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Modifying the Lantibiotic Mutacin 1140 for Increased Yield, Activity, and Stability

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Modifying the Lantibiotic Mutacin 1140 for Increased Yield, Activity, and Stability

Mengxin Geng et al. Appl Environ Microbiol.

Abstract

Mutacin 1140 belongs to the epidermin family of type AI lantibiotics. This family has a broad spectrum of activity against Gram-positive bacteria. The binding of mutacin 1140 to lipid II leads to the inhibition of cell wall synthesis. Pharmacokinetic experiments with type AI lantibiotics are generally discouraging for clinical applications due to the short half-life of these compounds. The unprotected dehydrated and protease-susceptible residues outside the lanthionine rings may play a role in the short half-life in physiological settings. Previous mutagenesis work on mutacin 1140 has been limited to the lanthionine-forming residues, the C-terminally decarboxylated residue, and single amino acid substitutions at residues Phe1, Trp4, Dha5, and Arg13. To study the importance of the dehydrated (Dha5 and Dhb14) and protease-susceptible (Lys2 and Arg13) residues within mutacin 1140 for stability and bioactivity, each of these residues was evaluated for its impact on production and inhibitory activity. More than 15 analogs were purified, enabling direct comparison of the activities against a select panel of Gram-positive bacteria. The efficiency of the posttranslational modification (PTM) machinery of mutacin 1140 is highly restricted on its substrate. Analogs in the various intermediate stages of PTMs were observed as minor products following single point mutations at the 2nd, 5th, 13th, and 14th positions. The combination of alanine substitutions at the Dha5 and Dhb14 positions abolished mutacin 1140 production, while the production was restored by substitution of a Gly residue at one of these positions. Analogs with improved activity, productivity, and proteolytic stability were identified.IMPORTANCE Our findings show that the efficiency of mutacin 1140 PTMs is highly dependent on the core peptide sequence. Analogs in various intermediate stages of PTMs can be transported by the bacterium, which indicates that PTMs and transport are finely tuned for the native mutacin 1140 core peptide. Only certain combinations of amino acid substitutions at the Dha5 and Dhb14 dehydrated residue positions were tolerated. Observation of glutamylated core peptide analogs shows that dehydrations occur in a glutamate-dependent manner. Interestingly, mutations at positions outside rings A and B, the lipid II binding domain, would interfere with lipid II binding. Purified mutacin 1140 analogs have various activities and selectivities against different genera of bacteria, supporting the effort to generate analogs with higher specificity against pathogenic bacteria. The discovery of analogs with improved inhibitory activity against pathogenic bacteria, increased stability in the presence of protease, and higher product yields may promote the clinical development of this unique antimicrobial compound.

Keywords: lanthipeptide; lantibiotics; mutacin 1140; mutagenesis.

Figures

FIG 1
FIG 1
Structural elements found in type AI lantibiotics. (A) Representation of the dehydration mechanism of the dehydratase LanB in type AI lanthipeptides. The mass difference between the intermediate in the dehydration process and the final product is 147 Da. (B) Primary sequence alignment of the nisin and epidermin families of type AI lantibiotics. The lipid II binding domain is shaded in gray. Lanthionine rings are shown by black lines. Epidermin and gallidermin are highly similar to mutacin 1140. The residues that are different between gallidermin and mutacin 1140 are underlined. (C) Representative covalent structure of mutacin 1140. The four lanthionine rings are labeled A, B, C, and D. The lipid II binding domain consists of rings A and B, while the lateral assembly domain consists of the hinge region (residues 12 to 15) and rings C and D. Protease-susceptible residues Lys and Arg are shown in bold, while dehydrated residues Dha and Dhb are shown in bold and italics.
FIG 2
FIG 2
Summary of the mutacin 1140 core peptide mutants generated in this study. Single core peptide amino acid substitutions and multiple amino acid substitutions in the hinge region are shown above the provided mutacin 1140 sequence, while multiple substitutions in both the N-terminal and hinge regions are shown below the mutacin 1140 sequence. Each lane represents one single core peptide mutant. Bold amino acid abbreviations represent mutant strains with significantly increased bioactivity, gray abbreviations represent mutant strains with significantly decreased bioactivity, and underlined abbreviations represent mutant strains that are inactive. The rest of the mutant strains presented had no statistically significant change in bioactivity against the indicator strain M. luteus ATCC 10240.
FIG 3
FIG 3
Production levels of mutant core peptides (mutA) compared to production of the wild-type core peptide. (A) Single point mutations in the core peptide. (B) Multiple amino acid substitution mutations in the hinge region. (C) Multiple amino acid substitution mutations within the N-terminal and hinge regions. Three independent extractions were quantified by measuring the RP-HPLC peak area at 220 nm. The ratios of the peak areas of the mutacin 1140 analogs to the peak area of wild-type mutacin 1140 are shown. The Student t test was used for statistical analyses, and asterisks signify statistical significance (P < 0.05).
FIG 4
FIG 4
Bioactivities of core peptide (mutA) mutant strains compared to that of wild-type S. mutans JH1140. (A) Single point mutations in the core peptide. (B) Multiple amino acid substitution mutations in the hinge region. (C) Multiple amino acid substitution mutations within the N-terminal and hinge regions. The bioactivities of the mutants were measured as the ratio of the area of the zone of inhibition of each mutant strain to the area of the zone of inhibition of the wild-type strain. A value of >1.0 indicates that the mutant strain is more active than the wild-type strain. M. luteus ATCC 10240 was used as the indicator strain for antimicrobial activity assays, and experiments were done in triplicate. The Student t test was used for statistical analysis, and asterisks signify statistical significance (P < 0.05).
FIG 5
FIG 5
Trypsin stability of selected mutacin 1140 analogs. Wild-type mutacin 1140 and the K2A, R13A, K2A:R13A, T14A, and S5A:T14G mutants were tested. The bioactivity following trypsin digestion was measured as the ratio of the areas of the zones of inhibition of the trypsin-treated analog and the untreated analog. A value of 1.0 would indicate that the analog retained 100% of activity following trypsin exposure. M. luteus ATCC 10240 was used as the indicator strain for antimicrobial activity assays. The experiments were done in duplicate.

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