Design and pharmacodynamics of recombinant NZ2114 histidine mutants with improved activity against methicillin-resistant Staphylococcus aureus

Currently, 2764 AMPs are registered in the antimicrobial peptide database (APD) (http://aps.unmc.edu/AP/main.php). However, only few AMPs entered into clinical trials. The use of AMPs was primarily hampered by their low oral or intravenous stability, high toxicity, serum binding activity, low activity in physiological condition and so on (Mohammad et al. 2015). Plectasin and its derived peptide NZ2114 had potent activity to S. aureus, which are the idea candidates for traditional drugs (Zasloff 2016). However, two histidine residues exist in the sequence of NZ2114, which are uncharged in the physiological condition (Kashiwada et al. 2016). To further improve the antimicrobial activity and properties of NZ2114, new derived peptides are needed.

Although AMPs vary widely in length, structure, and source, they have some important common traits, such as positive charges, presumed to be important for interaction with the negatively charged surface of pathogens, and amphipathicity, which enables better combine with the hydrophilic surface and interact with the hydrophobic part of the microbial membrane (Silva et al. 2014). The arginine and lysine are the key residues for AMPs which are stable charged and hydrophilic in physiological condition. Many studies showed that AMPs having appropriate proportion of arginine and lysine had an improved amphipathicity and higher activity (Veiga et al. 2012; Silva et al. 2014; Gopal et al. 2009; Taniguchi et al. 2014). As results, eight derived peptides (H1–H8) which H16 and H18 were replaced by arginine or lysine were generated.

All derived peptides were tried to express via P. pastoris but H4, H5 and H7 were not expressed. The antimicrobial activity of H1, H2, H3, H6 and H8 were assayed and H1, H2, and H3 had higher activity compared with original peptide NZ2114. However, the activity of mutants H6 and H8 which had more net positive charges did not significantly increase (Table 2), which indicated that the electrostatic interaction and cell-penetrating was not the all bactericidal mechanisms of mutants.

Unlike some AMPs with a wide antimicrobial spectrum, H1, H2, and H3 showed a narrow spectrum and they mainly killed the Gram-positive bacterium, such as S. aureus, and Streptococci, and showed a strong antimicrobial activity (0.007–0.454 ?M; Table 2), which was very stronger than the activities of plectasin and NZ2114 (Hara et al. 2008; Zhang et al. 2014). Especially, H1, H2, and H3 showed higher antimicrobial activity against MRSA (S. aureus ATCC43300) with MIC of 0.057, 0.114, and 0.057 ?M than NZ2114 (0.909 ?M) and vancomycin (0.714 ?M). Their characteristics of narrow-spectrum antibiotic and low MIC values are very attractive for developing them as candidate agent against MRSA infection.

The lack of economic feasibility to manufacture AMPs at large-scale is another roadblock in the clinical implementation of AMPs (Findlay et al. 2010). Majority of directly expressed AMPs, such as LL-37 (Hong et al. 2007), CecropinAD (Jin et al. 2009), and N2 (Yang et al. 2016), showed unsatisfactory yields. In our previous works, Agplectasin (Mao et al. 2013), NZ2114 (Zhang et al. 2014), and MP1106 (Cao et al. 2015) were expressed in P. pastoris X-33 in high level. Similarly, H1, H2, and H3 were expressed in P. pastoris X-33 with high yields, their total protein level in 5-l fermentation reached 1.70, 1.77, and 1.54 g/l, respectively (Fig. 2b, d, f). However, because the fermentation was performed in summer, the temperature cannot be controlled at 29 °C as previous operation. To maintain the dissolved oxygen content, we had to reduce the flow rate, which lead to the partial losing of yield. If the induction temperature can maintain at 29 °C, at which this key temperature is very important for its high expression in yeast (Li et al. 2001, 2007). the production of H1, H2, H3 may be further improved like their original peptide NZ2114 (2390 mg/l in 29 °C and 2310 mg/l in 25 °C) (Zhang et al. 2014).

The PAE is a very important pharmacodynamics parameter in choosing of antibiotic dosage regimens in clinical use (Pankuch and Appelbaum 2009a). Obviously observed, the PAEs of H1, H2, and H3 increased with the concentration from 1× MIC to 2× MIC (Table 3). They showed similar values to vancomycin (2× MIC: 1.72 h) and NZ2114 (2× MIC: 1.43 h). H1 also had comparative value (2× MIC: 2.94 h) to some conventional antibiotics, for instance, daptomycin (2.0 h), tigecycline (3.2 h), and arbekacin (3.0–3.2 h), respectively (Pankuch and Appelbaum 2009b; Pankuch et al. 2003; Watanabe et al. 1997). Their appropriate PAE is critical to lengthen the interval of administration, reduce the daily dosages, and thus potentially reduce potential drug resistance.

To combat antibiotic resistance, combination antibiotic therapy is practiced in the clinical use due to its advantages such as wider coverage, higher activity, bactericidal synergy and the inhibition on toxin production (Leibovici et al. 2010; Müller et al. 2013). Vancomycin, one of the most effective antibiotics against MRSA, is often combined used with rifampicin, gentamicin, dalfopristin, and ?-lactams to slow the development of resistance and enhance the antibacterial activity. The synergistic effect of H1, H2, and H3 is very different from their parent peptide NZ2114 (Zhang et al. 2014). The FICI of NZ2114 combined with ampicillin, and vancomycin to S. aureus ATCC43300 was 0.125, showing additivity effect (Zhang et al. 2014). However, the indifference effects (1.25 ? FICI ? 3) were observed for all combinations between H1, H2, H3 and four traditional antibiotics to S. aureus ATCC43300 (Table 4), which might result from the changed antimicrobial property and mechanism and its details should be studied in our next research.

The plectasin and NZ2114 showed no hemolysis in rabbit and human RBCs (Yang et al. 2011; Zhang et al. 2014). In this work, H1, H2, and H3 showed low hemolysis in mice RBCs. Although the values were higher than NZ2114 due to the high charge and hydrophilicity, they were still very low in the range of MICs. In addition, NZ2114 showed the highest activity in pH value of 8.0 and above 80% of initial activity was retained over a range of temperatures from 20 to 80 °C but maintained 20% activity at 100 °C (Zhang et al. 2014). Owing to contribution from the three pairs of disulfide bond into the stability of structure, H1, H2, and H3 all had high stability with minor differences. H1 was not sensitive to NaCl concentration, but sensitive to alkaline and high temperature environments. H2 was sensitive to high temperature and NaCl concentration, but not sensitive to different pHs environment. H3 was sensitive to high temperature and high NaCl concentration. Generally, no toxicity to erythrocytes and high stability of pH, temperature, proteases, and saline ions of H1, H2 and H3 meet the key requirements of new antimicrobial agents.

In summary, series of novel AMPs were designed and successfully expressed in P. pastoris. Among them, H1, H2, and H3 had high yields (1.70, 1.77 and 1.54 g/l) in 5-l fermentor level. H1, H2, and H3 also showed strong antimicrobial activity against S. aureus. They killed MRSA strain ATCC43300 in a short time with low concentrations and had long post antibiotic effect. Meanwhile, H1, H2, and H3 exhibited indifference effects when they were combined with conventional antibiotics. Furthermore, they had low toxicity to mice erythrocytes and high stability. All results indicate that H1, H2, and H3 have potential as candidates for the therapeutic agents with the better properties than their native peptide NZ2114.