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- Methods and Materials
- Supporting Information
Antimicrobial peptides are promising antibiotics as they possess strong antimicrobial activity and very broad spectra of activity. However, administration of an antibiotic with a very broad spectrum of activity disrupts normal microflora and increases the risks of other fatal infections. To solve the problem, we designed a novel antimicrobial peptide that is activated by virulent proteases of pathogenic organisms. We constructed a peptide composed of three domains, namely an antimicrobial peptide (lactoferricin) as the active center, a protective peptide (magainin intervening sequence) that suppresses antimicrobial activity, and a specific linker that joins these two components and is efficiently cleaved by virulent proteases. We utilized Candida albicans as a model organism that produces secreted aspartic proteases as a virulence attribute. We screened for a peptide sequence efficiently cleaved by secreted aspartic proteases isozymes and identified a GFIKAFPK peptide as the most favorable substrate. Subsequently, we chemically synthesized a peptide containing the GFIKAFPK sequence. The designed peptide possessed no antimicrobial activity until it was activated by secreted aspartic proteases isozymes. Furthermore, it demonstrated selective antimicrobial activity against C. albicans, but not against Saccharomyces cerevisiae. A designed peptide like the one described in this study may protect normal microflora, resulting in enhanced safety as a therapeutic.
The global concerns raised by drug-resistant microorganisms have encouraged exploration of novel antibiotics. Antimicrobial peptides (AMPs), which are evolutionarily conserved among many species for defense against pathogenic organisms, are promising therapeutic agents (1). Antimicrobial peptides have strong antimicrobial activity against a broad spectrum of microorganisms in vitro (2,3). They are also effective against pathogenic organisms that are resistant to conventional drugs (4). Therefore, AMPs have received much interest as a novel class of antibiotics.
Antimicrobial peptides are amphipathic peptides characterized by an overall positive charge and hydrophobicity. Antimicrobial peptides are electrostatically attracted to the negatively charged surface of the bacterial cytoplasmic membrane, and subsequent to the interaction with the membrane, they spontaneously oligomerize and form transmembrane pores that cause leakage of the cellular contents (5). Dissimilar to conventional antibiotics, acquisition of resistance against AMPs is very rare because the microorganism is killed by direct disruption of the microbial membrane (6,7).
In spite of their great potential, two major problems limit the development of AMPs as clinical therapeutics. One problem is the inherent instability and high metabolic turnover of AMPs (8). In general, peptides are rapidly metabolized by human cells and have a short half-life. Inversion of the stereochemistry is a promising way to resolve the rapid metabolic degradation of AMPs. Indeed, d-isomeric AMPs have excellent stability and a long plasma half-life (9). Importantly, they have similar activities compared with their corresponding l-amino acid enantiomers indicating that stereospecific recognition of AMPs is not required for cell disruption (10). d-Isomerization confers these favorable properties on AMPs, and some d-isomeric peptide drugs are currently undergoing clinical trials (11).
The other major problem is the exceptionally broad spectra of activity of AMPs. Broad-spectrum antibiotics disrupt the normal microflora and increase the risk of other fatal infections (12). Therefore, it is highly important to design therapeutics possessing temporally and spatially regulated antimicrobial activity. To solve the problem, we conceived a universal design of novel AMPs for the temporal and spatial regulation of their antimicrobial activity. Our design entailed inhibiting the antimicrobial activity until the peptide is cleaved by a particular protease (Figure 1A). We designed a compound composed of a positively charged AMP connected to a negatively charged protective peptide via a specific linker that is cleaved by a particular virulent protease. Selective killing of pathogenic microorganisms occurs following the release/activation of the protected AMP from the compound by particular virulent proteases (Figure 1B). In this study, we utilized Candida albicans as a model microorganism that possesses the secreted aspartic protease (Sap) family as an important virulence attribute (13,14).
Figure 1. Design of an antimicrobial peptide (AMP) activated by virulent proteases. (A) The protective peptide abolishes the microbicidal activity of the AMP via its negative charge. The AMP is activated and released by the action of virulent secreted aspartic proteases (Sap) isozymes. (B) A schematic representation of the selective killing of pathogens. Under normal conditions, our peptide possesses no antimicrobial activity. However, once the pathogen shows virulence and secretes virulent proteases, the peptide gets activated and induces cell death.
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- Methods and Materials
- Supporting Information
The Sap family is a major virulence attribute of C. albicans. There are 10 types of Sap isozymes that play individual roles at certain stages of infection (22), and Saps1–3 are representative virulence factors (13,17). C. albicans produces these proteins mainly in the yeast form, uses them to damage epithelial tissues, and degrades host proteins for nutritional acquisition (17,23,24). Using the FRETS-25Xaa libraries, we revealed that Saps1–3 preferred positively charged or hydrophobic amino acids for P1 specificity, which is consistent with previous studies (15,25), and hydrophobic amino acids for P2–P3 specificity (Figure 2C). It has been previously reported that proteases with such a substrate specificity degrade AMPs that are rich in positively charged and hydrophobic amino acids (26). As expected, the l-isomeric peptide was quickly degraded when mixed with Sap2 (Figure S3). Thus, the peptide was synthesized using d-amino acids for the LF11 region to increase stability.
We measured the kinetic values of Sap2 against the designed peptide because Sap2 plays a major role among Sap isozymes (27). As a result, we found that the KM = 310 μm and kcat/KM = 5.37 × 104/M/s. The kinetic values did not exceed our expectations, but were equivalent to that of pepsin degrading fluorescent peptides (28). In this study, we screened a peptide substrate optimized for the P1–P3 specificity of Sap isozymes. Further screening aimed at optimizing the specificity will be able to improve the kinetic values.
Based on the substrate specificity of the Sap isozymes, we synthesized a peptide with a GFIKAFPK-specific linker. As expected, considering that Sap1, Sap2, and Sap3 have proteolytic activities at pH values of <6.5, <6.0, and <6.0, respectively (15), this peptide showed Sap-dependent antimicrobial activity above pH 5 (Figure 3B). Thus, we hypothesized the peptide will be able to temporally and spatially regulate its own antimicrobial activity depending on the Sap isozymes present in the physiological conditions.
To our surprise, our peptide was also activated by low pH (Figure 3B) and showed a strong activity, similar to that of LF11D at pH values of <4. We, therefore, investigated the possibility that our peptide was unfavorably cleaved by the proteases of S. cerevisiae. We quantified the amount of intact peptide in the reaction solution by LC analysis and found no degradation in the samples (Figure 3C). This result indicates that the activation at low pH was not caused by the extracellular proteases of S. cerevisiae.
The isoelectric point (pI) of our peptide was calculated to be 4.8 by using the compute Mw/pI tool (29). Thus, the designed peptide is positively charged when the ambient pH is <4.8. The cationic charge of the AMPs is important for interactions with the cytoplasmic membrane (30), and therefore, we suggest that the positive charge at low pH induces the activation of the designed peptide. This result demonstrates the possibility of designing AMPs that are more strictly regulated by decreasing the pI value via the addition of more acidic amino acids such as E and D.
Depending on their surrounding environments, AMPs undergo structural changes that affect their antimicrobial activity (18). For example, LF has an α-helical structure in intact lactoferrin (31), but once liberated from lactoferrin, it loses the α-helical structure and forms β-sheets. In a membrane-mimetic solvent, LF becomes an amphipathic molecule (32,33) that interacts with lipid tails via its hydrophobic cluster and is capable of disrupting membrane integrity. This type of conformational transition was also seen in our peptide. The CD spectra demonstrated that our peptide formed a poly-l-proline II helix-like conformation (21) in a membranous environment at pH values of <pI (Figure 4). We suggest this structure possessed the maximum amphipathicity and induced the activation of the peptide without proteases. In addition, vegetating C. albicans actively acidifies its surrounding environments (34); this can enhance the efficacy of our peptide, allowing it to be activated by both the Sap isozymes and the low pH.
Our peptide selectively killed C. albicans, but did not affect the survival of S. cerevisiae (Figure 5C). We propose that specific cleavage of our peptide by the Sap isozymes induced selective cell death, because pepstatin A inhibited activity of our peptide, and we identified liberated LF11D in the culture supernatant of C. albicans. Although S. cerevisiae has several extracellular proteases (35), the proteolytic activity of C. albicans is significantly greater than that of S. cerevisiae because of the Sap family. In vitro, C. albicans can grow using proteins as a sole nitrogen source, whereas in vivo it survives in the blood stream utilizing serum albumin (36). In addition, the specific linker of our peptide was optimized for Sap isozymes. Therefore, our peptide succeeded in discriminating between C. albicans and S. cerevisiae with the aid of Sap isozyme production.
It is possible that other proteases produced by host or other non-harmful microflora could also activate our peptide. Further characterization of antimicrobial specificity and optimization of the linker sequence will enable more efficient targeting to C. albicans. In addition, we should investigate whether secreted Sap isozymes are widely distributed to the physiological environment or not. Rapid diffusion of active Sap isozymes may lower the antimicrobial specificity of the designed peptide.
In conclusion, we designed an AMP that is activated by virulent proteases and is capable of selective killing C. albicans. Such smart AMPs do not disrupt the normal microflora and are, therefore, safer for use as antimicrobial therapeutics (37,38). The design utilized in this work has universal applicability for many pathogenic organisms that produce proteases as virulence attributes.