Insights into lantibiotic immunity provided by

22 The lantibiotic lacticin 3147 has been the focus of much research due to its broad spectrum of 23 activity against many microbial targets, including drug resistant pathogens. In order to protect 24 itself, a lacticin 3147 producer must possess a cognate immunity mechanism. Lacticin 3147 25 immunity is provided by an ABC transporter, LtnFE, and a dedicated immunity protein, LtnI, 26 both of which are capable of independently providing a degree of protection. Here we carry out 27 an in-depth investigation of LtnI structure-function relationships through the creation of a series 28 of fusion proteins and LtnI-determinants that have been the subject of random and site directed 29 mutagenesis. We establish that LtnI is a transmembrane protein that contains a number of 30 individual residues and regions, such as those between amino acid 20-27 and 76-83, which are 31 essential for LtnI function. Finally, as a consequence of the screening of a bank of 28,000 strains 32 producing different LtnI derivatives, we identified one variant (LtnI I81V) that provides 33 enhanced protection. To our knowledge, this is the first report of a lantibiotic immunity protein 34 with enhanced functionality. 35 36 on July 6, 2017 by gest httpaac.asm .rg/ D ow nladed fom Introduction 37 Lantibiotics are post-translationally modified antimicrobial peptides produced by Gram-positive 38 bacteria. Many lantibiotics are active in nanomolar concentrations and have a broad spectrum of 39 activity against many bacteria, including drug resistant pathogens (5, 7, 18, 35, 43). As a 40 consequence, lantibiotics have been the subject of much investigation with respect to clinical 41 and/or food applications (3, 4, 7, 15, 30, 55). Because of the potency of lantibiotics, each 42 producer must provide immunity against its own lantibiotic. Lacticin 3147 is a type II lantibiotic 43 produced by rare strains of Lactococcus lactis (50). The lacticin 3147 producer employs two 44 systems to provide immunity (12, 13, 34). One system is comprised of an ABC-transporter 45 complex designated LtnFE, thought to function through the extrusion of lacticin 3147 from the 46 cytoplasmic membrane. Such immunity transporters have been identified in other lantibiotic 47 producers and are generically designated LanFE(G) (17, 41, 44, 48, 49). Immunity to lacticin 48 3147 is also provided by a dedicated immunity protein, LtnI. Generically designated LanI, these 49 heterogeneous proteins/lipoproteins can provide protection against an associated lantibiotic alone 50 or in combination with LanFE(G) (27, 31, 34, 40, 42). Immunity to a number of other 51 lantibiotics, including Pep5, epicidin 280, lactocin S and cytolysin, is provided solely by the 52 corresponding immunity proteins, PepI, EciI, LasI and CylI, respectively (6, 19, 21, 47). 53 Relatively little is known regarding the mechanism by which LtnI provides protection to lacticin 54 3147. Although this 116 amino acid protein is predicted to be membrane associated on the basis 55 of hydrophobicity profiling (34), to date, other insights into LtnI function have had to be inferred 56 from what is known about other LanI proteins. NisI and SpaI, proteins associated with immunity 57 to nisin and subtilin, respectively, differ from LtnI in that they are lipoproteins that are linked to 58 the membrane by a lipid moiety. These proteins have been investigated in some depth. For 59 on July 6, 2017 by gest httpaac.asm .rg/ D ow nladed fom example, a series of C-terminally truncated NisI proteins were created and expressed in L. lactis 60 in order to identify the region of NisI that interacts with nisin. A 21 amino acid C-terminal 61 deletion resulted in the retention of just 14% of the protective effect provided by native NisI, 62 whereas longer deletions (up to 74 aa) had no additional effect. When the corresponding 21 aa 63 region of SpaI was replaced with that of NisI and expressed in L. Lactis, the SpaI’-‘NisI fusion 64 protein provided immunity to nisin, confirming the nisin specific protective capabilities of these 65 C-terminally located amino acids (51). 66 Similar investigations have been carried out to identify essential domains within PepI, a LanI 67 protein associated with Pep5 immunity (37), and its homologue, EciI, which is responsible for 68 epicidin 280 immunity and cross-immunity to Pep5 (19). The introduction of charged amino 69 acids into the N-terminal hydrophobic 20 amino acid stretch of PepI impacted on the membrane 70 localisation of the protein. One such mutant protein, PepI-I17R, conferred substantially reduced 71 immunity to Pep5. The addition of an F13D change in this background, slightly increased 72 immunity levels compared to I17R alone, but also resulted in an enhanced susceptibility to 73 proteolysis (21). To investigate the importance of the C-terminal domain of PepI, a truncated 74 protein, PepI 1-65, was created that lacked the four C-terminally located charged amino acids. 75 The immunity provided by this truncated version was greatly reduced (42). A further study 76 focused on three other C-terminally truncated versions of PepI; PepI1-63, PepI 1-57 and PepI 1-53. 77 As each segment consisting of two positively charged residues next to one negatively charged 78 amino acid was removed, the level of protection was further reduced. The negative impact on 79 immunity was evident despite the fact that these proteins remained located in the membrane, 80 thereby suggesting that the C-terminus of PepI is also involved in target recognition. The 81 on July 6, 2017 by gest httpaac.asm .rg/ D ow nladed fom importance of charge distribution within this C-terminal region was also apparent from the 82 negative impact on immunity arising from the creation of truncated versions of PepI (21). 83 Finally, the structure and function of the LanH protein associated with immunity to the type II 84 lantibiotic nukacin ISK-1, NukH (92 amino acids), has been extensively investigated (1). NukH, 85 although distinct from LanI proteins in that it functions as an accessory protein to the ABC86 transporter immunity system NukFEG, has a transmembrane location. Through the creation of 87 truncated versions of NukH fused to an alkaline phosphatase (PhoA) reporter and by evaluating 88 their sensitivity to proteinase K, it was established that NukH contains 3 transmembrane 89 domains. The PhoA fusion sites of NukH(1-33)-PhoA and NukH(1-92)-PhoA were shown to be 90 extracellularly located in that they were subject to proteinase K degradation, whereas the PhoA 91 domain of NukH(1-64)-PhoA was not, thereby supporting in silico predictions that this 92 corresponded to a transmembrane domain (40). To identify functional domains within NukH, 93 amino acid substitutions, deletions and truncated versions were created. Deletion of either the N94 terminus (position 1-6) or the C-terminus (position 89-92) of NukH did not have any effect on its 95 Nukacin ISK-1 binding capabilities or immunity function. However, substituting the amino acids 96 of the internal or external loop to alanines abolished NukH function. It was revealed that the 97 external loop was of greatest importance with respect to target binding and that, while deletion of 98 the transmembrane regions abolished immunity completely, the truncated protein was still 99 capable of binding its target (40). 100 Here, to address a lack of knowledge with respect to the topology and functional domains of 101 LtnI, or indeed type II immunity proteins in general, a series of fusion proteins and site-directed 102 derivatives were created. We also created the first bank of randomly mutated LanI proteins and 103 identified the first LanI variant that provides enhanced lantibiotic protection. 104 on July 6, 2017 by gest httpaac.asm .rg/ D ow nladed fom Materials and Methods 105 Growth Conditions 106 Strains and plasmids utilized during this study are found in Table 1. Lactococci were routinely 107 grown at 30 ̊C without aeration in M17 broth (Oxoid Ltd., Basingstoke, Hampshire, England) 108 supplemented with 0.5% (wt/vol) glucose (GM17), GM17 supplemented with K2HPO4 (36 mM), 109 KH2PO4 (13.2 mM), sodium citrate (1.7 mM), MgSO4 (0.4 mM), (NH4)2SO4 (6.8 mM) and 4.4% 110 glycerol (GM17 freezing buffer) without aeration or GM17 agar unless otherwise stated. 111 Escherichia coli was grown in Luria-Bertani broth (LB broth; (45)) at 37 ̊C with vigorous 112 agitation. Antibiotics were used, where indicated, at the following concentrations: Ampicillin 113 (Amp) was used at a concentration of 100 μg ml for E. coli and chloramphenicol at a 114 concentration of 10 μg ml for E. coli and 5 μg ml for L. lactis. 115 116 General molecular biology techniques 117 Plasmid DNA was isolated from E. coli strains using the High Pure plasmid isolation kit as 118 recommended by the manufacturer (Roche Diagnostics, Mannhein, Germany). Plasmids isolated 119 from L. lactis were isolated in the same way following treatment with protoplast buffer (5mM 120 EDTA, 50 U ml mutanolysin, 10 mg ml lysozyme, 0.75M sucrose, 20mM Tris-HCl pH7.5). 121 Total cell DNA was isolated using Roche high pure PCR template preparation kit (Roche 122 Diagnostics, Mannheim, Germany). Chemically competent E. coli Top10 was used as an 123 immediate host for the plasmids pNZ44 following manufacturers guidelines for transformation. 124 L. lactis strains were made electrocompetent following the procedure described by Holo and Nes 125 (23). In both cases electrotransformation was performed with an Electro cell manipulator (BTX126 Harvard apparatus). PCR was performed according to standard procedures using BioTaq DNA 127 on July 6, 2017 by gest httpaac.asm .rg/ D ow nladed fom (Bioline), Vent polymerase (New England biolabs), KOD DNA polymerase (Novagen) and Pwo 128 DNA polymerase (Roche Diagnostics). For colony PCR genomic DNA was accessed through 129 lysis of cells in 10% Igepal CA-630 (Sigma-Alrich) at 94 ̊C for 10 mins. Extraction of DNA 130 from agarose gels were performed using the KeyPrep Spin Gel DNA Clean Up Kit (Anachem, 131 Bedfordshire, UK) as recommended by the manufacturer. DNA ligations were executed 132 according to established procedures using T4 ligase supplied by Roche Diagnostics. Restriction 133 enzymes were also used to manufacturer’s guidelines and were supplied by Roche Diagnostics. 134 DNA sequencing was performed by MWG Biotech AG or Beckman coulter genomics. 135