseudomallei to 10 antimicrobial peptides (Kanthawong et al. 2009), the present authors have shown previously that bovine LF peptides were only marginally active in comparison to LL-37. The reported striking increased antimicrobial activity of both LFchimera have stimulated the present investigation of these antimicrobial peptides against 7 isolates of B. pseudomallei, in comparison with their constituents and the conventional antibiotic CAZ.
Materials and methods Synthesis, purification and characterization of peptides The synthesis of the bovine lactoferrin peptides LFcin17–30, LFampin268–284 and LFampin265–284 was performed
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using fluoren-9-ylmethoxycarbonyl (Fmoc)-protected amino acids (Orpegen Pharma GmbH, Heidelberg, Germany) in a MilliGen 9050 peptide synthesizer (MilliGen-Biosearch, Bedford, USA) or a Syro II synthesizer (Biotage, Uppsala, Sweden) in the same manner as described previously (Van Der Kraan et al. 2004; Bolscher et al. 2009). The synthesis of the LFchimera was first described by Bolscher et al. (2009) and was accomplished using specific building blocks (NovaBiochemÒ, Merck Chemicals Ltd., Nottingham, United Kindom). Briefly, the synthesis was started by coupling Fmoc-Lys(ivDde)-OH to NovaSynÒTGR resin, followed by the sysntesis of LFcin 17–30, terminated with N-a-t.bocprotected Phe. Subsequently the ivDde-protecting group on the C-terminal Lys was released by hydrazinolysis followed by the synthesis of LFampin268–284 of LFampin265–284. In this way the chimerical peptides comprises a single C-terminal amidated lysine substituted at the a- and e-amino groups with the two peptides via the C-terminal site and leaving two N-termini as free ends. Peptides were purified by RP-HPLC (Jasco Corporation Tokyo, Japan) on a semipreparative Vydac C18-column (218MS510; Vydac, Hesperia, CA, USA) and the purity was tested on a analytic Vydac C18-column (218MS 5 l, 250 mm 9 4.6 mm) to be at least 95 % (Fig. 1). The authenticity of the peptides was confirmed by MALDI-TOF mass spectrometry on a Microflex LRF mass spectrometer equipped with an additional gridless reflectron (Bruker Daltonik, Bremen, Germany) as described previously (Bolscher et al. 2011). Amino acid sequences and characteristics of the 5 peptides investigated are shown in Table 1. Bacteria and growth conditions Seven isolates of B. pseudomallei were used in this study (Table 2). B. pseudomallei H777 was isolated from a patient admitted to Srinagarind Hospital (Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand). B. pseudomallei 1026b and SRM117 were kindly provided by Prof. D. E. Woods (University of Calgary Health Sciences Centre, Calgary, Canada). B. pseudomallei 979b was kindly provided by V. Wuthiekanun (Mahidol-Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand). Burkholderia pseudomallei were maintained on nutrient agar (NA) (CriterionTM; Hardy Diagnostics) except for B. pseudomallei M10, SRM117 and PPK mutants which were grown on Luria–Bertani (LB) agar (CriterionTM; Hardy Diagnostics) containing 15, 50 and 60 lg/ml tetracycline. B. pseudomallei isolates were cultured aerobically in brain–heart infusion (BHI) broth (CriterionTM; Hardy Diagnostics) at 37 °C overnight and, to yield a mid-logarithmic growth phase, then were subcultured at 37 °C in a 200 rpm shaker-incubator for 1.5 h.
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1219 Table 1 Sequences and characteristics of the peptides investigated Peptidea
Sequence
mol wt
Chargeb
LFcin17–30
FKCRRWQWRMKKLG
1,922
6?
LFampin265–284
DLIWKLLSKAQEKFGKNKSR
2,389
4?
LFampin268–284
WKLLSKAQEKFGKNKSR
2,047
5?
LFchimera1
FKCRRWQWRMKKLG—K
4,422
12?
4,081
13?
DLIWKLLSKAQEKFGKNKSR LFchimera2
FKCRRWQWRMKKLG—K WKLLSKAQEKFGKNKSR
a
The purity of the peptides was at least 95 % and the authenticity of the peptides was confirmed by ion trap mass spectrometry
b
Calculated net charge at neutral pH
Antimicrobial assay The killing activities of all peptides and CAZ against B. pseudomallei were determined by colony culturing assays as described previously (Kanthawong et al. 2009). Briefly, bacterial cells were washed three times and were re-suspended (approximately 105 CFU/ml) in 1 mM potassium phosphate buffer (PPB), pH 7.0. The bacterial suspension was then added to an equal volume of the tested agents to reach a final concentration of 1, 5, 10, 20, 50 and 100 lM. A bacterial suspension in PPB without peptide served as a control. Following incubation at 37 °C for 60 min, the incubation mixture was serially diluted in a physiological concentration of saline and plated in triplicate on NA. Colonies were counted after 24 h of incubation at 37 °C. The percentage killing or inhibiting effects of each agent were calculated using the formula [1 - (CFU sample/CFU control)] 9 100 %. Each assay was performed on two separate occasions, with triplicate determinations each time. Killing kinetic assay of LFchimera1 and LFchimera2
Fig. 1 RP-HPLC of LF-peptides and LFchimeras. The peptides were analyzed on a Vydac C18-column (218MS 5l, 250 mm 9 4.6 mm) developed with a linear gradient from 10 to 75 % Acetonitril (95 %, containing 0.1 % TFA) in 20 min at a flow rate of 1 ml/min. The absorbance of the column effluent was monitored at 220 nm
Killing kinetics were determined as described previously (Kanthawong et al. 2012). Bacterial cells were washed three times and were re-suspended in 1 mM of PPB (approximately 105 CFU/ml). Each peptide was added to the bacterial suspension to a final concentration of 5, 10, 20 and 50 lM and was incubated in a 200 rpm shaker-incubator at 37 °C. A bacterial suspension in PPB without peptide served as a control. At 0, 1, 2, 4, 6 and 24 h, samples were taken, serially diluted, plated in triplicate on NA and incubated for 24 h to allow colony counting.
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World J Microbiol Biotechnol (2013) 29:1217–1224 b Fig. 2 Antimicrobial activities of LF-peptides, LFchimeras and CAZ
against 7 isolates of B. pseudomallei. Bacterial suspensions were incubated with 1–100 lM of each agent and processed as described in materials and methods. A bacterial suspension without peptide served as a control. Percentage killing was calculated using the formula [1(CFU sample/CFU control)] 9 100 %. Data are the mean value of two independent experiments carried out in triplicate
1221 Table 2 Burkholderia pseudomallei isolates used in this study Isolate
Relevant characteristics
References
H777
Wild type, clinical isolate from blood
(Taweechaisupapong et al. 2005)
M10
Biofilm-defective mutant from a clinical isolated H777 wild type
(Taweechaisupapong et al. 2005)
NF10/38
Wild type, clinical isolate from blood
(Tunpiboonsak et al. 2010)
PPKM
A ppk mutant from a clinical isolated NF10/38 wild type
(Tunpiboonsak et al. 2010)
1026b
Wild type, clinical isolate
(DeShazer et al. 1997)
Results
SRM117
(DeShazer et al. 1998)
Antimicrobial activities of LF-peptides and LFchimera against B. pseudomallei
O-side chain LPS-defective mutant from a clinical isolated 1026b wild type
979b
Ceftazidime resistant isolate
(Kanthawong et al. 2009)
A bactericidal effect was defined as a C3 log10 reduction in CFU/ml compared with the initial inoculum. Each assay was performed on two separate occasions, with triplicated determinations each time.
The killing effect of the peptides at concentrations ranging from 1 to 100 lM against the B. pseudomallei isolates is shown in Fig. 2. The antimicrobial activities of each peptide or combinations thereof depended on the dose of the peptide and varied between the bacterial isolates. When comparing the peptides it is clear that LFampin265–284, which is three amino acids longer than LFampin268–284, exhibited somewhat higher antimicrobial activities than LFampin268–284 on most B. pseudomallei isolates and was comparable to LFcin17–30 as well as to CAZ. The same difference was observed when comparing the combination of LFcin17–30 and LFampin265–284 with the combination of LFcin17–30 and LFampin268–284. LFchimera1 (consisting of LFcin17–30 and LFampin265–284) and LFchimera2 (consisting of LFcin17–30 and LFampin268–284) displayed strikingly stronger antimicrobial activity than their constituent peptides and the combination of these peptides. Both LFchimeras showed also higher activities than CAZ. Comparison of the two LFchimeras, however, showed some differences depending on the bacterial isolate; for the wild-type B. pseudomallei 1026b and NF10/38 LFchimera1 seems a little more active while for the corresponding mutants B. pseudomallei SRM117 and PPKM, the LFchimara2 seems a little more potent. Comparison of the different strains indicated that B. pseudomallei M10 mutant appeared to be the least susceptible followed by the CAZ resistant B. pseudomallei 979b.
manner (Fig. 3). Without the addition of peptides, all isolates showed a growth of at least one log during the incubation period of 24 h. Addition of 10–50 lM LFchimera1 killed all 105 CFU/ml B. pseudomallei 979b within 2 h. A concentration of 5 lM LFchimera1was bactericidal, however did not reached the endpoint. In contrast, LFchimera2 never reached a bactericidal endpoint and B. pseudomallei 979b grew to ca. 106–107 CFU/ml after 24 h. LFchimera1 at all concentrations killed all 105 CFU/ ml B. pseudomallei 1026b within 2 h, whilst 4 h were needed for complete killing using 5 or 10 lM of the LFchimera2. For B. pseudomallei NF10/38 and H777, 5 lM LFchimera1 showed complete killing within 4 h, however, 5 lM LFchimera2 showed initial reduction in CFU/ml B. pseudomallei NF10/38 and H777 but it never reached a bactericidal endpoint. Moreover, at higher concentrations LFchimera1 killed all B. pseudomallei NF10/38 and H777 within 2 and 4 h. LFchimera2 showed only complete killing of B. pseudomallei NF10/38 at a concentration of 20 lM within 4 h while none of the concentrations of LFchimera2 reached a bactericidal endpoint for isolate B. pseudomallei H777. Overall, it is clear from these results that LFchimera1 has the highest potency with respect to killing of B. pseudomallei.
Discussion Long-term killing kinetics of LFchimera1 and LFchimera2 The two LFchimeras were further compared with respect to the long-term killing kinetics against 4 isolates of B. pseudomallei in a concentration- and time-dependent
The results from this study indicate that LFchimera possessed stronger killing activity against the tested B. pseudomallei isolates than their constituent peptides LFcin and LFampin and than the preferential antibiotic CAZ. Comparison of LFchimera1 and LFchimera2 in a long-term killing kinetics
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Fig. 3 Long-term killing kinetic of LFchimera1 and LFchimera2 against B. pseudomallei 1026b, NF10/38, H777 and 979b. Bacterial suspensions were incubated with the peptides (5–50 lM)) and samples were taken at the indicated time points (0, 1, 2, 4, 6 and 24 h). A bacterial suspension without peptide served as a control. The colonies were counted and a bactericidal effect was defined as a C3 log10 reduction in CFU/ ml compared with the initial inoculum. Data are the mean value of two independent experiments performed in duplicate. The LFcin17–30 and LFampin265–284 showed no killing in this assay (comparable to control, not shown)
assay using several isolates of B. pseudomallei revealed that LFchimera1 was the most powerful one. Moreover, the killing activity of LFchimera1 against B. pseudomallei 1026b was stronger than that of human cathelicidin peptides LL-37 and LL-31 studied in the previous report (Kanthawong et al. 2012). In contrast to 5 lM LFchimera1 which killed all 105 CFU/ml B. pseudomallei 1026b within 2 h, 5 lM LL-37 or LL-31 showed only an initial reduction in CFU/ml and as much as 20 lM was needed to reach a bactericidal endpoint (Kanthawong et al. 2012).
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This potent antimicrobial effect of LFchimera1, a chimerical structure containing LFcin17–30 and LFampin265–284, is in accordance with previous reports indicating strong bactericidal activity against several microorganisms, including Gram-positive and Gram-negative bacteria, fungi and parasites (Bolscher et al. 2009; LeonSicairos et al. 2009; Flores-Villasenor et al. 2010; Bolscher et al. 2012; Silva et al. 2012). In this study, all tested B. pseudomallei isolates including the CAZ resistant isolate were highly susceptible to LFchimera1. Although the exact
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mechanism by which the LFchimera1 kills B. pseudomallei is not clearly understood, previous studies have shown that LFchimera1 performs its bactericidal action toward antibiotic-resistant Staphylococcus aureus and Escherichia coli by causing permeabilization and disruption of the membrane (Flores-Villasenor et al. 2010). NMR analysis of LFchimera1 (Haney et al. 2012) indicated that both constituent peptides LFcin17–30 and LFampin265–284 were forced to a longer helical structure by the lysine linker. This structural conformation may play a role in the power of antimicrobial activity of LFchimera. Calcein leakage and vesicle fusion experiments with anionic liposomes revealed that LFchimera had enhanced membrane perturbing properties compared to the individual peptides. The increase in membrane permeability induced by the LFchimera1 is correlated with an increase positive charge of the LFchimera1 (?12) compared to LFcin17–30 and LFampin265–284 (?6 and ?4, respectively) which results in a stronger electrostatic attraction to the negatively charged surface of bacterial cells (Haney et al. 2012). LFchimera2 with a positive charge of 13, however, possessed lower killing activity. Comparison of the HPLC profiles of both LFchimeras (Fig. 1) indicate that LFchimera1 has a higher hydrophobic nature than LFchimera2, most probably due to the LFampin part of the molecule as similar shift in hydrophobicity is seen between LFampin 265–284 and LFampin268–284. This suggests that in this case hydrophobicity is a greater discriminant than charge. Comparison of NMR analysis of both LFchimera is needed to elucidate the relative importance of helicity of these peptides. In bacteria, polyphosphate (polyP) has a crucial role in stress responses and stationary-phase survival. Polyphosphate kinase (PPK) is the principal enzyme that catalyses the synthesis of polyP in bacteria (Zhu et al. 2005). In the present study, B. pseudomallei PPKM, a ppk mutant, was shown to be more susceptible to all tested peptides and CAZ than its wild type NF10/38. This is in agreement with role of PPK in motility, quorum sensing and biofilm formation, important contributors to antibiotic resistance of clinically important pathogens (Rashid et al. 2000). B. pseudomallei SRM117 was not susceptible to LFchimera1 and its constituent peptides at low concentration. This is consistent with the findings of a previous study that B. pseudomallei SRM117, the mutant lacking only the LPS type II O-polysaccharide, was not susceptible to the action of polymyxin B (PMB) and suggested that in order to achieve a PMB-susceptible phenotype in this organism, both outer core and O-antigen moieties of LPS type II must be absent (Burtnick and Woods 1999). Recently, therapeutic effects of LFchimera1 on the mortality rate, the CFU excreted in the feces, sepsis, and kidney and liver damage caused by enterohaemorrhagic E. coli O157:H7 in a mouse model were reported (FloresVillasenor et al. 2012). A mouse model of melioidosis will
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be used to test whether comparable results can be found for B. pseudomallei. In conclusion, the findings of this study suggest that LFchimera1 is very potent against B. pseudomallei and therefore could be considered as an excellent candidate to combat B. pseudomallei. Acknowledgments This work was supported by the Commission on Higher Education granting under the CHE-Ph.D.-SW and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Health Cluster (SHeP-GMS), Khon Kaen University. JGMB, KN and ECIV are supported by a grant from the University of Amsterdam for research into the focal point Oral Infections and Inflammation. We thank Emeritus Professor James A Will, University of Wisconsin-Madison for his time editing English of the manuscript.
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