Antimicrobial peptide has the potential to be developed as new kind of antimicrobial agents with novel action mechanism. However, the susceptibility to protease is a drawback for potential peptides to be clinical used. d-amino acid substitution can be one way to increase the proteolytic stability of peptides. In the present study, we synthesized the d-lysines substituted analog (d-lys-MPI) and the d-enantiomer of polybia-MPI (D-MPI) to improve the proteolytic resistance of polybia-MPI. Our results showed that, the stability of its d-amino acid partially substituted analog d-lys-MPI was increased. However, it lost antimicrobial activity at the tested concentration with the loss of α-helix content. As shown in the CD spectra, after substitution, the spectra of D-MPI is symmetrical to MPI, indicated it turned into left hand α-helical conformation. Excitingly, the stability of D-MPI toward the tested protease was improved greatly. Notably, the antimicrobial activity of D-MPI was comparable to its L-counterpart MPI, even improved. In addition, the hemolytic activity of D-MPI was lowered. This also indicated that the action target of antimicrobial peptide polybia-MPI was not chiral specific. So, D-MPI may offer a therapeutic strategy to defend the infection of microbes, considering its stability to protease and relatively lower cytotoxicity to human erythrocytes.

With the increasing emergence of resistant microbes toward conventional antimicrobial agents, there is an urgent need for the development of antimicrobial agents with novel action mode. Antimicrobial peptides (AMPs) are believed to be one kind of ideal alternatives. However, AMPs can be easily degraded by protease, which limited their therapeutic use. In the present study, D-amino acid substitution strategy was employed to enhance the stability of polybia-CP. We investigated the stability of peptides against the degradation of trypsin and chymotrypsin by determining the antimicrobial activity or determining the HPLC profile of peptides after incubation with proteases. Our results showed that both the all D-amino acid derivative (D-CP) and partial D-lysine substitution derivative (D-lys-CP) have an improved stability against trypsin and chymotrypsin. Although D-CP takes left-hand α-helical conformation and D-lys-CP loses some α-helical content, both of the D-amino acid-substituted derivatives maintain their parental peptides' membrane active action mode. In addition, D-lys-CP showed a slight weaker antimicrobial activity than polybia-CP, but the hemolytic activity decreased greatly. These results suggest that D-CP and D-lys-CP can offer strategy to improve the property of AMPs and may be leading compounds for the development of novel antimicrobial agents.

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To develop an effective subunit vaccine which could target tubercle bacilli with different metabolic states and provide effective protective immunity, we fused antigens ESAT6, Ag85B, peptide 190-198 of MPT64, and Mtb8.4 mainly expressed by proliferating bacteria and latency-associated antigen Rv2626c together to construct a multistage protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-Rv2626c (LT70 for short) with the molecular weight of 70 kDa. The human T-cell responses to LT70 and other antigens were analyzed. The immune responses of LT70 in the adjuvant of DDA and Poly I:C and its protective efficacy against Mycobacterium tuberculosis (M. tuberculosis) infection in C57BL/6 mice were evaluated. The results showed that LT70 was stably produced in Escherichia coli and could be purified by successive salting-out and chromatography. LT70 could be strongly recognized by human T cells from TB patients and persons who are supposed latently infected with M. tuberculosis. The subunit vaccine LT70 generated strong antigen-specific humoral and cell-mediated immunity, and induced higher protective efficacy (5.41±0.37 Log10 CFU in lung) than traditional vaccine Bacillus Calmette-Guerin (6.01±0.33 Log10 CFU) and PBS control (6.53±0.26 Log10 CFU) at 30 weeks post vaccination (10 weeks post-challenge) against M. tuberculosis infection (p < 0.05). These findings suggested that LT70 would be a promising subunit vaccine candidate against M. tuberculosis infection.

Recently, the mortality of life-threatening fungal infections increased dramatically. However, there are few antifungals existed. Antimicrobial peptides (AMPs) as promising antifungal candidates have attracted much attention. Here, we present a small antimicrobial peptide Jelleine-I that had potent in vitro and in vivo antifungal activity. Negligible hemolytic activity and in vivo toxicity were observed. Selectivity index (SI) of Jelleine-I is at least 4.6 times higher than amphotericin B. Jelleine-I could increase the production of cellular ROS and bind with genome DNA. This may contribute to its antifungal activity. Furthermore, drug resistance is not induced when the fungal cells were repeatedly treated by Jelleine-I. In conclusion, our results suggest that Jelleine-I may have the potential to be developed as a novel antifungal agent.