Biometals (2014) 27:949–956 DOI 10.1007/s10534-014-9760-5
Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis Sakawrat Kanthawong • Aekkalak Puknun • Jan G. M. Bolscher • Kamran Nazmi • Jan van Marle • Johannes J. de Soet • Enno C. I. Veerman Surasakdi Wongratanacheewin • Suwimol Taweechaisupapong
•
Received: 14 February 2014 / Accepted: 6 June 2014 / Published online: 25 June 2014 Ó Springer Science+Business Media New York 2014
Abstract LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265–284 and lactoferricin17–30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and
below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.
S. Kanthawong A. Puknun S. Wongratanacheewin Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
J. van Marle Department of Electron Microscopy, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
S. Kanthawong A. Puknun S. Wongratanacheewin S. Taweechaisupapong Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand S. Kanthawong A. Puknun J. G. M. Bolscher K. Nazmi E. C. I. Veerman Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands
Keywords Burkholderia pseudomallei Burkholderia thailandensis Antimicrobial peptide LFchimera Lactoferrin
J. J. de Soet Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands S. Taweechaisupapong (&) Biofilm Research Group, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected]
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Table 1 Sequences of peptides tested Peptides
a
Sequence
LFchimera
FKCRRWQWRMKKLG—K
LL-37
LLGDFFRKSKEKIGKEFKRIVQRI KDFLRNLVPRTES
DLIWKLLSKAQEKFGKNKSR
a
The purity of peptides was at least 95 % and the authenticity of the peptides was confirmed by ion trap mass spectrometry
Introduction Melioidosis is a term describing a collection of serious diseases, often fatal in humans and animals, arising from infection by Burkholderia pseudomallei, a Gram-negative, facultative anaerobic, motile bacillus found in soil and water in the areas of endemic infection. This disease is widespread in Northern Australia, the Indian subcontinent, Iran, Central and South America and Southeast Asia (Wiersinga et al. 2013). The clinical manifestations can range from a silent infection that does not result in clinical diseases to acute infection with fulminate septicemias or chronic infection. Long periods of latency and persisting infections after antibiotic treatment are characteristic of melioidosis (Limmathurotsakul and Peacock 2011). Moreover, B. pseudomallei is intrinsically resistant to diverse groups of antibiotics, including third-generation cephalosporins, penicillins, rifamycins, aminoglycosides, quinolones and macrolides (Wuthiekanun et al. 2011; Schweizer 2012; Wiersinga et al. 2013), which is the reason why so much cases of relapse are found in melioidosis patients (Wiersinga et al. 2006). Increasing antibiotic resistance of this bacteria necessitates the development of novel anti-infective agents. Antimicrobial peptides (AMPs) are considered as potential agents to overcome the problem of the increasing resistance of bacterial strains to conventional antibiotics (Brogden 2005). Lactoferrin (LF) is an 80 kDa iron binding glycoprotein of the transferrin family found in the secretary fluids of mammals such as milk, tears, saliva, bronchial mucus and seminal plasma. It contains two antimicrobial domains: lactoferricin (LFcin) which is generated by pepsin digestion (Bellamy et al. 1992) and lactoferrampin (LFampin) (van der Kraan et al. 2004). Synthetic
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peptides derived from both domains showed more potent antibacterial activity than the native protein (van der Kraan et al. 2005; Haney et al. 2007; Bolscher et al. 2009; Leon-Sicairos et al. 2009). Moreover, joining parts of either domain (LFcin17–30, LFampin265–284) with a lysine residue resulting in the designated LFchimera enhanced strongly the bactericidal activity (Bolscher et al. 2009; Leon-Sicairos et al. 2009). We have found that LFchimera possesses a strong killing activity against seven isolates of B. pseudomallei, stronger than the conventional antibiotic ceftazidime (CAZ) and stronger than a shorter LFchimera variant consisting of LFcin17–30 and LFampin268–284 instead of LFampin265–284 (Puknun et al. 2013). However, the action mechanisms of LFchimera on B. pseudomallei and Burkholderia thailandensis are not well known yet. In this study, we evaluated the long term killing kinetics and mechanisms of action of LFchimera against B. pseudomallei and the closely related nonvirulent species, B. thailandensis, often used as modelorganism for the category B pathogen B. pseudomallei (Haraga et al. 2008). The results showed that B. thailandensis E264 was more susceptible to LFchimera than B. pseudomallei 1026b and that the antimicrobial activities of LFchimera were pH dependent. The mechanisms of action of LFchimera seem similar for both species: plasma membrane perturbations that cause the leakage of intracellular nucleotides leading to cell dead.
Materials and methods Peptide synthesis, purification and labeling The synthetic peptides LFchimera and LL-37 as well as the fluorescent labeled peptides (Table 1) were synthesized using fluoren-9- fluorenylmethoxycarbonyl (Fmoc)-protect amino acids in a MilliGen 9050 peptide synthesizer (MilliGen/Biosearch, Bedford, MA) and purified with RP-HPLC (Jasco Corperation, Japan) to a purity of at least 95 % as previously described (van der Kraan et al. 2004; Bolscher et al. 2009). The authenticity of the peptides was confirmed by MALDI-TOF mass spectrometry on a Microflex LRF mass spectrometer equipped with an additional gridless reflection (Bruker Daltonik,
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Fig. 1 The effect of pH on antimicrobial activity of LFchimera against B. thailandensis E264. (a) Bacterial suspension was incubated with various concentrations of LFchimera. (b) Bacterial suspension was incubated with 150 lM LFchimera at various pH. Samples were taken at the indicated time points (0, 1, 2, 4, 6 and 24 h). Colonies were counted and a bactericidal effect was defined as [3 log10 reduction in colony-forming unit (CFU)/ ml compared with the initial inoculums. Data are the mean value of two independent experiments carried out in duplicate
Bremen, Germany) as described previously (Bolscher et al. 2011).
Killing kinetics of LFchimera against B. pseudomallei and B. thailandensis
Cultures and media used
Approximately 105 colony-forming units (CFU)/ml of bacterial cells in potassium phosphate buffer (PPB, 1 mM, pH 7.0), were incubated with LFchimera at final concentration and different pH as indicated, in a 200 rpm shaker-incubator at 37 °C. A bacterial suspension in PPB without peptide served as a control. Samples were taken at 0, 1, 2, 4, 6 and 24 h, serially diluted, plated in triplicate on nutrient agar (Difco) and incubated at 37 °C for 24 h to allow colony counting. The reduction of bacterial cells as a C3 log10 in CFU/ ml compared with the initial inoculums was defined as bactericidal effect. Each assay was performed on two separate occasions, with duplicated determinations each time.
B. pseudomallei isolate 1026b and B. thailandensis E264 (kindly provided by Mahidol-Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand) were grown aerobically in Luria–Bertani (LB) agar (Difco, Becton–Dickinson Microbiology Systems, USA) at 37 °C for 24 h. In contrast to the mandated B. pseudomallei working conditions, B. thailandensis does not require a biosafety level 3 containment facility and has been used previously as a model to study certain aspects of B. pseudomallei biology (Haraga et al. 2008). For all experiments, both bacterial isolates were cultured in Brain heart infusion (BHI) broth (Difco, Becton– Dickinson Microbiology Systems) at 37 °C, overnight and, to yield a mid-logarithmic growth phase, subcultured at 37 °C in 200 rpm shaker-incubator for 1.5 h. In each experiment, bacterial cells were resuspended to the densities as indicated below.
Localization of LFchimera in bacteria The localization of LFchimera in B. pseudomallei 1026b and B. thailandensis E264 was determined using fluorescence microscopy and performed
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Fig. 2 The killing kinetics of LFchimera against B. thailandensis E264 (a) and B. pseudomallei 1026b (b): Bacterial suspensions were incubated with LFchimera (5-20 lM) and samples were taken at the indicated time points (0, 1, 2, 6 and
24 h). Colonies were counted and a bactericidal effect was defined as a C3 log10 reduction in colony forming units (CFU)/ml compared with the initial inoculum. Data are the mean value of two independent experiments carried out in duplicate
essentially as described previously (Ruissen et al. 2001). Bacterial suspension (approximately 108 CFU/ml) in 1 mM PPB was incubated with 20 lM FITClabeled LFchimera for 15–30 min, examined by fluorescence microscope (Leica DMRA, Germany) and photographed with a Leica DMDL camera. From our previous observation, we found that FITC-labeled LL-37 bounded to B. thailandensis membrane (unpublished data). Thus, bacterial suspension incubated with 20 lM FITC-labeled LL-37 was used as control.
5 min. The leakage of the nucleotides AMP, ADP and ATP from bacteria was analyzed by capillary zone electrophoresis with a BioFocus 2000 Capillary Electrophoresis System (Bio-Rad Laboratories, Hercules, CA) equipped with an uncoated fused silica capillary of internal diameter 50 lm and length 50 cm. Samples were monitored continuously at 260 nm. Peaks were quantified against the nucleotide standard containing a mixture of 10 lM AMP, ADP and ATP (den Hertog et al. 2006).
Freeze-fracture electron microscopy B. pseudomallei 1026b and B. thailandensis E264 (approximately 108 CFU/ml) were incubated with 20 lM LFchimera in PPB for 1 h, then centrifuged and resuspended in fixative solution (2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). Freeze-fracture electron microscopy was performed as described previously (den Hertog et al. 2006). A transmission electron microscope (Philips EM-420; Philips, The Netherlands) operated at 100 kV and equipped with a SIS MegaView II camera (Olympus Soft Imaging Solutions GmbH, Germany) was used to examine replicas. Release of nucleotides B. pseudomallei 1026b and B. thailandensis E264 (approximately 108 CFU/ml) were incubated with 20 lM LFchimera. Samples were collected at several time points (1 and 2 h), and centrifuged at 5,0009g for
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Hemolysis assay The hemolytic activity of LFchimera was tested against human red blood cells (hRBCs) essentially as described previously (Oren et al. 1999). Fresh hRBCs were rinsed three times and resuspended in PPB, then 400 lL of the hRBC suspension was incubated with 20 lM LFchimera at 37 °C for 1 h. Samples were centrifuged at 8009g for 10 min and the release of hemoglobin was monitored by measuring the absorbance of the supernatant at 540 nm (A540). Controls for zero hemolysis and 100 % hemolysis consisted of hRBCs suspended in PPB and 2 % Triton X-100 (v/v), respectively. The hemolysis percentage was calculated using the equation: % Hemolysis ¼ 100 ½A540 in the peptide solution A540 in PPB ½A540 in 2 % Triton X 100 A540 in PPB
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Fig. 3 Fluorescence micrographs of B. thailandensis E264 and B. pseudomallei 1026b treated with FITC-labeled LFchimera and LL-37. B. thailandensis E264 (a) and B. pseudomallei 1026b (b) incubated with 20 lM FITC-labeled LFchimera
showed a rapid staining of cytoplasm after 15 min; (c) B. thailandensis E264 showed localization of FITC-labeled LL-37 at the plasma membrane after 15 min. Magnification 91000
Statistical analysis
within 2 h, whilst 150 lM of LFchimera at pH 5, 6 and 6.4 showed only initial reduction in CFU/ml in first 6 h, and then bacteria grew to 104–106 CFU/ml after 24 h of incubation (Fig. 1b). Next, long term killing kinetics against B. pseudomallei and B. thailandensis were determined using different concentrations of LFchimera adjusted to pH 7.0. LFchimera at concentrations of 15 and 20 lM killed all 105 CFU/ml B. thailandensis E264 within 6 and 2 h, respectively (Fig. 2a), whilst only 20 lM LFchimera reached the bactericidal endpoint for B. pseudomallei 1026b within 2 h (Fig. 2b). However, 5 and 10 lM of LFchimera showed initial reduction of CFU/ml of B. thailandensis E264 within the first 6 h in a concentration-dependent way, but these concentrations never reached a bactericidal endpoint and bacteria grew to ca. 103 CFU/ml after 24 h. Furthermore, B. pseudomallei 1026b exhibited less susceptibility to LFchimera. At concentrations of 5, 10 and 15 lM, LFchimera reduced number of B. pseudomallei 1026b within 4 h and the bacteria grew again to [104 CFU/ml within 24 h.
The leakage of nucleotides are expressed as mean ± standard deviation of two independent experiments carried out in duplication. The statistical significance of differences between the leakage of nucleotides from LFchimera-treated and control groups was then tested using Student’s t test. The level required for statistical significance was P \ 0.05.
Results Antimicrobial activities of LFchimera Initially, various concentrations of LFchimera (20–150 lM) were used to analyze the antimicrobial activities against 105 CFU/ml B. thailandensis E264 during a 24 h incubation. LFchimera at concentration 20, 40, 60 lM reached the bactericidal endpoint within 2 h, while 80 and 100 lM of LFchimera showed complete killing within 4 h. In contrast, 150 lM of LFchimera never reached the bactericidal endpoint and the bacteria grew to 105 CFU/ml after 24 h of incubation (Fig. 1a). This seemingly reciprocal inhibition appeared to be caused by pH changes of the incubation mixture due to the high peptide concentration, being 7.12, 6.92, 6.86, 6.71, 6.54 and 6.4, for 20 up to 150 lM, respectively for LFchimera solubilized in water. To control this, the effect of 150 lM of LFchimera was tested at a pH varying from 5 to 7. At pH 7 all 105 CFU/ml of bacteria were killed
Localization of LFchimera in B. pseudomallei and B. thailandensis The interaction between FITC-labeled LFchimera and bacteria was analyzed using fluorescence microscopy. After incubation with 20 lM FITC-labeled peptide for 15 min with B. pseudomallei 1026b and B. thailandensis E264, a rapid staining of the cytoplasm was observed and this pattern continued up to 30 min (Fig. 3a, b). No accumulation of FITC-labeled
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LFchimera was found on the membrane of both Burkholderia strains. In contrast FITC-labeled cathelicidin LL-37 did accumulate on the membrane after 15 min (Fig. 3c). The effects of LFchimera on bacterial morphology, membrane permeability and hemolytic effects The effects of LFchimera on the membrane morphology of B. pseudomallei 1026b and B. thailandensis E264 were examined using freeze-fracture electron microscopy. The micrographs of untreated bacteria in Fig. 4a, c showed a rod like shape, smooth surface and a regular periplasmic area between the inner (black
Fig. 4 Freeze-fracture electron micrographs of B. thailandensis E264 and B. pseudomallei 1026b cells. B. thailandensis E264 (a) and B. pseudomallei 1026b (c) incubated with potassium phosphate buffer (PPB) showed normal inner (black arrow head) and outer membrane (white arrow head); (b) B. thailandensis E264 incubated with 20 lM LFchimera showed clearly irregular membranes and shapes of bacteria (black arrow); (d) B. pseudomallei 1026b incubated with 20 lM LFchimera demonstrated the collapse on the bacterial cell membrane (black arrow), and blebs were also found. Scale bars, 500 nm
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arrow head) and outer membrane (white arrow head). Following incubation with 20 lM LFchimera, the membrane and shape of B. pseudomallei 1026b and B. thailandensis E264 were dramatically disturbed. Irregularities and blebs were observed on the cell membrane of both bacterial isolates (Fig. 4b, d, black arrows). LFchimera treatment caused fracture along cytoplasmic membrane in approximately 80 % of B. pseudomallei 1026b and [95 % of B. thailandensis E264 of (data not shown). Occasionally even total collapse of the B. pseudomallei 1026b membrane was observed (Fig. 4d). LFchimera-induced membrane permeabilization was further investigated by measuring the intracellular level of nucleotides (AMP, ADP and ATP). After treatment with 20 lM LFchimera for 1 h, a drastic reduction of intracellular nucleotides was observed in both bacterial isolates. After 2 h of incubation a further decrease in intracellular level of nucleotides occurred (Fig. 5a, b).
Fig. 5 LFchimera-induced leakage of nucleotides and protein from bacterial cells. (a) B. thailandensis E264 and (b) B. pseudomallei 1026b cells were incubated with 20 lM LFchimera for 1 and 2 h. Concentrations of nucleotides in the lysis fractions were measured by capillary zone electrophoresis. Data are presented as the mean and standard deviation of two independent experiments. *P \ 0.05 compared to control
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Next, the cytotoxicity of 20 lM LFchimera to human erythrocytes was determined in a hemolysis assay. LFchimera induced only minimal release of hemoglobin (around 9 % compared with the control treatment with Triton X-100; data not shown).
Discussion The present study shows that LFchimera exhibits bactericidal activity against both B. pseudomallei and B. thailandensis. The B. thailandensis strain being a model for the severe pathogen B. pseudomallei was somewhat more susceptible (Fig. 2). Remarkable however was the striking pH sensitivity. Lowering the pH from 7.0 to 6.4 resulted in a shift in the dose– response curve to a two-fold higher concentration of the peptide (Fig. 1b). This indicates the importance of the pH, which have to be pH 7.0 for optimal killing activity by AMPs like LFchimera. Killing kinetic assay demonstrated that B. thailandensis E264 and B. pseudomallei 1026b were sensitive to LFchimera in a dose-dependent manner. Similar results have been found for various Grampositive and Gram-negative bacteria such as Vibrio parahaemolyticus, fungi and parasites (Bolscher et al. 2009; Leon-Sicairos et al. 2009; Flores-Villasenor et al. 2010, 2012; Silva et al. 2012). B. thailandensis E264 appeared to be more susceptible to LFchimera than B. pseudomallei 1026b comparable to the difference previously found with human cathelicidin LL-37 (Kanthawong et al. 2010, 2012). This might be due to differences in lipopolysaccharide (LPS) as the major lipid A species identified in B. pseudomallei is capped with 4-amino-4-deoxy-arabinose (Ara4N) residues, and is different from B. thailandensis LPS (Novem et al. 2009). Ara4N lipid A modifications reduce the negative charge of the bacterial membrane resulting in increased bacterial resistance to cationic AMPs and cationic antibiotics by altering the binding of these peptides to the membrane (Gunn et al. 1998; Kawasaki et al. 2005). In the present study, localization of FITC-labeled LFchimera in bacteria revealed a rapid staining of the cytoplasm within 15 min and this pattern continued up to 30 min while the accumulation of FITC-labeled LL-37 was observed on the membrane for more than 30 min. Furthermore, freeze-fracture electron microscopy of bacterial cells demonstrated that LFchimera
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caused dramatic destruction, collapse and blebbing on the cell membrane of both bacterial strains, and resulting in the leakage of vital constituents such as nucleotides from the cell. Also for other bacterial species, it was shown that LFchimera directly interacts with biological membranes and enhances membrane disruption (Leon-Sicairos et al. 2009; Flores-Villasenor et al. 2010; Silva et al. 2012). The antimicrobial activities of many cationic peptides are diminished in physiological salt concentrations (Bellamy et al. 1992). In contrast, LFchimera, which exhibits more potent bactericidal activity than the constituent peptides, is not sensitive to ionic strength (Bolscher et al. 2009; Leon-Sicairos et al. 2009). On the other hand, concentrations up to 20 lM LFchimera do not induce significant lysis or permeabilization of human red blood, nor cytotoxicity on rat hepatocytes (Leon-Sicairos et al. 2009). In conclusion these results merit further investigation and exploration of LFchimera for the therapy of melioidosis. Acknowledgments This work was supported by the Commission on Higher Education granting under the CHEPh.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.
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956 Bactericidal effect of bovine lactoferrin, LFcin, LFampin and LFchimera on antibiotic-resistant Staphylococcus aureus and Escherichia coli. Biometals 23:569–578 Flores-Villasenor H, Canizalez-Roman A, de la Garza M, Nazmi K, Bolscher JG, Leon-Sicairos N (2012) Lactoferrin and lactoferrin chimera inhibit damage caused by enteropathogenic Escherichia coli in HEp-2 cells. Biochimie 94:1935–1942 Gunn JS, Lim KB, Krueger J, Kim K, Guo L, Hackett M et al (1998) PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance. Mol Microbiol 27:1171–1182 Haney EF, Lau F, Vogel HJ (2007) Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin. Biochim Biophys Acta 1768:2355–2364 Haraga A, West TE, Brittnacher MJ, Skerrett SJ, Miller SI (2008) Burkholderia thailandensis as a model system for the study of the virulence-associated type III secretion system of Burkholderia pseudomallei. Infect Immun 76:5402–5411 Kanthawong S, Bolscher JG, Veerman EC, van Marle J, Nazmi K, Wongratanacheewin S et al (2010) Antimicrobial activities of LL-37 and its truncated variants against Burkholderia thailandensis. Int J Antimicrob Agents 36:447–452 Kanthawong S, Bolscher JG, Veerman EC, van Marle J, de Soet HJ, Nazmi K et al (2012) Antimicrobial and antibiofilm activity of LL-37 and its truncated variants against Burkholderia pseudomallei. Int J Antimicrob Agents 39:39–44 Kawasaki K, Ernst RK, Miller SI (2005) Inhibition of Salmonella enterica serovar Typhimurium lipopolysaccharide deacylation by aminoarabinose membrane modification. J Bacteriol 187:2448–2457 Leon-Sicairos N, Canizalez-Roman A, de la Garza M, ReyesLopez M, Zazueta-Beltran J, Nazmi K et al (2009) Bactericidal effect of lactoferrin and lactoferrin chimera against halophilic Vibrio parahaemolyticus. Biochimie 91:133–140 Limmathurotsakul D, Peacock SJ (2011) Melioidosis: a clinical overview. Br Med Bull 99:125–139 Novem V, Shui G, Wang D, Bendt AK, Sim SH, Liu Y et al (2009) Structural and biological diversity of lipopolysaccharides from Burkholderia pseudomallei and Burkholderia thailandensis. Clin Vaccine Immunol 16:1420–1428
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Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis Sakawrat Kanthawong • Aekkalak Puknun • Jan G. M. Bolscher • Kamran Nazmi • Jan van Marle • Johannes J. de Soet • Enno C. I. Veerman Surasakdi Wongratanacheewin • Suwimol Taweechaisupapong
•
Received: 14 February 2014 / Accepted: 6 June 2014 / Published online: 25 June 2014 Ó Springer Science+Business Media New York 2014
Abstract LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265–284 and lactoferricin17–30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and
below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.
S. Kanthawong A. Puknun S. Wongratanacheewin Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
J. van Marle Department of Electron Microscopy, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
S. Kanthawong A. Puknun S. Wongratanacheewin S. Taweechaisupapong Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand S. Kanthawong A. Puknun J. G. M. Bolscher K. Nazmi E. C. I. Veerman Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands
Keywords Burkholderia pseudomallei Burkholderia thailandensis Antimicrobial peptide LFchimera Lactoferrin
J. J. de Soet Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands S. Taweechaisupapong (&) Biofilm Research Group, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected]
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Table 1 Sequences of peptides tested Peptides
a
Sequence
LFchimera
FKCRRWQWRMKKLG—K
LL-37
LLGDFFRKSKEKIGKEFKRIVQRI KDFLRNLVPRTES
DLIWKLLSKAQEKFGKNKSR
a
The purity of peptides was at least 95 % and the authenticity of the peptides was confirmed by ion trap mass spectrometry
Introduction Melioidosis is a term describing a collection of serious diseases, often fatal in humans and animals, arising from infection by Burkholderia pseudomallei, a Gram-negative, facultative anaerobic, motile bacillus found in soil and water in the areas of endemic infection. This disease is widespread in Northern Australia, the Indian subcontinent, Iran, Central and South America and Southeast Asia (Wiersinga et al. 2013). The clinical manifestations can range from a silent infection that does not result in clinical diseases to acute infection with fulminate septicemias or chronic infection. Long periods of latency and persisting infections after antibiotic treatment are characteristic of melioidosis (Limmathurotsakul and Peacock 2011). Moreover, B. pseudomallei is intrinsically resistant to diverse groups of antibiotics, including third-generation cephalosporins, penicillins, rifamycins, aminoglycosides, quinolones and macrolides (Wuthiekanun et al. 2011; Schweizer 2012; Wiersinga et al. 2013), which is the reason why so much cases of relapse are found in melioidosis patients (Wiersinga et al. 2006). Increasing antibiotic resistance of this bacteria necessitates the development of novel anti-infective agents. Antimicrobial peptides (AMPs) are considered as potential agents to overcome the problem of the increasing resistance of bacterial strains to conventional antibiotics (Brogden 2005). Lactoferrin (LF) is an 80 kDa iron binding glycoprotein of the transferrin family found in the secretary fluids of mammals such as milk, tears, saliva, bronchial mucus and seminal plasma. It contains two antimicrobial domains: lactoferricin (LFcin) which is generated by pepsin digestion (Bellamy et al. 1992) and lactoferrampin (LFampin) (van der Kraan et al. 2004). Synthetic
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peptides derived from both domains showed more potent antibacterial activity than the native protein (van der Kraan et al. 2005; Haney et al. 2007; Bolscher et al. 2009; Leon-Sicairos et al. 2009). Moreover, joining parts of either domain (LFcin17–30, LFampin265–284) with a lysine residue resulting in the designated LFchimera enhanced strongly the bactericidal activity (Bolscher et al. 2009; Leon-Sicairos et al. 2009). We have found that LFchimera possesses a strong killing activity against seven isolates of B. pseudomallei, stronger than the conventional antibiotic ceftazidime (CAZ) and stronger than a shorter LFchimera variant consisting of LFcin17–30 and LFampin268–284 instead of LFampin265–284 (Puknun et al. 2013). However, the action mechanisms of LFchimera on B. pseudomallei and Burkholderia thailandensis are not well known yet. In this study, we evaluated the long term killing kinetics and mechanisms of action of LFchimera against B. pseudomallei and the closely related nonvirulent species, B. thailandensis, often used as modelorganism for the category B pathogen B. pseudomallei (Haraga et al. 2008). The results showed that B. thailandensis E264 was more susceptible to LFchimera than B. pseudomallei 1026b and that the antimicrobial activities of LFchimera were pH dependent. The mechanisms of action of LFchimera seem similar for both species: plasma membrane perturbations that cause the leakage of intracellular nucleotides leading to cell dead.
Materials and methods Peptide synthesis, purification and labeling The synthetic peptides LFchimera and LL-37 as well as the fluorescent labeled peptides (Table 1) were synthesized using fluoren-9- fluorenylmethoxycarbonyl (Fmoc)-protect amino acids in a MilliGen 9050 peptide synthesizer (MilliGen/Biosearch, Bedford, MA) and purified with RP-HPLC (Jasco Corperation, Japan) to a purity of at least 95 % as previously described (van der Kraan et al. 2004; Bolscher et al. 2009). The authenticity of the peptides was confirmed by MALDI-TOF mass spectrometry on a Microflex LRF mass spectrometer equipped with an additional gridless reflection (Bruker Daltonik,
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Fig. 1 The effect of pH on antimicrobial activity of LFchimera against B. thailandensis E264. (a) Bacterial suspension was incubated with various concentrations of LFchimera. (b) Bacterial suspension was incubated with 150 lM LFchimera at various pH. Samples were taken at the indicated time points (0, 1, 2, 4, 6 and 24 h). Colonies were counted and a bactericidal effect was defined as [3 log10 reduction in colony-forming unit (CFU)/ ml compared with the initial inoculums. Data are the mean value of two independent experiments carried out in duplicate
Bremen, Germany) as described previously (Bolscher et al. 2011).
Killing kinetics of LFchimera against B. pseudomallei and B. thailandensis
Cultures and media used
Approximately 105 colony-forming units (CFU)/ml of bacterial cells in potassium phosphate buffer (PPB, 1 mM, pH 7.0), were incubated with LFchimera at final concentration and different pH as indicated, in a 200 rpm shaker-incubator at 37 °C. A bacterial suspension in PPB without peptide served as a control. Samples were taken at 0, 1, 2, 4, 6 and 24 h, serially diluted, plated in triplicate on nutrient agar (Difco) and incubated at 37 °C for 24 h to allow colony counting. The reduction of bacterial cells as a C3 log10 in CFU/ ml compared with the initial inoculums was defined as bactericidal effect. Each assay was performed on two separate occasions, with duplicated determinations each time.
B. pseudomallei isolate 1026b and B. thailandensis E264 (kindly provided by Mahidol-Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand) were grown aerobically in Luria–Bertani (LB) agar (Difco, Becton–Dickinson Microbiology Systems, USA) at 37 °C for 24 h. In contrast to the mandated B. pseudomallei working conditions, B. thailandensis does not require a biosafety level 3 containment facility and has been used previously as a model to study certain aspects of B. pseudomallei biology (Haraga et al. 2008). For all experiments, both bacterial isolates were cultured in Brain heart infusion (BHI) broth (Difco, Becton– Dickinson Microbiology Systems) at 37 °C, overnight and, to yield a mid-logarithmic growth phase, subcultured at 37 °C in 200 rpm shaker-incubator for 1.5 h. In each experiment, bacterial cells were resuspended to the densities as indicated below.
Localization of LFchimera in bacteria The localization of LFchimera in B. pseudomallei 1026b and B. thailandensis E264 was determined using fluorescence microscopy and performed
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Fig. 2 The killing kinetics of LFchimera against B. thailandensis E264 (a) and B. pseudomallei 1026b (b): Bacterial suspensions were incubated with LFchimera (5-20 lM) and samples were taken at the indicated time points (0, 1, 2, 6 and
24 h). Colonies were counted and a bactericidal effect was defined as a C3 log10 reduction in colony forming units (CFU)/ml compared with the initial inoculum. Data are the mean value of two independent experiments carried out in duplicate
essentially as described previously (Ruissen et al. 2001). Bacterial suspension (approximately 108 CFU/ml) in 1 mM PPB was incubated with 20 lM FITClabeled LFchimera for 15–30 min, examined by fluorescence microscope (Leica DMRA, Germany) and photographed with a Leica DMDL camera. From our previous observation, we found that FITC-labeled LL-37 bounded to B. thailandensis membrane (unpublished data). Thus, bacterial suspension incubated with 20 lM FITC-labeled LL-37 was used as control.
5 min. The leakage of the nucleotides AMP, ADP and ATP from bacteria was analyzed by capillary zone electrophoresis with a BioFocus 2000 Capillary Electrophoresis System (Bio-Rad Laboratories, Hercules, CA) equipped with an uncoated fused silica capillary of internal diameter 50 lm and length 50 cm. Samples were monitored continuously at 260 nm. Peaks were quantified against the nucleotide standard containing a mixture of 10 lM AMP, ADP and ATP (den Hertog et al. 2006).
Freeze-fracture electron microscopy B. pseudomallei 1026b and B. thailandensis E264 (approximately 108 CFU/ml) were incubated with 20 lM LFchimera in PPB for 1 h, then centrifuged and resuspended in fixative solution (2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). Freeze-fracture electron microscopy was performed as described previously (den Hertog et al. 2006). A transmission electron microscope (Philips EM-420; Philips, The Netherlands) operated at 100 kV and equipped with a SIS MegaView II camera (Olympus Soft Imaging Solutions GmbH, Germany) was used to examine replicas. Release of nucleotides B. pseudomallei 1026b and B. thailandensis E264 (approximately 108 CFU/ml) were incubated with 20 lM LFchimera. Samples were collected at several time points (1 and 2 h), and centrifuged at 5,0009g for
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Hemolysis assay The hemolytic activity of LFchimera was tested against human red blood cells (hRBCs) essentially as described previously (Oren et al. 1999). Fresh hRBCs were rinsed three times and resuspended in PPB, then 400 lL of the hRBC suspension was incubated with 20 lM LFchimera at 37 °C for 1 h. Samples were centrifuged at 8009g for 10 min and the release of hemoglobin was monitored by measuring the absorbance of the supernatant at 540 nm (A540). Controls for zero hemolysis and 100 % hemolysis consisted of hRBCs suspended in PPB and 2 % Triton X-100 (v/v), respectively. The hemolysis percentage was calculated using the equation: % Hemolysis ¼ 100 ½A540 in the peptide solution A540 in PPB ½A540 in 2 % Triton X 100 A540 in PPB
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Fig. 3 Fluorescence micrographs of B. thailandensis E264 and B. pseudomallei 1026b treated with FITC-labeled LFchimera and LL-37. B. thailandensis E264 (a) and B. pseudomallei 1026b (b) incubated with 20 lM FITC-labeled LFchimera
showed a rapid staining of cytoplasm after 15 min; (c) B. thailandensis E264 showed localization of FITC-labeled LL-37 at the plasma membrane after 15 min. Magnification 91000
Statistical analysis
within 2 h, whilst 150 lM of LFchimera at pH 5, 6 and 6.4 showed only initial reduction in CFU/ml in first 6 h, and then bacteria grew to 104–106 CFU/ml after 24 h of incubation (Fig. 1b). Next, long term killing kinetics against B. pseudomallei and B. thailandensis were determined using different concentrations of LFchimera adjusted to pH 7.0. LFchimera at concentrations of 15 and 20 lM killed all 105 CFU/ml B. thailandensis E264 within 6 and 2 h, respectively (Fig. 2a), whilst only 20 lM LFchimera reached the bactericidal endpoint for B. pseudomallei 1026b within 2 h (Fig. 2b). However, 5 and 10 lM of LFchimera showed initial reduction of CFU/ml of B. thailandensis E264 within the first 6 h in a concentration-dependent way, but these concentrations never reached a bactericidal endpoint and bacteria grew to ca. 103 CFU/ml after 24 h. Furthermore, B. pseudomallei 1026b exhibited less susceptibility to LFchimera. At concentrations of 5, 10 and 15 lM, LFchimera reduced number of B. pseudomallei 1026b within 4 h and the bacteria grew again to [104 CFU/ml within 24 h.
The leakage of nucleotides are expressed as mean ± standard deviation of two independent experiments carried out in duplication. The statistical significance of differences between the leakage of nucleotides from LFchimera-treated and control groups was then tested using Student’s t test. The level required for statistical significance was P \ 0.05.
Results Antimicrobial activities of LFchimera Initially, various concentrations of LFchimera (20–150 lM) were used to analyze the antimicrobial activities against 105 CFU/ml B. thailandensis E264 during a 24 h incubation. LFchimera at concentration 20, 40, 60 lM reached the bactericidal endpoint within 2 h, while 80 and 100 lM of LFchimera showed complete killing within 4 h. In contrast, 150 lM of LFchimera never reached the bactericidal endpoint and the bacteria grew to 105 CFU/ml after 24 h of incubation (Fig. 1a). This seemingly reciprocal inhibition appeared to be caused by pH changes of the incubation mixture due to the high peptide concentration, being 7.12, 6.92, 6.86, 6.71, 6.54 and 6.4, for 20 up to 150 lM, respectively for LFchimera solubilized in water. To control this, the effect of 150 lM of LFchimera was tested at a pH varying from 5 to 7. At pH 7 all 105 CFU/ml of bacteria were killed
Localization of LFchimera in B. pseudomallei and B. thailandensis The interaction between FITC-labeled LFchimera and bacteria was analyzed using fluorescence microscopy. After incubation with 20 lM FITC-labeled peptide for 15 min with B. pseudomallei 1026b and B. thailandensis E264, a rapid staining of the cytoplasm was observed and this pattern continued up to 30 min (Fig. 3a, b). No accumulation of FITC-labeled
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LFchimera was found on the membrane of both Burkholderia strains. In contrast FITC-labeled cathelicidin LL-37 did accumulate on the membrane after 15 min (Fig. 3c). The effects of LFchimera on bacterial morphology, membrane permeability and hemolytic effects The effects of LFchimera on the membrane morphology of B. pseudomallei 1026b and B. thailandensis E264 were examined using freeze-fracture electron microscopy. The micrographs of untreated bacteria in Fig. 4a, c showed a rod like shape, smooth surface and a regular periplasmic area between the inner (black
Fig. 4 Freeze-fracture electron micrographs of B. thailandensis E264 and B. pseudomallei 1026b cells. B. thailandensis E264 (a) and B. pseudomallei 1026b (c) incubated with potassium phosphate buffer (PPB) showed normal inner (black arrow head) and outer membrane (white arrow head); (b) B. thailandensis E264 incubated with 20 lM LFchimera showed clearly irregular membranes and shapes of bacteria (black arrow); (d) B. pseudomallei 1026b incubated with 20 lM LFchimera demonstrated the collapse on the bacterial cell membrane (black arrow), and blebs were also found. Scale bars, 500 nm
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arrow head) and outer membrane (white arrow head). Following incubation with 20 lM LFchimera, the membrane and shape of B. pseudomallei 1026b and B. thailandensis E264 were dramatically disturbed. Irregularities and blebs were observed on the cell membrane of both bacterial isolates (Fig. 4b, d, black arrows). LFchimera treatment caused fracture along cytoplasmic membrane in approximately 80 % of B. pseudomallei 1026b and [95 % of B. thailandensis E264 of (data not shown). Occasionally even total collapse of the B. pseudomallei 1026b membrane was observed (Fig. 4d). LFchimera-induced membrane permeabilization was further investigated by measuring the intracellular level of nucleotides (AMP, ADP and ATP). After treatment with 20 lM LFchimera for 1 h, a drastic reduction of intracellular nucleotides was observed in both bacterial isolates. After 2 h of incubation a further decrease in intracellular level of nucleotides occurred (Fig. 5a, b).
Fig. 5 LFchimera-induced leakage of nucleotides and protein from bacterial cells. (a) B. thailandensis E264 and (b) B. pseudomallei 1026b cells were incubated with 20 lM LFchimera for 1 and 2 h. Concentrations of nucleotides in the lysis fractions were measured by capillary zone electrophoresis. Data are presented as the mean and standard deviation of two independent experiments. *P \ 0.05 compared to control
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Next, the cytotoxicity of 20 lM LFchimera to human erythrocytes was determined in a hemolysis assay. LFchimera induced only minimal release of hemoglobin (around 9 % compared with the control treatment with Triton X-100; data not shown).
Discussion The present study shows that LFchimera exhibits bactericidal activity against both B. pseudomallei and B. thailandensis. The B. thailandensis strain being a model for the severe pathogen B. pseudomallei was somewhat more susceptible (Fig. 2). Remarkable however was the striking pH sensitivity. Lowering the pH from 7.0 to 6.4 resulted in a shift in the dose– response curve to a two-fold higher concentration of the peptide (Fig. 1b). This indicates the importance of the pH, which have to be pH 7.0 for optimal killing activity by AMPs like LFchimera. Killing kinetic assay demonstrated that B. thailandensis E264 and B. pseudomallei 1026b were sensitive to LFchimera in a dose-dependent manner. Similar results have been found for various Grampositive and Gram-negative bacteria such as Vibrio parahaemolyticus, fungi and parasites (Bolscher et al. 2009; Leon-Sicairos et al. 2009; Flores-Villasenor et al. 2010, 2012; Silva et al. 2012). B. thailandensis E264 appeared to be more susceptible to LFchimera than B. pseudomallei 1026b comparable to the difference previously found with human cathelicidin LL-37 (Kanthawong et al. 2010, 2012). This might be due to differences in lipopolysaccharide (LPS) as the major lipid A species identified in B. pseudomallei is capped with 4-amino-4-deoxy-arabinose (Ara4N) residues, and is different from B. thailandensis LPS (Novem et al. 2009). Ara4N lipid A modifications reduce the negative charge of the bacterial membrane resulting in increased bacterial resistance to cationic AMPs and cationic antibiotics by altering the binding of these peptides to the membrane (Gunn et al. 1998; Kawasaki et al. 2005). In the present study, localization of FITC-labeled LFchimera in bacteria revealed a rapid staining of the cytoplasm within 15 min and this pattern continued up to 30 min while the accumulation of FITC-labeled LL-37 was observed on the membrane for more than 30 min. Furthermore, freeze-fracture electron microscopy of bacterial cells demonstrated that LFchimera
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caused dramatic destruction, collapse and blebbing on the cell membrane of both bacterial strains, and resulting in the leakage of vital constituents such as nucleotides from the cell. Also for other bacterial species, it was shown that LFchimera directly interacts with biological membranes and enhances membrane disruption (Leon-Sicairos et al. 2009; Flores-Villasenor et al. 2010; Silva et al. 2012). The antimicrobial activities of many cationic peptides are diminished in physiological salt concentrations (Bellamy et al. 1992). In contrast, LFchimera, which exhibits more potent bactericidal activity than the constituent peptides, is not sensitive to ionic strength (Bolscher et al. 2009; Leon-Sicairos et al. 2009). On the other hand, concentrations up to 20 lM LFchimera do not induce significant lysis or permeabilization of human red blood, nor cytotoxicity on rat hepatocytes (Leon-Sicairos et al. 2009). In conclusion these results merit further investigation and exploration of LFchimera for the therapy of melioidosis. Acknowledgments This work was supported by the Commission on Higher Education granting under the CHEPh.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.
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