Insights into Lantibiotic Immunity Provided by Bioengineering of LtnI

Abstract

The lantibiotic lacticin 3147 has been the focus of much research due to its broad spectrum of activity against many microbial targets, including drug-resistant pathogens. In order to protect itself, a lacticin 3147 producer must possess a cognate immunity mechanism. Lacticin 3147 immunity is provided by an ABC transporter, LtnFE, and a dedicated immunity protein, LtnI, both of which are capable of independently providing a degree of protection. In the study described here, we carried out an in-depth investigation of LtnI structure-function relationships through the creation of a series of fusion proteins and LtnI determinants that have been the subject of random and site-directed mutagenesis. We establish that LtnI is a transmembrane protein that contains a number of individual residues and regions, such as those between amino acids 20 and 27 and amino acids 76 and 83, which are essential for LtnI function. Finally, as a consequence of the screening of a bank of 28,000 strains producing different LtnI derivatives, we identified one variant (LtnI I81V) that provides enhanced protection. To our knowledge, this is the first report of a lantibiotic immunity protein with enhanced functionality.

Fig 1

LtnI topology predictions. Membrane topology models of LtnI using TM prediction sites. TMHMM (A) and SPLIT (version 4.0) (B) analysis. (A) Dark grey, amino acids predicted to be in the membrane; black, amino acids inside; light grey, amino acids outside. (B) Dark grey hatched boxes indicate amino acids in the membrane. (C) On the basis of the study by Cuthbertson et al. (10), the findings obtained with different algorithms were compared to indicate LtnI transmembrane regions: TMHMM, SPLIT (version 4.0), SOUSI, and TMpred. Protein sequence of LtnI also indicates residues fused to reporter enzymes LacZ and PhoA (residues numbered and with asterisks).

Fig 2

Investigation of LtnI homologues. LtnI and its closest homologues derived from a variety of Bacillus strains. Highlighted are the amino acids that were involved in random or site-directed mutagenesis. The amino acids between W20 and N27 have also been investigated for homology due to the importance of this region in immunity. Where homology is conserved among homologues alone (highlighted in black) is of particular interest, as conversion of the amino acid at this position in LtnI to the corresponding conserved amino acid diminishes immunity.

Fig 3

Activity of LtnI-LacZ and LtnI-PhoA fusions in E. coli CC118. Activity of ltnI-lacZ and ltnI-phoA fusions in E. coli CC118. A21, F45, etc., represent the residue of LtnI fused to lacZ or phoA. Activity is representative of the average of three independent triplicate experiments.

Fig 4

Prediction of LtnI topology with overview of mutagenesis and truncation positions. Predicted here is the membrane location of LtnI. Indicated are positions at which random mutagenesis due to amino acid changes, frameshifts, and stop codon introduction resulted in diminished immunity, or as for I81V, in which the amino acid change resulted in a functionally enhanced LtnI peptide. Positions at which truncated LtnI proteins were created are also present.

Fig 5

Growth curve demonstrating the enhanced immunity of LtnI (I81V). MG1363/pNZ44ltnI and MG1363/pNZ44ltnI (I81V) were grown in the presence and absence of 1.1 μM lacticin 3147. Each strain was grown in triplicate, and readings of the absorbance at 600 nm were taken hourly.