Jun-ichi Nagao

Evaluation of essential and variable residues of nukacin ISK‐1 by NNK scanning

Nukacin ISK‐1, a type‐A(II) lantibiotic, comprises 27 amino acids with a distinct linear N‐terminal and a globular C‐terminal region. To identify the positional importance or redundancy of individual residues responsible for nukacin ISK‐1 antimicrobial activity, we replaced the native codons of the parent peptide with NNK triplet oligonucleotides in order to generate a bank of nukacin ISK‐1 variants. The bioactivity of each peptide variant was evaluated by colony overlay assay, and hence we identified three Lys residues (Lys1, Lys2 and Lys3) that provided electrostatic interactions with the target membrane and were significantly variable. The ring structure of nukacin ISK‐1 was found to be crucially important as replacing the ring‐forming residues caused a complete loss of bioactivity. In addition to the ring‐forming residues, Gly5, His12, Asp13, Met16, Asn17 and Gln20 residues were found to be essential for antimicrobial activity; Val6, Ile7, Val10, Phe19, Phe21, Val22, Phe23 and Thr24 were relatively variable; and Ser4, Pro8, His15 and Ser27 were extensively variable relative to their positions. We obtained two variants, Asp13Glu and Val22Ile, which exhibited a twofold higher specific activity compared with the wild‐type and are the first reported type‐A(II) lantibiotic mutant peptides with increased potency.

Ring A of nukacin ISK-1: a lipid II-binding motif for type-A(II) lantibiotic.

Ring A of nukacin ISK-1, which is also present in different type-A(II) lantibiotics, resembles a lipid II-binding motif (TxS/TxD/EC, x denotes undefined residues) similar to that present in mersacidin (type-B lantibiotics), which suggests that nukacin ISK-1 binds to lipid II as a docking molecule. Results from our experiments on peptidoglycan precursor (UDP-MurNAc-pp) accumulation and peptide antagonism assays clearly indicated that nukacin ISK-1 inhibits cell-wall biosynthesis, accumulating lipid II precursor inside the cell, and the peptide activity can be repressed by lipid I and lipid II. Interaction analysis of nukacin ISK-1 and different ring A variants with lipid II revealed that nukacin ISK-1 and nukacin D13E (a more active variant) have a high affinity (K(D) = 0.17 and 0.19 μM, respectively) for lipid II, whereas nukacin D13A (a less active variant) showed a lower affinity, and nukacin C14S (a negative variant lacking the ring A structure) exhibited no interaction. Therefore, on the basis of the structural similarity and positional significance of the amino acids in this region, we concluded that nukacin ISK-1 binds lipid II via its ring A region and may lead to the inhibition of cell-wall biosynthesis.

Antimicrobial mechanism of lantibiotics.

Lantibiotics are ribosomally synthesized antimicrobial peptides that commonly target the cell wall precursor lipid II during their antimicrobial mechanism and exert their inhibitory activity by (i) inhibition of cell wall biosynthesis, and (ii) stable pore formation in the target membrane. Type-A(I) (i.e. nisin) and two-component (i.e. lacticin 3147) lantibiotics initially interact with lipid II to stabilize the complex, which then proceeds to inhibit cell wall biosynthesis and pore formation. Type-A(II) (i.e. nukacin ISK-1) and type-B (i.e. mersacidin) lantibiotics also use lipid II as a docking molecule, but can only inhibit cell wall biosynthesis without forming pores. In the present paper, we review the antimicrobial mechanism of different types of lantibiotics, their current progress and future prospect.

Three Distinct Two-Component Systems Are Involved in Resistance to the Class I Bacteriocins, Nukacin ISK-1 and Nisin A, in Staphylococcus aureus

Staphylococcus aureus uses two-component systems (TCSs) to adapt to stressful environmental conditions. To colonize a host, S. aureus must resist bacteriocins produced by commensal bacteria. In a comprehensive analysis using individual TCS inactivation mutants, the inactivation of two TCSs, graRS and braRS, significantly increased the susceptibility to the class I bacteriocins, nukacin ISK-1 and nisin A, and inactivation of vraSR slightly increased the susceptibility to nukacin ISK-1. In addition, two ABC transporters (BraAB and VraDE) regulated by BraRS and one transporter (VraFG) regulated by GraRS were associated with resistance to nukacin ISK-1 and nisin A. We investigated the role of these three TCSs of S. aureus in co-culture with S. warneri, which produces nukacin ISK-1, and Lactococcus lactis, which produces nisin A. When co-cultured with S. warneri or L. lactis, the braRS mutant showed a significant decrease in its population compared with the wild-type, whereas the graRS and vraSR mutants showed slight decreases. Expression of vraDE was elevated significantly in S. aureus co-cultured with nisin A/nukacin ISK-1-producing strains. These results suggest that three distinct TCSs are involved in the resistance to nisin A and nukacin ISK-1. Additionally, braRS and its related transporters played a central role in S. aureus survival in co-culture with the strains producing nisin A and nukacin ISK-1.

Nukacin ISK-1, a Bacteriostatic Lantibiotic

ABSTRACT We determined the mode of action of nukacin ISK-1. It did not cause membrane potential dissipation or the efflux of ATP or K+ ions from the cells of a sensitive bacterial strain; however, it blocked the membrane depolarization activity of nisin. Nukacin ISK-1-treated cells had single arrangements of cells without the formation of a complete septum. A remarkable reduction in cell wall width was also observed, but cytoplasmic content was unaffected. We concluded that nukacin ISK-1 is bacteriostatic.

Lysine-Oriented Charges Trigger the Membrane Binding and Activity of Nukacin ISK-1

ABSTRACT The antibacterial activities and membrane binding of nukacin ISK-1 and its fragments and mutants were evaluated to delineate the determinants governing structure-function relationships. The tail region (nukacin1-7) and ring region (nukacin7-27) were shown to have no antibacterial activity and also had no synergistic effect on each other or even on nukacin ISK-1. Both a fragment with three lysines in the N terminus deleted (nukacin4-27) and a mutant with three lysines in the N terminus replaced with alanine (K1-3A nukacin ISK-1) imparted very low activity (32-fold lower than nukacin ISK-1) and also exhibited a similar antagonistic effect on nukacin ISK-1. Addition of two lysine residues at the N terminus (+2K nukacin ISK-1) provided no further increased antibacterial activity. Surface plasmon resonance sensorgrams and kinetic rate constants determined by a BIAcore biosensor revealed that nukacin ISK-1 has remarkably higher binding affinity to anionic model membrane than to zwitterionic model membrane. Similar trends of strong binding responses and kinetics were indicated by the high affinities of nukacin ISK-1 and +2K nukacin ISK-1, but there was no binding of tail region, ring region, nukacin4-27, and K1-3A nukacin ISK-1 to the anionic model membrane. Our findings therefore suggest that the complete structure of nukacin ISK-1 is necessary for its full activity, in which the N-terminus three lysine residues play a crucial role in electrostatic binding to the target membrane and therefore nukacin ISK-1s ability to exert its potent antibacterial activity.

A poly(γ-glutamic acid)–amphiphile complex as a novel nanovehicle for drug delivery system

Recently, many studies have focused on biomedical and pharmaceutical applications of self-assembled nanoparticles. In addition, several biodegradable nanoparticles have been reported to possess poor dispersion stability and poor size-controllability. However, these nanoparticles require complicated fabrication procedures using synthesis techniques. We developed an efficient method for producing nanoparticles derived from a biological origin of molecule poly(γ-glutamic acid) (γ-PGA), a cationic lipid, and doxorubicin (Dox). The complex had a size of 510u2009nm and was able to encapsulate over 90% of the added Dox. An in vivo assay of antitumor activity demonstrated that the complex had significant antitumor activity in sarcoma 180–bearing mice, and was effectively accumulated in solid tumors based on the EPR effect. The data suggested that this complex is a promising formulation of γ-PGA for targeted delivery to solid tumors. γ-PGA–12GP2 complexes may possess several unique advantages, including simplicity of nanoparticle preparation, high drug-carrying capacity, appropriate size to allow deeper penetration based on EPR effect into solid tumors, and lack of necessity to modify the chemical structure of the drugs. These data indicate that the γ-PGA–12GP2 complexes are potentially useful in cancer chemotherapy.

Lantibiotic Transporter Requires Cooperative Functioning of the Peptidase Domain and the ATP Binding Domain

Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics that contain unusual amino acids such as dehydro and lanthionine residues. Nukacin ISK-1 is a class II lantibiotic, whose precursor peptide (NukA) is modified by NukM to form modified NukA. ATP-binding cassette (ABC) transporter NukT is predicted to cleave off the N-terminal leader peptide of modified NukA and secrete the mature peptide. Multiple sequence alignments revealed that NukT has an N-terminal peptidase domain (PEP) and a C-terminal ATP binding domain (ABD). Previously, in vitro reconstitution of NukT has revealed that NukT peptidase activity depends on ATP hydrolysis. Here, we constructed a series of NukT mutants and investigated their transport activity in vivo and peptidase activity in vitro. Most of the mutations of the conserved residues of PEP or ABD resulted in failure of nukacin ISK-1 production and accumulation of modified NukA inside the cells. NukT(N106D) was found to be the only mutant capable of producing nukacin ISK-1. Asn106 is conserved as Asp in other related ABC transporters. Additionally, an in vitro peptidase assay of NukT mutants demonstrated that PEP is on the cytosolic side and all of the ABD mutants as well as PEP (with the exception of NukT(N106D)) did not have peptidase activity in vitro. Taken together, these observations suggest that the leader peptide is cleaved off inside the cells before peptide secretion; both PEP and ABD are important for NukT peptidase activity, and cooperation between these two domains inside the cells is indispensable for proper functioning of NukT.

Involvement of the Novel Two-Component NsrRS and LcrRS Systems in Distinct Resistance Pathways against Nisin A and Nukacin ISK-1 in Streptococcus mutans

ABSTRACT The novel two-component systems NsrRS and LcrRS are individually associated with resistance against the distinct lantibiotics nisin A and nukacin ISK-1 in Streptococcus mutans. NsrRS regulates the expression of NsrX, which is associated with nisin A binding, and LcrRS regulates the expression of the ABC transporter LctFEG.

ATP-dependent leader peptide cleavage by NukT, a bifunctional ABC transporter, during lantibiotic biosynthesis

NukT, a possible ABC transporter maturation and secretion (AMS) protein, may contribute to the cleavage of the leader peptide of NukA, which is the prepeptide of the lantibiotic nukacin ISK-1, and to nukacin ISK-1 transport. In this study, we reconstituted in vitro peptidase activity of the full-length NukT overexpressed in inside-out membrane vesicles of Staphylococcus carnosus TM300. We found that the presence of unusual amino acids in NukA is required for leader peptide cleavage. Furthermore, NukT peptidase activity was inhibited by phenylmethylsulfonyl fluoride, a serine/cysteine protease inhibitor; this finding strongly suggests that NukT, like other AMS proteins, is a cysteine protease. Interestingly, NukT peptidase activity depended on ATP hydrolysis. These results suggest that the N-terminal peptidase domain of NukT may cooperatively function with the C-terminal ATP-binding domain. This is the first in vitro study on lantibiotics that reports the processing mechanism of a full-length bifunctional ABC transporter.