Impact of the nisin modification machinery on the transport kinetics of NisT

Abstract

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides containing dehydrated amino acids and (methyl-)lanthionine rings. one of the best-studied examples is nisin produced by Lactococcus lactis. nisin is synthesized as a precursor peptide comprising of an n-terminal leader peptide and a c-terminal core peptide. Amongst others, the leader peptide is crucial for enzyme recognition and acts as a secretion signal for the ABc transporter nist that secretes nisin in a proposed channeling mechanism. Here, we present an in vivo secretion analysis of this process in the presence and absence of the nisin maturation machinery, consisting of the dehydratase nisB and the cyclase nisc. our determined apparent secretion rates of nist show how nisB and nisc modulate the transport kinetics of nisA. Additional in vitro studies of the detergent-solubilized nist revealed how these enzymes and the substrates again influence the activity of transporter. In summary, this study highlights the pivotal role of nisB for nist in the secretion process. Abbreviations ABC transporter ATP binding cassette transporter CP Core peptide Dha Didehydroalanine Dhb Didehydrobutyrine Lan Lanthionine LP Leader peptide MeLan Methyl-lanthionine MS Mass spectrometry NBD Nucleotide-binding domain NisA Precursor peptide of nisin NisB Dehydratase of nisin precursor peptide NisC Cyclase of nisin precursor peptide NisT Transporter of nisin precursor peptide PTM Post-translational modificationn RP-HPLC Reverse-phase HPLC SEC Size-exclusion chromatography Many natural products (NP) produced as secondary metabolites by microorganisms can be used as pharmaceuticals (e.g. as anticancer, antibacterial or antiviral drugs) 1. One class of these NPs are ribosomally synthesized and post-translationally modified peptides (RiPPs). The RiPP family of lanthipeptides, especially those with antimicrobial activity (lantibiotics), is gaining interest as a potential alternative for antibiotics to treat harmful multidrug resistance strains such as methicillin-resistant Staphylococcus aureus or vancomycin-resistant Enterococci 2,3. Lanthipeptides (LanA) are produced as precursor peptides with an N-terminal leader peptide (LP) and a C-terminal core peptide (CP) 4. The LP serves as a signal sequence and recognition site for the modification enzymes and the export protein 5-8. Furthermore, the LP keeps the modified peptide (mLanA) inactive in the cytosol 9 , while the post-translational modifications (PTM) are installed within the CP and not found in the LP 10 .

Self‐immunity to antibacterial peptides by ABC transporters

“…Class V contains a triprotein synthetase (LanK, LanX and LanY) [17,18]. The different structures and mechanisms of the enzymes from class I‐IV have been reviewed in detail in references [19,20].…”

Importance of the leader peptide sequence on the lanthipeptide secretion level

“…If all aa in the ERS were mutated to alanine ( F‐18 AAAA D‐15 ), the secretion had a value of 0.13 ± 0.02 µ m , which was similar to the single mutants F‐18A and L‐16A. As a control, the ATPase‐deficient H‐loop mutant NisT H551A was used to determine the background, since this mutant is not able to secrete uNisA [32], which had a value of 0.09 ± 0.07 µ m (Fig. 2A).…”

“…There, the multiple sequence alignment is performed with MUSCULE algorithm using the default settings. The accession number is derived from Uniprot:BovA (Q83ZN8), Entianin (G0Z2H4), Epidermin (P86047), Epilancin 15x (P86047), Epilancin K7 (Q57312), Ericina ( Q93GH5), Ericin S ( Q93GH3), Gallidermin As a control, the ATPase-deficient H-loop mutant NisT H551A was used to determine the background, since this mutant is not able to secrete uNisA [32], which had a value of 0.09 AE 0.07 µM (Fig. 2A).…”

“…0.5 L GMM (Erm + Cm) was inoculated to OD 600 of 0.3 and incubated at 30°C. Secretion and quantification were performed as in [32].…”

Potential Novel Food-Related and Biomedical Applications of Nanomaterials Combined with Bacteriocins

“…Different functionalization methods have been reported to address stability for bacteriocins; cyclization by the incorporation of d -amino acids is one such process [ 21 ]. The stability of nisin (i.e., NisB and NisC) can also be increased by modifying unrelated peptides via the modification machinery used for the incorporation of lanthionine ring structures in nisin [ 22 ]. Peng et al [ 23 ] enhanced the stability and bactericidal activity of nisin by conjugation with gellan gum.…”