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The ATPase activity of the mycobacterial plasma membrane is inhibited by the LL37-analogous peptide LLAP
Peptides 71 (2015) 222–228
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The ATPase activity of the mycobacterial plasma membrane is inhibited by the LL37-analogous peptide LLAP Sandra Chingaté a , Gabriela Delgado b , Luz Mary Salazar a,∗ , Carlos-Yesid Soto a a b
Chemistry Department, Faculty of Sciences, Universidad Nacional de Colombia, Carrera 30# 45-03, Ciudad Universitaria, Bogotá, Colombia Pharmacy Department, Faculty of Sciences, Universidad Nacional de Colombia, Carrera 30# 45-03, Ciudad Universitaria, Bogotá, Colombia
a r t i c l e
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Article history: Received 24 April 2015 Received in revised form 22 July 2015 Accepted 22 July 2015 Available online 26 July 2015 Keywords: Mycobacteria Antimicrobial peptides LL37 Cell membrane ATPase activity
a b s t r a c t The emergence of multidrug-resistant Mycobacterium tuberculosis strains has led to the development of new antituberculous agents. In this context, antimicrobial targeting proteins to the cell membrane are interesting due to the avoidance of the plasma membrane permeation. Through this study, the antimicrobial activity, cellular toxicity, as well as the effect on the mycobacterial cell membrane ATPase activity of a cathelicidin-analogous peptide were assessed. By using bioinformatics analyses, a 15 amino acid LL37analogous peptide called LLAP, which has the amino acid sequence: GRKSAKKIGKRAKRI, was designed to improve its helical structure and antibacterial activity compared to the native sequence. The LLAP peptide was synthesized, purified by RP-HPLC and its structural characteristics were determined by MALDI-TOF MS and circular dichroism. Compared to the native amino acid sequence, the minimum inhibitory concentration and cytotoxic activity of LLAP were 4.0 and 5.6-fold lower, respectively. In addition, the hemolytic activity of LLAP was lower than 1.1% and the cytotoxic activity of peptides was similar for both peptides. Interestingly, the LLAP peptide displayed approximately 50% inhibition of basal ATPase activity of the mycobacterial plasma membrane, which could in turn be associated with the impaired cell viability. The results suggest that LLAP could be considered as potential antimycobacterial compounds against cell membrane targeting ATPases. However, this antimycobacterial activity can be improved. It is expected further applications to be found for other antimicrobial peptides families based on the implemented methodology. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Tuberculosis (TB) is the infectious disease that produces the largest number of deaths around the world caused by a bacterial infectious agent [1]. According to the World Health Organization (WHO), there were 9.0 million new cases and 1.5 million deaths caused for TB in 2013 [1]. The emergence of multidrug resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains, and the co-infection with HIV has led to the discovery of alternative antimycobacterial compounds. The highly hydrophobic cell envelope provides the mycobacteria a barrier to many antimicrobial compounds [2]. Thus, the study of the cell membrane
Abbreviations: AMPs, antimicrobial peptides; CD, circular dichroism; IC50, median cytotoxic concentration; HC50, median hemolytic concentration; LLAP,LL37, -analogous peptide; MDR, multidrug resistant; MIC, minimum inhibitory concentration; RBC, red blood cells; TB, tuberculosis; XDR, extensively drug-resistant. ∗ Corresponding author: Tel.: +57 1 3165000x14449; fax: +57 1316 5220. E-mail address: [email protected] (L.M. Salazar). http://dx.doi.org/10.1016/j.peptides.2015.07.021 0196-9781/© 2015 Elsevier Inc. All rights reserved.
antimicrobial targeting proteins could be interesting, since they avoid issues related with the plasma membrane permeation [3]. The mycobacterial cell membrane contains different kinds of pumps associated with the cell detoxification, as well as enzymes capable of hydrolyze or modify the antimicrobial compounds [4]. P-type ATPases are cell membrane transporters which hydrolyze ATP releasing energy used to generate electrochemical gradients [5]. This kind of enzymes use these electrochemical gradients to transport metal ions and other solutes across the plasma membrane [5,6]. The large number of P-type ATPases in the M. tuberculosis genome strongly suggests that these transporters might be essential for the mycobacterial viability and virulence [7]. For instance, the M. tuberculosis CtpF is a putative alkali/alkaline earth cation metal transporter activated in the presence of toxic substances as isoniazid, tetrahydrolipstatin, isoxyl, and sodium hypochlorite; moreover, CtpF is the only M. tuberculosis P-type ATPase regulated by the global latency regulator dosR and also over expressed during the infection of human macrophages [8]. The potential of antimicrobial peptides (AMPs) as antimycobacterial compounds has not yet been deeply explored. AMPs are able
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to disrupt bacterial membranes via non-specific electrostatic interactions with the phospholipids [9,10]. Some studies have shown that net charge, amphipathicity and structural tendency are important parameters that explain the AMPs ability to interact with cell membranes [11,12]. Cathelicidins are a family of the structurally diverse AMPs from several mammalian species, including humans, monkeys, horses, cattle, sheep, goats, pigs, rabbits, mice and guinea pigs that are part of the innate immunity response [13]. The hCAP-18 cathelicidin protein is expressed in human neutrophils and proteolysed within the c-terminal end to produce a 37 amino acid peptide known as LL-37 [14]. This cathelicidin LL37 peptide has an amphipathic alpha-helical structure that shows membrane-directed antimicrobial, antiviral, antifungal and anticancer activity [15–18], and its interaction with the cell membrane is affected by the oligomerization degree [19]. In addition, LL-37 has an important role in the human being innate immune system displaying functions of chemotaxis, immune modulation and wound healing [20]. Information related with the cathelicidins upregulation in J7774 macrophages infected with M. smegmatis [21] has been reported, suggesting that this type of antimicrobial peptides may play an important role in controlling intracellular survival of mycobacteria. Nevertheless, the interaction between cathelicidins and the mycobacterial cell envelope is not well known. In a previous work we established that the magainin-analogous peptide MIAP inhibits the F1 F0 ATPase activity of mycobacterial plasma membrane, suggesting the mycobacterial ATPases as alternative for the identification of new therapeutic targets of mycobacteria [22]. In this work in silico modifications of the LL37 cathelicidin generated a 15 amino acid analogous peptide with improved antimycobacterial activity and low cytotoxicity and hemolysis, which inhibit the basal ATPase activity on the mycobacterial plasma membrane.
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by Houghten [25] using Rink amide solid resin as support with a substitution of 0.52mmol/g. Peptides were analyzed by RP-HPLC in a column of ODS in a gradient A/B from 0 to 70% B (A: Deionized water 0.05% TFA, B: acetonitrile 0.05% TFA, flow 1.0 mL/min) for 45 min and then purified through a preparative technique. The molecular weight of peptides was determined by MALDI-TOF MS (Autoflex Bruker Daltonics, Germany). Circular dichroism (CD) assays were performed in a Jasco J-815CD Spectropolarimeter to determine the pattern of peptides secondary structure. Spectra were performed in triplicate at a scan rate of 20 nm/min, using peptide dissolved in 0.30% aqueous 2,2,2-trifluoroethanol (2 mM final concentration) to avoid induction of secondary structure. Spectra deconvolution was performed by using CDPro (Continll, Selcon3 and CDsst) tools (http://lamar.colostate.edu/∼sreeram/ CDPro/main.html). 2.4. Antimicrobial susceptibility testing The MIC of peptides was estimated on M. smegmatis mc2 155 cells using resazurin as an indicator of cell viability. Briefly, 100 L of serial dilutions of peptides (from 50 mg/mL) were mixed in 96-well microtiter plates with 100 L of a 1:100 dilution of M. smegmatis mc2 155 cells growth until OD600 = 0.5. Mycobacterial cells were incubated at 37 ◦ C for 4 days; then, 30 L of resazurin (5 mg/mL) were added and after that, cells were subsequently incubated for 24 h. Bacterial growth was shown by a change in the broth color from yellow to violet, and the MIC was defined as the lowest peptide concentration avoiding this color change. Mycobacterial cells supplemented without peptide, and kanamycin (final concentration: 25 g/mL) were used as positive and negative controls, respectively. 2.5. Assays of cytotoxicity
2. Materials and methods 2.1. Mycobacterial strains and culture conditions The fast-growing M. smegmatis mc2 155 strain [23] was used in this work. Mycobacteria were grown in LB broth with agitation at 70 rpm and 37 ◦ C for 4–6 days until an OD600 of 0.4–0.5 approximately for the minimum inhibitory concentration (MIC) of peptides and for mycobacterial plasma membrane isolation. 2.2. Bioinformatic design of LL37-analogous peptides The server (http://www.imtech.res.in/raghava/antibp/index. html) and database (APD, http://aps.unmc.edu/AP/main.php) were used to identify positive charged LL37-amino acid sequences (15 amino acids), which displayed the best antimicrobial score. The antimicrobial scores calculated by the anti-BP server were predicted by the SVM (support vector machine) algorithm, which displays the highest values of accuracy, sensitivity and specificity for each 15 amino acid sequence [24]. The prediction of the structural and chemical characteristics of the selected peptides were determined by Protparam (http://web.expasy.org/protparam/) for the primary structure, and PSSpred for the secondary structure (http://zhanglab.ccmb.med.umich.edu/PSSpred). The primary amino acid sequence in the selected peptide was manually changed to increase its antimicrobial score, its cationic charge and the proportion of ␣-helices in the secondary structures as predicted by Jpred (http://www.compbio.dundee.ac.uk/www-jpred/). 2.3. Peptide synthesis and characterization The 15 amino acid native and the LL37-analogous peptide (LLAP) were synthesized by the Fmoc solid phase methodology proposed
The cytotoxicity of peptides was determined in duplicate on 100 L of J774 macrophages suspension (1.0 × 104 cells in RPMI supplemented with 5% fetal calf serum) seeded on 96-well microtiter plates (Corning, USA). Cells were incubated at 37 ◦ C for 24 h under 5% CO2 atmosphere and subsequently supplemented with 100 L of serial dilutions of peptide (from 2500 to 19.53 g/mL final concentration). Incubation of cells continued for 72 h; after that, resazurin (Sigma Aldrich, USA) was added to a final concentration of 44 mM. Finally, the cells were incubated for additional 4 h and the fluorescence units were read by using a spectrofluorometer (Tecan Genios Plate Reader) and Magellan® Software. Samples with kanamycin and mycobacterial cells without peptide were used as negative and positive controls, respectively. Florescence units were processed in Microsoft Excel 2000 to estimate cell viability percentages. For this purpose, fluorescence from samples of cells supplemented without peptide was considered as 100% feasibility. The percentage of viability was considered as follows: (fluorescence units at X peptide concentration × 100) /(fluorescence units from cells which have not been exposed to any treatment). In order to obtain the median cytotoxic concentration (IC50 ) – considered as the peptide concentration that produces death in 50% of the treated cells – the calculated percentages were plotted (concentration vs. response) by using GraphPad Prism 5 Demo Software (GraphPad Software, Inc., La Jolla, CA, USA). 2.6. Hemolysis assays The hemolytic activity was performed in duplicate. Briefly, 0.1 mL of O Rh + red blood cells (RBC) from healthy human donors suspended in 0.9% saline solution (Baxter, Colombia) to obtain a final hematocrit concentration of 2%, were mixed with 0.1 mL of serial peptide dilutions (from 50 to 0.19 g/mL) in V-bottom, 96-
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well plates (Corning, USA). After 2 h of incubation at 37 ◦ C with 5% of CO2 , the plates were centrifuged at 3500 rpm and the supernatants were collected to determine the concentration of hemoglobin by measuring OD540 in a digital spectrophotometer (Ultramark Microplate Reader, Bio-Rad, USA). The assay included control wells containing erythrocytes without peptide in saline solution (corresponding to 0% of hemolysis), erythrocyte in sterile distilled water (control for 100% of hemolysis), insulin as unrelated peptide, and kanamycin at inhibitory concentration. Absorbance units from saline-diluted RBC that were not exposed to any substance were used as a negative control (100% of viability). Absorbance readings of supernatants from RBC solutions exposed to each peptide and kanamycin were normalized regarding the absorbance reading obtained for the RBC solutions exposed to sterile water, which were considered as 100% of hemolysis. The percentage of hemolysis was calculated in a MS Excel Worksheet, then analyzed with the GraphPad Prism 5.0 Trial Version® program (LA Jolla, CA, USA), and the median hemolytic concentration (HC50 ) was calculated as equivalent to the concentration of peptide that causes 50% of RBC lysis.
Table 1 In silico analysis of 15 amino acid sequences from LL37 using the AntiBP tool.
2.7. Plasma membrane isolation Plasma membranes were isolated by using the protocol reported by Basu and co-workers with some modifications [26–28]. In brief, harvested mycobacteria were centrifuged and resuspended in a pH 7.4 buffer solution (10 mM MOPS, 1 mM EDTA and 0.3 mM phenylmethylsulfonyl fluoride) and lysed in a Mini Beadbeater-16 (Biospec, USA) for 7 min. Samples were centrifuged at 25,000 × g for 30 min at 4 ◦ C and the supernatants containing the plasma membranes were again centrifuged at 100,000 × g for 90 min at 4 ◦ C. The resulting pellets (plasma membrane fraction) were frozen for further use at −80 ◦ C in pH 7.4 buffer containing 10 mM MOPS and 0.08 g/mL sucrose. Protein concentration was determined by the Bradford-Zor-Selinger method [29]. 2.8. ATPase activity The ATPase activity was determined by using the FiskeSubbarow method [30,31] with some modifications, incorporating bismuth citrate to stabilize the molybdenum complexes [27]. The ATPase assay was performed in 96-well microtiter plates in which reactions (50 L) included 100 mM MOPS (pH 7.4), 3 mM MgCl2 , 150 mM NaCl, 0.02% (w/v) Brij-58 and 38 g of M. smegmatis plasma membrane vesicles. Samples were pre-incubated at 37 ◦ C for 5 min. The enzymatic reactions started by adding 3 mM Tris-ATP (final concentration) and incubated at 37 ◦ C for 30 min. Reactions were stopped by adding 100 L of stop solution (3% ascorbic acid, 0.5% ammonium molybdate, 3% SDS in 0.5 M HCl), and additional incubation of samples on ice were performed for 10 min. The complex concentration was determined at 690 nm in a Microplate absorbance iMarkTM Reader (BioRad). To estimate the peptides effect on the basal ATPase activity of mycobacterial plasma membrane, enzymatic reactions were separately supplemented with peptide (50 M) by using reactions without plasma membrane protein as negative controls. Reactions were assessed in triplicate from three independent experiments. The entire enzymatic activity was reported as U/mg of protein (nmol of Pi released/mg × min). 3. Results 3.1. In silico selection of LL37-analogous peptides with increased antimicrobial activity AntiBP server and database APD2 were used to estimate the antibacterial score of amino acid sequences derived from the cathe-
licidin LL37. Specifically, 15 amino acid sequences were analyzed by walking from the N- to C of the antibacterial peptide. Thus, the 15 amino acids fragment with the best antibacterial score (native peptide) was identified as shown in Table 1. Then, some residues from the primary amino acid sequence of the native peptide were manually replaced and the antimicrobial scores were again in silico estimated. We observed that the replacement of Phe-1 → Gly, Lys5 → Ala, Glu6 → Lys, Glu11 → Arg and Phe12 → Ala in the native peptide originated an analogous sequence (the LLAP peptide) which allowed the greatest increase of the antimicrobial score; specifically, from 2108 to 3120 (Fig. 1). In addition, LLAP exhibited a high content of the basic amino acids: Arg (20%) and Lys (33.3%). The predicted isoelectric point and molecular weight for LLAP were 12, 32 and 1697,1 respectively (Fig. 1). 3.2. LLAP is an amphipathic and cationic peptide which displays low helical structure The RP-HPLC analyses indicated high purity of the synthetized peptides, evidenced by the presence of only one peak in the obtained chromatograms. In addition, the peptides molecular mass determined by mass-spectrometry analysis, corresponded to the expected theoretical masses (Fig. 2). The LLAP has a charge of +8, and according to the helical wheel distribution, it displayed an amphipathic distribution of amino acids (Fig. 1). In this context, the structural profile of the native and the LLAP peptides by CD, showed two maximum: one at 190 nm (positive –* transition) with low molar ellipticity and other at 222 nm with an ellipticity near to zero which suggested an undefined structure with low tendency to helicity for both peptides (Fig 3). In addition, spectra deconvolution showed that peptides displayed approximately 37% of the alpha-helical content. 3.3. The LLAP peptide displays an increased antimycobacterial and low toxicity compared to the native amino acid sequence To compare the antimycobacterial activity between the designed and the native amino acid sequence, an antimicrobial susceptibility test was performed by using the environmental and non-pathogenic strain M. smegmatis mc2 155. This experiment demonstrated that the analogue peptide LLAP showed a MIC
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Fig. 1. Insilico peptides analysis. The antimicrobial score predicted by AntiBP of the 15 amino acid native sequences (a) and the analogue LLAP peptide (b); amino acids with positive charge and hydrophobic are shown in pink and black respectively. The helical wheel of peptides (c) is also presented.
Fig.2. Peptides purity.
MALDI-TOF MS indicating the purity and molecular weight of the 15 amino acid native peptide (A) and LLAP peptide (B).
approximately 4-fold, lower than the one showed for the parental peptide (Table 2). The peptides cytotoxicity was estimated by comparing the fluorescence released by the samples supplemented with peptides, and the fluorescence released by cells without supplemented peptide. In general, the peptides cytotoxicity was low. LLAP and native peptides showed specifically IC50 of 11,226 g/mL and 7727 g/mL for J774 cell; that was approximately 13.8 and 9.5-fold lower than the positive control (kanamycin), respectively. Regarding the peptides hemolytic activity on human erythrocytes,
0 190 -10000
200
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the LLAP peptide displayed a hemolytic activity 4-fold (1.1%) lower than the one showed for the native amino acid sequence (Table 2). 3.4. LLAP inhibits the basal ATPasa activity of mycobacterial plasma membrane In order to estimate the peptides harmful effect on the ATPase activity of mycobacterial plasma membrane, the basal ATPase
0 190 -20000
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-50000 -60000
M O L A R E L L IPT .
M O L A R EL L IPT .
-40000
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-60000 -80000
-100000 -120000
-70000 -80000 -90000
-140000 -160000 Fig. 3. Circular dichroism (CD) spectra for the native (A) and LLAP (B) peptides.
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Table 2 Effect of native peptide and LLAP peptides on the viability of Mycobacterium smegmatis mc2 155, J774 macrophages and hemolysis of human erythrocytes. The median cytotoxic concentration (IC50 ) is considered as the peptide concentration that produces death in 50% of the treated cells, and the median hemolytic concentration (HC50 ) is equivalent to the concentration of peptide that causes 50% of red blood cells lysis. The minimum inhibitory concentration (MIC) of kanamycin on M. smegmatis cells (2 g/mL) has been previously reported [44]. PEPTIDE
MIC (g/mL)
IC50 (g/mL)
% Hemolysis
Charge
Native* LLAP Kanamycin
2400 600 ND
11226 7727 813
4,0 1,1 ND
7 8 ND
*15 amino acid native sequence from LL37; ND = non determined.
activity (stimulated by Mg2+ ) of plasma membrane vesicles from M. smegmatis (control) was compared with the enzymatic activity estimated in presence of lethal concentration of peptides (MIC). The enzymatic reactions were supplemented with 0.02% (w/v) Brij-58 to ensure that most of the vesicles were right-side out [22]. Thus, the cell membrane ATPase proteins were exposed to the reaction media and adequately stimulated by Mg2+ (cofactor) allowing the released Pi to the reaction media be easily quantified. Reactions were supplemented with 50 M of peptide (final concentration) due to the estimation as an optimal concentration for assaying ATPase activity. Plasma membrane vesicles of M. smegmatis mc2 155 showed a basal ATPase activity of 1.46 ± 0.14 nmol Pi/min × mg corresponding to 100% of the activity. Nevertheless, when reactions were performed in the presence of native and LLAP peptides, the enzymatic activity diminished from 1.31 to ± 0.03 and from 0.72 to ± 0.08 nmol Pi/min × mg, respectively, which was related to an inhibition of enzymatic activity of 10.1 and 50.6%, respectively (Fig. 4). 4. Discussion In this study, an LL37-analogous peptide was in silico designed. This displayed increased antimycobacterial activity, low toxicity, and specially, inhibition of the basal ATPase activity of mycobacterial plasma membrane, compared with the native amino-acid sequence. LL37 belongs to the cathelidicin antimicrobial peptide family, as part of the innate immune response in humans [13]. We decided to evaluate the potential of cathelicidins as antimycobacterial compounds because it is known that this AMPs family has an interesting role in the intracellular killing of M. tuberculo-
% Inhibion ATPase acvity
60 50,6 ± 0,08
50 40 30 20 10,1 ± 0,03
10 0
0,0 ± 0,14 Basal
LL37-Nave Pepde
LLAP
Fig. 4. Inhibition of the plasma membrane basal ATPase activity, mediated by peptides. The inhibition percentage corresponds to the difference between ATPase activity of plasma membrane vesicles in the presence and absence of peptides (LL37-A Native peptide and LL37 Analogue peptide LLAP). Error bars correspond to the standard deviation.
sis; therefore, their molecular mechanisms of action may be useful to develop strategies for the M. tuberculosis control. Cathelicidins are present in different human epithelia, and interact with the cell membrane by inducing pores formation and cell death [19], which could be induced by D vitamin and mediated by IFN-␥ [32–36]. Previous studies have shown that AMPs have limited applications as antituberculous agents, mainly explained by the hydrophobic nature of the mycobacterial cell envelope that reduces permeability for antimicrobial compounds [2]. For instance, wellknown AMPs such as beta-defensin, human neutrophil peptide-1 (HNP1), granulysins, protegrins and cathelicidins, display low in vitro activity against M. tuberculosis, which increases in the case of protegrin-1 and beta-defensin-1, only when combined with typical antituberculous drugs as isoniazid, [32]. However in a previous work, we reported both, in silico and manually strategies which allowed the improvement of the AMPs antimicrobial activity which originally displayed low antimycobacterial activity [22]. In this study, the viability of non-pathogenic and fast-growing M. smegmatis mc2 155 strain was estimated in the presence of the designed LLAP peptide by using resazurin as an indicator of cell viability. M. smegmatis is an environmental mycobacteria that shares a high percentage of genomic and physiological characteristics with M. tuberculosis [37–39]. Proteomic studies show that approximately 70% of M. tuberculosis proteins have conserved orthologues in M. smegmatis; for instance, M. smegmatis contains secretory systems necessary to over-express M. tuberculosis proteins involved in mycobacterial virulence, and also contains the same genomic background part of the M. tuberculosis resistance to typical anti-TB drugs (http://mycobrowser.epfl.ch/smegmalist. html). In addition, M. smegmatis has technical benefits such as a shorter culture time and minor laboratory risks compared to other standard strains used to evaluate anti-TB drugs, such as M. bovis BCG. From this perspective, M. smegmatis is a suitable model to evaluate the mycobacterial-surviving processes, which can be extrapolated to the natural behavior of the tubercle bacilli. Regarding the design of the analogous peptide, the bioinformatic analyses suggested an improved antibacterial activity for LLAP on M. smegmatis mc2 155 cells compared to the native amino acid sequence. LLAP exhibited a high content of the basic amino acids Arg and Lys and the presence of Lys in C-terminal, which – as expected – improved the positive charge and the pore forming characteristics. Helical wheel projection and secondary structure prediction suggest that the amino acid sequence of both, the native and LLAP peptides, should adopt an alpha helix structure. It also suggests that LLAP should have an amphipathic helical structure; however, according to CD, both peptides display low tendency of helicity. The spectra deconvolution results showed that both molecules tend to be alpha-helical structures rather than other structural possibilities. Although the percentages of this structural element are lower than 36%, we consider that this percentage of helical structure is enough to partially explain the LLAP antimycobacterial activity. We also consider that the native and LLAP peptides may have flexibility to configure amphipathic regions despite they do not display full helical structure. Therefore, there is the possibility that positively charged amino acids containing long side chains, such as lysine, could display positive charge repulsion, producing distortion of the alpha-helix structure. Nevertheless, this structural characteristic does not avoid an adequate interaction between the peptides positive charges and the negatively charged phospholipids of mycobacterial plasma membrane. In addition, a comparison of the positive charge shown for peptides, could partially explain why the LLAP peptide is 4-fold more active than the native peptide. According to the well-known AMPs action mechanisms, a high positive charge of peptides enables the interaction with the cell membrane phospholipids. Besides, the peptides insertion in the cell
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lipid bilayer is facilitated by interactions of peptides hydrophobic amino acids with the cell membrane phospholipids, inducing the formation of pores and affecting the cell homeostasis and viability [9]. LLAP contains several amino acid substitutions for Gly, Ala, Lys and Arg residues, which increase its positive charge and amphipathicity, as indicated by the helical Wheel. In this study, the Phe of the native amino acid sequence was replaced for Gly in N-terminal of LLAP, facilitating the interaction between the analogous peptide and the mycobacterial cell membrane, as it has been also observed before [24,40]. According to previous studies, the Phe in positions 1 and 12 of native peptide (Fig. 1) are important for the interaction between LL-37 derived peptides and the phosphatidylglycerols of bacterial cell membranes [40]. Conversely, we observed that substitution of Phe for Gly and Ala in these positions respectively, did not affect the antimicrobial activity of the designed peptide LLAP, reinforcing the prediction for optimal antimicrobial scores given by the AntiBP server. We consider that Gly and Ala are convenient for the design of peptides, as well as the preservation of the peptide hydrophobicity. In addition, the substitution of Glu for Arg in the native peptide, produced the Lys-Arg motive in the LLAP peptide, which has been reported to have an important role in the LL-37 derived peptides antibacterial activity [41]. AMPs must not exhibit cytotoxicity against mammalian cells to be used as therapeutic agents. Fortunately, the IC50 of peptides was comparable between them, and the hemolytic activity of LLAP was lower than the LL37 native amino acid sequence, which was correlated with a significant inhibition of the basal ATPase activity on the mycobacterial plasma membrane. This enzymatic inhibition affects biological processes essential for the cell viability, such as ions and other substances transported across the plasma membrane [42]. In this particular there are two possibilities: (1) AMPs such as LLAP are able to create pores across the plasma membrane, promoting the leaching of essential substances for the mycobacterial surviving. We cannot rule out the possibility that, (2) AMPs can be anchored to the cell surface chelating cations or another potentially translocated substances by ATPases enzymes across the plasma membrane, producing an unbalance of the delicate cell ion homeostasis. However, further experiments using crystallography and other analytical techniques are necessary to confirm any of these possibilities. Although, the MIC value showed for LLAP (600 g/mL) is too high compared with the displayed one for common first and second-line antituberculous drugs: rifampicin, isoniazid, ethambutol, capreomycin, cycloserin (8–16, 0.025, 0.5, 2 and 25 g/mL, respectively), and the drug used as positive control in this study, kanamycin (2 g/mL) [43–44]; the activity of peptides can be improved. To achieve this purpose in a later step of this research, we will apply the strategy proposed in this work in other wellknown antimicrobial peptides, including cathelicidins. However, the obtained results allow us to say that the methodological approach used is able to design peptides with improved antimycobacterial activity, as well as LL37 derived amino acid sequences could be considered in the future as a potential antimycobacterial agent.
Conflict of interest We have no conflict of interest.
Authors’ contributions L.M.-S. and C.Y.-S. wrote the manuscript, validated the tools and carried out the data analysis and interpretation. S.C., G.D., L.M.-S. and C.Y.-S. contributed to the methodological design and super-
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vised its development. All authors read and approved the final version of the manuscript. Acknowledgment We want to express our special gratitude to José Julián PerezCordero from the Inmunotoxicology Research Group (Universidad Nacional de Colombia), for his collaboration in hemolysis and cytotoxicity experiments. This work was supported by the “División de Investigación Bogotá” (DIB)–Universidad Nacional de Colombia, grants 13592 and 23667. References [1] World Health Organization. Global tuberculosis report 2014. 2014. [2] P.A. Lambert, Cellular impermeability and uptake of biocides and antibiotics in gram-positive bacteria and mycobacteria, Symp. Ser. Soc. Appl. Microbiol. 92 (2002) 46S–54S, http://dx.doi.org/10.1046/j.1365-2672.92.5s1.19.x [3] L. Novoa-Aponte, C.Y. Soto Ospina, Mycobacterium tuberculosis p-type atpases: possible targets for drug or vaccine development, Biomed. Res. Int. 201 (2014) 4, http://dx.doi.org/10.1155/2014/296986 [4] L.S. Ligon, J.D. Hayden, M. Braunstein, The ins and outs of Mycobacterium tuberculosis protein export, Tuberculosis (Edinb.) 92 (2012) 121–132, http:// dx.doi.org/10.1016/j.tube.2011.11.005 [5] M.G. Palmgren, P. Nissen, P-type ATPases, Annu. Rev. Biophys. 40 (2011) 243–266, http://dx.doi.org/10.1146/annurev.biophys.093008.131331 [6] H. Chan, V. Babayan, E. Blyumin, C. Gandhi, K. Hak, D. Harake, et al., The p-type ATPase superfamily, J. Mol. Microbiol. Biotechnol. 19 (2010) 5–104, http://dx.doi.org/10.1159/000319588 ˜ [7] L. Novoa-Aponte, A. León-Torres, M. Patino-Ruiz, J. Cuesta-Bernal, L.-M. Salazar, D. Landsman, et al., In silico identification and characterization of the ion transport specificity for P-type ATPases in the Mycobacterium tuberculosis complex, BMC Struct. Biol. 12 (2012) 25, http://dx.doi.org/10. 1186/1472-6807-12-25 [8] P.A. Pulido, L. Novoa-Aponte, N. Villamil, C.Y. Soto, The DosR dormancy regulator of mycobacterium tuberculosis stimulates the Na+ /K+ and Ca2+ atpase activities in plasma membrane vesicles of mycobacteria, Curr. Microbiol. 69 (2014) 604–610, http://dx.doi.org/10.1007/s00284-014-0632-6 [9] H.-T. Chou, H.-W. Wen, T.-Y. Kuo, C.-C. Lin, W.-J. Chen, Interaction of cationic antimicrobial peptides with phospholipid vesicles and their antibacterial activity, Peptides 31 (2010) 1811–1820, http://dx.doi.org/10.1016/j.peptides. 2010.06.021 [10] L.T. Nguyen, E.F. Haney, H.J. Vogel, The expanding scope of antimicrobial peptide structures and their modes of action, Trends Biotechnol. 29 (2011) 464–472, http://dx.doi.org/10.1016/j.tibtech.2011.05.001 [11] D. Takahashi, S.K. Shukla, O. Prakash, G. Zhang, Structural determinants of host defense peptides for antimicrobial activity and target cell selectivity, Biochimie 92 (2010) 1236–1241, http://dx.doi.org/10.1016/j.biochi.2010.02. 023 [12] V. Teixeira, M.J. Feio, M. Bastos, Role of lipids in the interaction of antimicrobial peptides with membranes, Prog. Lipid Res. 51 (2012) 149–177, http://dx.doi.org/10.1016/j.plipres.2011.12.005 [13] B. Ramanathan, E.G. Davis, C.R. Ross, F. Blecha Cathelicidins, Microbicidal activity, mechanisms of action, and roles in innate immunity.;:72, Microbes. Infect. 4 (2002) 361–372, http://dx.doi.org/10.1016/S1286-4579(02)1549-6 [14] R. Rico-Mata, L.M. De Leon-Rodriguez, E.E. Avila, Effect of antimicrobial peptides derived from human cathelicidin LL-37 on Entamoeba histolytica trophozoites, Exp. Parasitol. 133 (2013) 300–306, http://dx.doi.org/10.1016/j. exppara.2012.12.009 [15] Y.-Y. Jiang, W. Xiao, M.-X. Zhu, Z.-H. Yang, X.-J. Pan, Y. Zhang, et al., The effect of human antibacterial peptide LL-37 in the pathogenesis of chronic obstructive pulmonary disease, Respir. Med. 106 (2012) 1680–1689, http:// dx.doi.org/10.1016/j.rmed.2012.08.018 [16] D.W. Hoskin, A. Ramamoorthy, Studies on anticancer activities of antimicrobial peptides, Biochim. Biophys. Acta — Biomembr. 1778 (2008) 357–375, http://dx.doi.org/10.1016/j.bbamem.2007.11.008 [17] J. Von Haussen, R. Koczulla, R. Shaykhiev, C. Herr, O. Pinkenburg, D. Reimer, et al., The host defence peptide LL-37/hCAP-18 is a growth factor for lung cancer cells, Lung Cancer 59 (2008) 12–23, http://dx.doi.org/10.1016/j. lungcan.2007.07.014 [18] J.H. Wong, T.B. Ng, A. Legowska, K. Rolka, M. Hui, C.H. Cho, Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans, Peptides 32 (2011) 1996–2002, http://dx.doi.org/10.1016/j.peptides.2011.08.018 [19] P. Méndez-Samperio, The human cathelicidin hCAP18/LL-37: A multifunctional peptide involved in mycobacterial infections, Peptides 31 (2010) 1791–1798, http://dx.doi.org/10.1016/j.peptides.2010.06.016 [20] D. Vandamme, B. Landuyt, W. Luyten, L. Schoofs, A comprehensive summary of LL-37, the factoctum human cathelicidin peptide, Cell Immunol. 280 (2012) 22–35, http://dx.doi.org/10.1016/j.cellimm.2012.11.009 [21] A. Sonawane, J.C. Santos, B.B. Mishra, P. Jena, C. Progida, O.E. Sorensen, et al., Cathelicidin is involved in the intracellular killing of mycobacteria in
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The ATPase activity of the mycobacterial plasma membrane is inhibited by the LL37-analogous peptide LLAP Sandra Chingaté a , Gabriela Delgado b , Luz Mary Salazar a,∗ , Carlos-Yesid Soto a a b
Chemistry Department, Faculty of Sciences, Universidad Nacional de Colombia, Carrera 30# 45-03, Ciudad Universitaria, Bogotá, Colombia Pharmacy Department, Faculty of Sciences, Universidad Nacional de Colombia, Carrera 30# 45-03, Ciudad Universitaria, Bogotá, Colombia
a r t i c l e
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Article history: Received 24 April 2015 Received in revised form 22 July 2015 Accepted 22 July 2015 Available online 26 July 2015 Keywords: Mycobacteria Antimicrobial peptides LL37 Cell membrane ATPase activity
a b s t r a c t The emergence of multidrug-resistant Mycobacterium tuberculosis strains has led to the development of new antituberculous agents. In this context, antimicrobial targeting proteins to the cell membrane are interesting due to the avoidance of the plasma membrane permeation. Through this study, the antimicrobial activity, cellular toxicity, as well as the effect on the mycobacterial cell membrane ATPase activity of a cathelicidin-analogous peptide were assessed. By using bioinformatics analyses, a 15 amino acid LL37analogous peptide called LLAP, which has the amino acid sequence: GRKSAKKIGKRAKRI, was designed to improve its helical structure and antibacterial activity compared to the native sequence. The LLAP peptide was synthesized, purified by RP-HPLC and its structural characteristics were determined by MALDI-TOF MS and circular dichroism. Compared to the native amino acid sequence, the minimum inhibitory concentration and cytotoxic activity of LLAP were 4.0 and 5.6-fold lower, respectively. In addition, the hemolytic activity of LLAP was lower than 1.1% and the cytotoxic activity of peptides was similar for both peptides. Interestingly, the LLAP peptide displayed approximately 50% inhibition of basal ATPase activity of the mycobacterial plasma membrane, which could in turn be associated with the impaired cell viability. The results suggest that LLAP could be considered as potential antimycobacterial compounds against cell membrane targeting ATPases. However, this antimycobacterial activity can be improved. It is expected further applications to be found for other antimicrobial peptides families based on the implemented methodology. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Tuberculosis (TB) is the infectious disease that produces the largest number of deaths around the world caused by a bacterial infectious agent [1]. According to the World Health Organization (WHO), there were 9.0 million new cases and 1.5 million deaths caused for TB in 2013 [1]. The emergence of multidrug resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains, and the co-infection with HIV has led to the discovery of alternative antimycobacterial compounds. The highly hydrophobic cell envelope provides the mycobacteria a barrier to many antimicrobial compounds [2]. Thus, the study of the cell membrane
Abbreviations: AMPs, antimicrobial peptides; CD, circular dichroism; IC50, median cytotoxic concentration; HC50, median hemolytic concentration; LLAP,LL37, -analogous peptide; MDR, multidrug resistant; MIC, minimum inhibitory concentration; RBC, red blood cells; TB, tuberculosis; XDR, extensively drug-resistant. ∗ Corresponding author: Tel.: +57 1 3165000x14449; fax: +57 1316 5220. E-mail address: [email protected] (L.M. Salazar). http://dx.doi.org/10.1016/j.peptides.2015.07.021 0196-9781/© 2015 Elsevier Inc. All rights reserved.
antimicrobial targeting proteins could be interesting, since they avoid issues related with the plasma membrane permeation [3]. The mycobacterial cell membrane contains different kinds of pumps associated with the cell detoxification, as well as enzymes capable of hydrolyze or modify the antimicrobial compounds [4]. P-type ATPases are cell membrane transporters which hydrolyze ATP releasing energy used to generate electrochemical gradients [5]. This kind of enzymes use these electrochemical gradients to transport metal ions and other solutes across the plasma membrane [5,6]. The large number of P-type ATPases in the M. tuberculosis genome strongly suggests that these transporters might be essential for the mycobacterial viability and virulence [7]. For instance, the M. tuberculosis CtpF is a putative alkali/alkaline earth cation metal transporter activated in the presence of toxic substances as isoniazid, tetrahydrolipstatin, isoxyl, and sodium hypochlorite; moreover, CtpF is the only M. tuberculosis P-type ATPase regulated by the global latency regulator dosR and also over expressed during the infection of human macrophages [8]. The potential of antimicrobial peptides (AMPs) as antimycobacterial compounds has not yet been deeply explored. AMPs are able
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to disrupt bacterial membranes via non-specific electrostatic interactions with the phospholipids [9,10]. Some studies have shown that net charge, amphipathicity and structural tendency are important parameters that explain the AMPs ability to interact with cell membranes [11,12]. Cathelicidins are a family of the structurally diverse AMPs from several mammalian species, including humans, monkeys, horses, cattle, sheep, goats, pigs, rabbits, mice and guinea pigs that are part of the innate immunity response [13]. The hCAP-18 cathelicidin protein is expressed in human neutrophils and proteolysed within the c-terminal end to produce a 37 amino acid peptide known as LL-37 [14]. This cathelicidin LL37 peptide has an amphipathic alpha-helical structure that shows membrane-directed antimicrobial, antiviral, antifungal and anticancer activity [15–18], and its interaction with the cell membrane is affected by the oligomerization degree [19]. In addition, LL-37 has an important role in the human being innate immune system displaying functions of chemotaxis, immune modulation and wound healing [20]. Information related with the cathelicidins upregulation in J7774 macrophages infected with M. smegmatis [21] has been reported, suggesting that this type of antimicrobial peptides may play an important role in controlling intracellular survival of mycobacteria. Nevertheless, the interaction between cathelicidins and the mycobacterial cell envelope is not well known. In a previous work we established that the magainin-analogous peptide MIAP inhibits the F1 F0 ATPase activity of mycobacterial plasma membrane, suggesting the mycobacterial ATPases as alternative for the identification of new therapeutic targets of mycobacteria [22]. In this work in silico modifications of the LL37 cathelicidin generated a 15 amino acid analogous peptide with improved antimycobacterial activity and low cytotoxicity and hemolysis, which inhibit the basal ATPase activity on the mycobacterial plasma membrane.
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by Houghten [25] using Rink amide solid resin as support with a substitution of 0.52mmol/g. Peptides were analyzed by RP-HPLC in a column of ODS in a gradient A/B from 0 to 70% B (A: Deionized water 0.05% TFA, B: acetonitrile 0.05% TFA, flow 1.0 mL/min) for 45 min and then purified through a preparative technique. The molecular weight of peptides was determined by MALDI-TOF MS (Autoflex Bruker Daltonics, Germany). Circular dichroism (CD) assays were performed in a Jasco J-815CD Spectropolarimeter to determine the pattern of peptides secondary structure. Spectra were performed in triplicate at a scan rate of 20 nm/min, using peptide dissolved in 0.30% aqueous 2,2,2-trifluoroethanol (2 mM final concentration) to avoid induction of secondary structure. Spectra deconvolution was performed by using CDPro (Continll, Selcon3 and CDsst) tools (http://lamar.colostate.edu/∼sreeram/ CDPro/main.html). 2.4. Antimicrobial susceptibility testing The MIC of peptides was estimated on M. smegmatis mc2 155 cells using resazurin as an indicator of cell viability. Briefly, 100 L of serial dilutions of peptides (from 50 mg/mL) were mixed in 96-well microtiter plates with 100 L of a 1:100 dilution of M. smegmatis mc2 155 cells growth until OD600 = 0.5. Mycobacterial cells were incubated at 37 ◦ C for 4 days; then, 30 L of resazurin (5 mg/mL) were added and after that, cells were subsequently incubated for 24 h. Bacterial growth was shown by a change in the broth color from yellow to violet, and the MIC was defined as the lowest peptide concentration avoiding this color change. Mycobacterial cells supplemented without peptide, and kanamycin (final concentration: 25 g/mL) were used as positive and negative controls, respectively. 2.5. Assays of cytotoxicity
2. Materials and methods 2.1. Mycobacterial strains and culture conditions The fast-growing M. smegmatis mc2 155 strain [23] was used in this work. Mycobacteria were grown in LB broth with agitation at 70 rpm and 37 ◦ C for 4–6 days until an OD600 of 0.4–0.5 approximately for the minimum inhibitory concentration (MIC) of peptides and for mycobacterial plasma membrane isolation. 2.2. Bioinformatic design of LL37-analogous peptides The server (http://www.imtech.res.in/raghava/antibp/index. html) and database (APD, http://aps.unmc.edu/AP/main.php) were used to identify positive charged LL37-amino acid sequences (15 amino acids), which displayed the best antimicrobial score. The antimicrobial scores calculated by the anti-BP server were predicted by the SVM (support vector machine) algorithm, which displays the highest values of accuracy, sensitivity and specificity for each 15 amino acid sequence [24]. The prediction of the structural and chemical characteristics of the selected peptides were determined by Protparam (http://web.expasy.org/protparam/) for the primary structure, and PSSpred for the secondary structure (http://zhanglab.ccmb.med.umich.edu/PSSpred). The primary amino acid sequence in the selected peptide was manually changed to increase its antimicrobial score, its cationic charge and the proportion of ␣-helices in the secondary structures as predicted by Jpred (http://www.compbio.dundee.ac.uk/www-jpred/). 2.3. Peptide synthesis and characterization The 15 amino acid native and the LL37-analogous peptide (LLAP) were synthesized by the Fmoc solid phase methodology proposed
The cytotoxicity of peptides was determined in duplicate on 100 L of J774 macrophages suspension (1.0 × 104 cells in RPMI supplemented with 5% fetal calf serum) seeded on 96-well microtiter plates (Corning, USA). Cells were incubated at 37 ◦ C for 24 h under 5% CO2 atmosphere and subsequently supplemented with 100 L of serial dilutions of peptide (from 2500 to 19.53 g/mL final concentration). Incubation of cells continued for 72 h; after that, resazurin (Sigma Aldrich, USA) was added to a final concentration of 44 mM. Finally, the cells were incubated for additional 4 h and the fluorescence units were read by using a spectrofluorometer (Tecan Genios Plate Reader) and Magellan® Software. Samples with kanamycin and mycobacterial cells without peptide were used as negative and positive controls, respectively. Florescence units were processed in Microsoft Excel 2000 to estimate cell viability percentages. For this purpose, fluorescence from samples of cells supplemented without peptide was considered as 100% feasibility. The percentage of viability was considered as follows: (fluorescence units at X peptide concentration × 100) /(fluorescence units from cells which have not been exposed to any treatment). In order to obtain the median cytotoxic concentration (IC50 ) – considered as the peptide concentration that produces death in 50% of the treated cells – the calculated percentages were plotted (concentration vs. response) by using GraphPad Prism 5 Demo Software (GraphPad Software, Inc., La Jolla, CA, USA). 2.6. Hemolysis assays The hemolytic activity was performed in duplicate. Briefly, 0.1 mL of O Rh + red blood cells (RBC) from healthy human donors suspended in 0.9% saline solution (Baxter, Colombia) to obtain a final hematocrit concentration of 2%, were mixed with 0.1 mL of serial peptide dilutions (from 50 to 0.19 g/mL) in V-bottom, 96-
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well plates (Corning, USA). After 2 h of incubation at 37 ◦ C with 5% of CO2 , the plates were centrifuged at 3500 rpm and the supernatants were collected to determine the concentration of hemoglobin by measuring OD540 in a digital spectrophotometer (Ultramark Microplate Reader, Bio-Rad, USA). The assay included control wells containing erythrocytes without peptide in saline solution (corresponding to 0% of hemolysis), erythrocyte in sterile distilled water (control for 100% of hemolysis), insulin as unrelated peptide, and kanamycin at inhibitory concentration. Absorbance units from saline-diluted RBC that were not exposed to any substance were used as a negative control (100% of viability). Absorbance readings of supernatants from RBC solutions exposed to each peptide and kanamycin were normalized regarding the absorbance reading obtained for the RBC solutions exposed to sterile water, which were considered as 100% of hemolysis. The percentage of hemolysis was calculated in a MS Excel Worksheet, then analyzed with the GraphPad Prism 5.0 Trial Version® program (LA Jolla, CA, USA), and the median hemolytic concentration (HC50 ) was calculated as equivalent to the concentration of peptide that causes 50% of RBC lysis.
Table 1 In silico analysis of 15 amino acid sequences from LL37 using the AntiBP tool.
2.7. Plasma membrane isolation Plasma membranes were isolated by using the protocol reported by Basu and co-workers with some modifications [26–28]. In brief, harvested mycobacteria were centrifuged and resuspended in a pH 7.4 buffer solution (10 mM MOPS, 1 mM EDTA and 0.3 mM phenylmethylsulfonyl fluoride) and lysed in a Mini Beadbeater-16 (Biospec, USA) for 7 min. Samples were centrifuged at 25,000 × g for 30 min at 4 ◦ C and the supernatants containing the plasma membranes were again centrifuged at 100,000 × g for 90 min at 4 ◦ C. The resulting pellets (plasma membrane fraction) were frozen for further use at −80 ◦ C in pH 7.4 buffer containing 10 mM MOPS and 0.08 g/mL sucrose. Protein concentration was determined by the Bradford-Zor-Selinger method [29]. 2.8. ATPase activity The ATPase activity was determined by using the FiskeSubbarow method [30,31] with some modifications, incorporating bismuth citrate to stabilize the molybdenum complexes [27]. The ATPase assay was performed in 96-well microtiter plates in which reactions (50 L) included 100 mM MOPS (pH 7.4), 3 mM MgCl2 , 150 mM NaCl, 0.02% (w/v) Brij-58 and 38 g of M. smegmatis plasma membrane vesicles. Samples were pre-incubated at 37 ◦ C for 5 min. The enzymatic reactions started by adding 3 mM Tris-ATP (final concentration) and incubated at 37 ◦ C for 30 min. Reactions were stopped by adding 100 L of stop solution (3% ascorbic acid, 0.5% ammonium molybdate, 3% SDS in 0.5 M HCl), and additional incubation of samples on ice were performed for 10 min. The complex concentration was determined at 690 nm in a Microplate absorbance iMarkTM Reader (BioRad). To estimate the peptides effect on the basal ATPase activity of mycobacterial plasma membrane, enzymatic reactions were separately supplemented with peptide (50 M) by using reactions without plasma membrane protein as negative controls. Reactions were assessed in triplicate from three independent experiments. The entire enzymatic activity was reported as U/mg of protein (nmol of Pi released/mg × min). 3. Results 3.1. In silico selection of LL37-analogous peptides with increased antimicrobial activity AntiBP server and database APD2 were used to estimate the antibacterial score of amino acid sequences derived from the cathe-
licidin LL37. Specifically, 15 amino acid sequences were analyzed by walking from the N- to C of the antibacterial peptide. Thus, the 15 amino acids fragment with the best antibacterial score (native peptide) was identified as shown in Table 1. Then, some residues from the primary amino acid sequence of the native peptide were manually replaced and the antimicrobial scores were again in silico estimated. We observed that the replacement of Phe-1 → Gly, Lys5 → Ala, Glu6 → Lys, Glu11 → Arg and Phe12 → Ala in the native peptide originated an analogous sequence (the LLAP peptide) which allowed the greatest increase of the antimicrobial score; specifically, from 2108 to 3120 (Fig. 1). In addition, LLAP exhibited a high content of the basic amino acids: Arg (20%) and Lys (33.3%). The predicted isoelectric point and molecular weight for LLAP were 12, 32 and 1697,1 respectively (Fig. 1). 3.2. LLAP is an amphipathic and cationic peptide which displays low helical structure The RP-HPLC analyses indicated high purity of the synthetized peptides, evidenced by the presence of only one peak in the obtained chromatograms. In addition, the peptides molecular mass determined by mass-spectrometry analysis, corresponded to the expected theoretical masses (Fig. 2). The LLAP has a charge of +8, and according to the helical wheel distribution, it displayed an amphipathic distribution of amino acids (Fig. 1). In this context, the structural profile of the native and the LLAP peptides by CD, showed two maximum: one at 190 nm (positive –* transition) with low molar ellipticity and other at 222 nm with an ellipticity near to zero which suggested an undefined structure with low tendency to helicity for both peptides (Fig 3). In addition, spectra deconvolution showed that peptides displayed approximately 37% of the alpha-helical content. 3.3. The LLAP peptide displays an increased antimycobacterial and low toxicity compared to the native amino acid sequence To compare the antimycobacterial activity between the designed and the native amino acid sequence, an antimicrobial susceptibility test was performed by using the environmental and non-pathogenic strain M. smegmatis mc2 155. This experiment demonstrated that the analogue peptide LLAP showed a MIC
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Fig. 1. Insilico peptides analysis. The antimicrobial score predicted by AntiBP of the 15 amino acid native sequences (a) and the analogue LLAP peptide (b); amino acids with positive charge and hydrophobic are shown in pink and black respectively. The helical wheel of peptides (c) is also presented.
Fig.2. Peptides purity.
MALDI-TOF MS indicating the purity and molecular weight of the 15 amino acid native peptide (A) and LLAP peptide (B).
approximately 4-fold, lower than the one showed for the parental peptide (Table 2). The peptides cytotoxicity was estimated by comparing the fluorescence released by the samples supplemented with peptides, and the fluorescence released by cells without supplemented peptide. In general, the peptides cytotoxicity was low. LLAP and native peptides showed specifically IC50 of 11,226 g/mL and 7727 g/mL for J774 cell; that was approximately 13.8 and 9.5-fold lower than the positive control (kanamycin), respectively. Regarding the peptides hemolytic activity on human erythrocytes,
0 190 -10000
200
210
220
230
240
250
the LLAP peptide displayed a hemolytic activity 4-fold (1.1%) lower than the one showed for the native amino acid sequence (Table 2). 3.4. LLAP inhibits the basal ATPasa activity of mycobacterial plasma membrane In order to estimate the peptides harmful effect on the ATPase activity of mycobacterial plasma membrane, the basal ATPase
0 190 -20000
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M O L A R E L L IPT .
M O L A R EL L IPT .
-40000
210
-40000
-20000 -30000
200
-60000 -80000
-100000 -120000
-70000 -80000 -90000
-140000 -160000 Fig. 3. Circular dichroism (CD) spectra for the native (A) and LLAP (B) peptides.
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Table 2 Effect of native peptide and LLAP peptides on the viability of Mycobacterium smegmatis mc2 155, J774 macrophages and hemolysis of human erythrocytes. The median cytotoxic concentration (IC50 ) is considered as the peptide concentration that produces death in 50% of the treated cells, and the median hemolytic concentration (HC50 ) is equivalent to the concentration of peptide that causes 50% of red blood cells lysis. The minimum inhibitory concentration (MIC) of kanamycin on M. smegmatis cells (2 g/mL) has been previously reported [44]. PEPTIDE
MIC (g/mL)
IC50 (g/mL)
% Hemolysis
Charge
Native* LLAP Kanamycin
2400 600 ND
11226 7727 813
4,0 1,1 ND
7 8 ND
*15 amino acid native sequence from LL37; ND = non determined.
activity (stimulated by Mg2+ ) of plasma membrane vesicles from M. smegmatis (control) was compared with the enzymatic activity estimated in presence of lethal concentration of peptides (MIC). The enzymatic reactions were supplemented with 0.02% (w/v) Brij-58 to ensure that most of the vesicles were right-side out [22]. Thus, the cell membrane ATPase proteins were exposed to the reaction media and adequately stimulated by Mg2+ (cofactor) allowing the released Pi to the reaction media be easily quantified. Reactions were supplemented with 50 M of peptide (final concentration) due to the estimation as an optimal concentration for assaying ATPase activity. Plasma membrane vesicles of M. smegmatis mc2 155 showed a basal ATPase activity of 1.46 ± 0.14 nmol Pi/min × mg corresponding to 100% of the activity. Nevertheless, when reactions were performed in the presence of native and LLAP peptides, the enzymatic activity diminished from 1.31 to ± 0.03 and from 0.72 to ± 0.08 nmol Pi/min × mg, respectively, which was related to an inhibition of enzymatic activity of 10.1 and 50.6%, respectively (Fig. 4). 4. Discussion In this study, an LL37-analogous peptide was in silico designed. This displayed increased antimycobacterial activity, low toxicity, and specially, inhibition of the basal ATPase activity of mycobacterial plasma membrane, compared with the native amino-acid sequence. LL37 belongs to the cathelidicin antimicrobial peptide family, as part of the innate immune response in humans [13]. We decided to evaluate the potential of cathelicidins as antimycobacterial compounds because it is known that this AMPs family has an interesting role in the intracellular killing of M. tuberculo-
% Inhibion ATPase acvity
60 50,6 ± 0,08
50 40 30 20 10,1 ± 0,03
10 0
0,0 ± 0,14 Basal
LL37-Nave Pepde
LLAP
Fig. 4. Inhibition of the plasma membrane basal ATPase activity, mediated by peptides. The inhibition percentage corresponds to the difference between ATPase activity of plasma membrane vesicles in the presence and absence of peptides (LL37-A Native peptide and LL37 Analogue peptide LLAP). Error bars correspond to the standard deviation.
sis; therefore, their molecular mechanisms of action may be useful to develop strategies for the M. tuberculosis control. Cathelicidins are present in different human epithelia, and interact with the cell membrane by inducing pores formation and cell death [19], which could be induced by D vitamin and mediated by IFN-␥ [32–36]. Previous studies have shown that AMPs have limited applications as antituberculous agents, mainly explained by the hydrophobic nature of the mycobacterial cell envelope that reduces permeability for antimicrobial compounds [2]. For instance, wellknown AMPs such as beta-defensin, human neutrophil peptide-1 (HNP1), granulysins, protegrins and cathelicidins, display low in vitro activity against M. tuberculosis, which increases in the case of protegrin-1 and beta-defensin-1, only when combined with typical antituberculous drugs as isoniazid, [32]. However in a previous work, we reported both, in silico and manually strategies which allowed the improvement of the AMPs antimicrobial activity which originally displayed low antimycobacterial activity [22]. In this study, the viability of non-pathogenic and fast-growing M. smegmatis mc2 155 strain was estimated in the presence of the designed LLAP peptide by using resazurin as an indicator of cell viability. M. smegmatis is an environmental mycobacteria that shares a high percentage of genomic and physiological characteristics with M. tuberculosis [37–39]. Proteomic studies show that approximately 70% of M. tuberculosis proteins have conserved orthologues in M. smegmatis; for instance, M. smegmatis contains secretory systems necessary to over-express M. tuberculosis proteins involved in mycobacterial virulence, and also contains the same genomic background part of the M. tuberculosis resistance to typical anti-TB drugs (http://mycobrowser.epfl.ch/smegmalist. html). In addition, M. smegmatis has technical benefits such as a shorter culture time and minor laboratory risks compared to other standard strains used to evaluate anti-TB drugs, such as M. bovis BCG. From this perspective, M. smegmatis is a suitable model to evaluate the mycobacterial-surviving processes, which can be extrapolated to the natural behavior of the tubercle bacilli. Regarding the design of the analogous peptide, the bioinformatic analyses suggested an improved antibacterial activity for LLAP on M. smegmatis mc2 155 cells compared to the native amino acid sequence. LLAP exhibited a high content of the basic amino acids Arg and Lys and the presence of Lys in C-terminal, which – as expected – improved the positive charge and the pore forming characteristics. Helical wheel projection and secondary structure prediction suggest that the amino acid sequence of both, the native and LLAP peptides, should adopt an alpha helix structure. It also suggests that LLAP should have an amphipathic helical structure; however, according to CD, both peptides display low tendency of helicity. The spectra deconvolution results showed that both molecules tend to be alpha-helical structures rather than other structural possibilities. Although the percentages of this structural element are lower than 36%, we consider that this percentage of helical structure is enough to partially explain the LLAP antimycobacterial activity. We also consider that the native and LLAP peptides may have flexibility to configure amphipathic regions despite they do not display full helical structure. Therefore, there is the possibility that positively charged amino acids containing long side chains, such as lysine, could display positive charge repulsion, producing distortion of the alpha-helix structure. Nevertheless, this structural characteristic does not avoid an adequate interaction between the peptides positive charges and the negatively charged phospholipids of mycobacterial plasma membrane. In addition, a comparison of the positive charge shown for peptides, could partially explain why the LLAP peptide is 4-fold more active than the native peptide. According to the well-known AMPs action mechanisms, a high positive charge of peptides enables the interaction with the cell membrane phospholipids. Besides, the peptides insertion in the cell
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lipid bilayer is facilitated by interactions of peptides hydrophobic amino acids with the cell membrane phospholipids, inducing the formation of pores and affecting the cell homeostasis and viability [9]. LLAP contains several amino acid substitutions for Gly, Ala, Lys and Arg residues, which increase its positive charge and amphipathicity, as indicated by the helical Wheel. In this study, the Phe of the native amino acid sequence was replaced for Gly in N-terminal of LLAP, facilitating the interaction between the analogous peptide and the mycobacterial cell membrane, as it has been also observed before [24,40]. According to previous studies, the Phe in positions 1 and 12 of native peptide (Fig. 1) are important for the interaction between LL-37 derived peptides and the phosphatidylglycerols of bacterial cell membranes [40]. Conversely, we observed that substitution of Phe for Gly and Ala in these positions respectively, did not affect the antimicrobial activity of the designed peptide LLAP, reinforcing the prediction for optimal antimicrobial scores given by the AntiBP server. We consider that Gly and Ala are convenient for the design of peptides, as well as the preservation of the peptide hydrophobicity. In addition, the substitution of Glu for Arg in the native peptide, produced the Lys-Arg motive in the LLAP peptide, which has been reported to have an important role in the LL-37 derived peptides antibacterial activity [41]. AMPs must not exhibit cytotoxicity against mammalian cells to be used as therapeutic agents. Fortunately, the IC50 of peptides was comparable between them, and the hemolytic activity of LLAP was lower than the LL37 native amino acid sequence, which was correlated with a significant inhibition of the basal ATPase activity on the mycobacterial plasma membrane. This enzymatic inhibition affects biological processes essential for the cell viability, such as ions and other substances transported across the plasma membrane [42]. In this particular there are two possibilities: (1) AMPs such as LLAP are able to create pores across the plasma membrane, promoting the leaching of essential substances for the mycobacterial surviving. We cannot rule out the possibility that, (2) AMPs can be anchored to the cell surface chelating cations or another potentially translocated substances by ATPases enzymes across the plasma membrane, producing an unbalance of the delicate cell ion homeostasis. However, further experiments using crystallography and other analytical techniques are necessary to confirm any of these possibilities. Although, the MIC value showed for LLAP (600 g/mL) is too high compared with the displayed one for common first and second-line antituberculous drugs: rifampicin, isoniazid, ethambutol, capreomycin, cycloserin (8–16, 0.025, 0.5, 2 and 25 g/mL, respectively), and the drug used as positive control in this study, kanamycin (2 g/mL) [43–44]; the activity of peptides can be improved. To achieve this purpose in a later step of this research, we will apply the strategy proposed in this work in other wellknown antimicrobial peptides, including cathelicidins. However, the obtained results allow us to say that the methodological approach used is able to design peptides with improved antimycobacterial activity, as well as LL37 derived amino acid sequences could be considered in the future as a potential antimycobacterial agent.
Conflict of interest We have no conflict of interest.
Authors’ contributions L.M.-S. and C.Y.-S. wrote the manuscript, validated the tools and carried out the data analysis and interpretation. S.C., G.D., L.M.-S. and C.Y.-S. contributed to the methodological design and super-
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vised its development. All authors read and approved the final version of the manuscript. Acknowledgment We want to express our special gratitude to José Julián PerezCordero from the Inmunotoxicology Research Group (Universidad Nacional de Colombia), for his collaboration in hemolysis and cytotoxicity experiments. This work was supported by the “División de Investigación Bogotá” (DIB)–Universidad Nacional de Colombia, grants 13592 and 23667. References [1] World Health Organization. Global tuberculosis report 2014. 2014. [2] P.A. Lambert, Cellular impermeability and uptake of biocides and antibiotics in gram-positive bacteria and mycobacteria, Symp. Ser. Soc. Appl. Microbiol. 92 (2002) 46S–54S, http://dx.doi.org/10.1046/j.1365-2672.92.5s1.19.x [3] L. Novoa-Aponte, C.Y. Soto Ospina, Mycobacterium tuberculosis p-type atpases: possible targets for drug or vaccine development, Biomed. Res. Int. 201 (2014) 4, http://dx.doi.org/10.1155/2014/296986 [4] L.S. Ligon, J.D. Hayden, M. Braunstein, The ins and outs of Mycobacterium tuberculosis protein export, Tuberculosis (Edinb.) 92 (2012) 121–132, http:// dx.doi.org/10.1016/j.tube.2011.11.005 [5] M.G. Palmgren, P. Nissen, P-type ATPases, Annu. Rev. Biophys. 40 (2011) 243–266, http://dx.doi.org/10.1146/annurev.biophys.093008.131331 [6] H. Chan, V. Babayan, E. Blyumin, C. Gandhi, K. Hak, D. Harake, et al., The p-type ATPase superfamily, J. Mol. Microbiol. Biotechnol. 19 (2010) 5–104, http://dx.doi.org/10.1159/000319588 ˜ [7] L. Novoa-Aponte, A. León-Torres, M. Patino-Ruiz, J. Cuesta-Bernal, L.-M. Salazar, D. Landsman, et al., In silico identification and characterization of the ion transport specificity for P-type ATPases in the Mycobacterium tuberculosis complex, BMC Struct. Biol. 12 (2012) 25, http://dx.doi.org/10. 1186/1472-6807-12-25 [8] P.A. Pulido, L. Novoa-Aponte, N. Villamil, C.Y. Soto, The DosR dormancy regulator of mycobacterium tuberculosis stimulates the Na+ /K+ and Ca2+ atpase activities in plasma membrane vesicles of mycobacteria, Curr. Microbiol. 69 (2014) 604–610, http://dx.doi.org/10.1007/s00284-014-0632-6 [9] H.-T. Chou, H.-W. Wen, T.-Y. Kuo, C.-C. Lin, W.-J. Chen, Interaction of cationic antimicrobial peptides with phospholipid vesicles and their antibacterial activity, Peptides 31 (2010) 1811–1820, http://dx.doi.org/10.1016/j.peptides. 2010.06.021 [10] L.T. Nguyen, E.F. Haney, H.J. Vogel, The expanding scope of antimicrobial peptide structures and their modes of action, Trends Biotechnol. 29 (2011) 464–472, http://dx.doi.org/10.1016/j.tibtech.2011.05.001 [11] D. Takahashi, S.K. Shukla, O. Prakash, G. Zhang, Structural determinants of host defense peptides for antimicrobial activity and target cell selectivity, Biochimie 92 (2010) 1236–1241, http://dx.doi.org/10.1016/j.biochi.2010.02. 023 [12] V. Teixeira, M.J. Feio, M. Bastos, Role of lipids in the interaction of antimicrobial peptides with membranes, Prog. Lipid Res. 51 (2012) 149–177, http://dx.doi.org/10.1016/j.plipres.2011.12.005 [13] B. Ramanathan, E.G. Davis, C.R. Ross, F. Blecha Cathelicidins, Microbicidal activity, mechanisms of action, and roles in innate immunity.;:72, Microbes. Infect. 4 (2002) 361–372, http://dx.doi.org/10.1016/S1286-4579(02)1549-6 [14] R. Rico-Mata, L.M. De Leon-Rodriguez, E.E. Avila, Effect of antimicrobial peptides derived from human cathelicidin LL-37 on Entamoeba histolytica trophozoites, Exp. Parasitol. 133 (2013) 300–306, http://dx.doi.org/10.1016/j. exppara.2012.12.009 [15] Y.-Y. Jiang, W. Xiao, M.-X. Zhu, Z.-H. Yang, X.-J. Pan, Y. Zhang, et al., The effect of human antibacterial peptide LL-37 in the pathogenesis of chronic obstructive pulmonary disease, Respir. Med. 106 (2012) 1680–1689, http:// dx.doi.org/10.1016/j.rmed.2012.08.018 [16] D.W. Hoskin, A. Ramamoorthy, Studies on anticancer activities of antimicrobial peptides, Biochim. Biophys. Acta — Biomembr. 1778 (2008) 357–375, http://dx.doi.org/10.1016/j.bbamem.2007.11.008 [17] J. Von Haussen, R. Koczulla, R. Shaykhiev, C. Herr, O. Pinkenburg, D. Reimer, et al., The host defence peptide LL-37/hCAP-18 is a growth factor for lung cancer cells, Lung Cancer 59 (2008) 12–23, http://dx.doi.org/10.1016/j. lungcan.2007.07.014 [18] J.H. Wong, T.B. Ng, A. Legowska, K. Rolka, M. Hui, C.H. Cho, Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans, Peptides 32 (2011) 1996–2002, http://dx.doi.org/10.1016/j.peptides.2011.08.018 [19] P. Méndez-Samperio, The human cathelicidin hCAP18/LL-37: A multifunctional peptide involved in mycobacterial infections, Peptides 31 (2010) 1791–1798, http://dx.doi.org/10.1016/j.peptides.2010.06.016 [20] D. Vandamme, B. Landuyt, W. Luyten, L. Schoofs, A comprehensive summary of LL-37, the factoctum human cathelicidin peptide, Cell Immunol. 280 (2012) 22–35, http://dx.doi.org/10.1016/j.cellimm.2012.11.009 [21] A. Sonawane, J.C. Santos, B.B. Mishra, P. Jena, C. Progida, O.E. Sorensen, et al., Cathelicidin is involved in the intracellular killing of mycobacteria in
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