Both IIC and IID Components of Mannose Phosphotransferase System Are Involved in the Specific Recognition between Immunity Protein PedB and Bacteriocin-Receptor Complex

Upon exposure to exogenous pediocin-like bacteriocins, immunity proteins specifically bind to the target receptor of the mannose phosphotransferase system components (man-PTS IIC and IID), therefore preventing bacterial cell death. However, the specific recognition of immunity proteins and its associated target receptors remains poorly understood. In this study, we constructed hybrid receptors to identify the domains of IIC and/or IID recognized by the immunity protein PedB, which confers immunity to pediocin PA-1. Using Lactobacillus plantarum man-PTS EII mutant W903, the IICD components of four pediocin PA-1-sensitive strains (L. plantarum WQ0815, Leuconostoc mesenteroides 05–43, Lactobacillus salivarius REN and Lactobacillus acidophilus 05–172) were respectively co-expressed with the immunity protein PedB. Well-diffusions assays showed that only the complex formed by LpIICD from L. plantarum WQ0815 with pediocin PA-1 could be recognized by PedB. In addition, a two-step PCR approach was used to construct hybrid receptors by combining LpIIC or LpIID recognized by PedB with the other three heterologous IID or IIC compounds unrecognized by PedB, respectively. The results showed that all six hybrid receptors were recognized by pediocin PA-1. However, when IIC or IID of L. plantarum WQ0815 was replaced with any corresponding IIC or IID component from L. mesenteroides 05–43, L. salivarius REN and L. acidophilus 05–172, all the hybrid receptors could not be recognized by PedB. Taken altogether, we concluded that both IIC and IID components of the mannose phosphotransferase system play an important role in the specific recognition between the bacteriocin-receptor complex and the immunity protein PedB.


Introduction
Bacteriocins, a class of antimicrobial peptides that are produced by bacteria, can inhibit growth or kill bacteria, while having diverse spectra of activity, i.e. from targeting only strains that are closely related to the producer strains or to a broader range of bacteria [1]. Pediocin-like bacteriocins are typically characterized by the presence of a conserved YGNGVXCXXXXCXV peptide motif within their N-terminal domains and also by a strong inhibitory effect on Listeria [2]. Pediocin-like bacteriocins contain two domains: a cationic N-terminal domain and a more hydrophobic C-terminal domain determining the target cell specificity [3]. The strong inhibitory effect of pediocin-like bacteriocins on food pathogens, such as Listeria, suggests multiple applications as antimicrobials in food and feed industry [4].
Generally, genes encoding pediocin-like bacteriocins are co-transcribed with or in the vicinity of a gene encoding a cognate immunity protein that protects the bacteriocin-producer from being killed by their own bacteriocins [5]. Although immunity proteins show remarkably high degrees of specificity to the bacteriocin they recognize, some of these immunity proteins may also provide immunity to other pediocin-like bacteriocins [6]. The C-terminal domains of pediocin-like immunity proteins specifically recognize the C-terminal hairpin domain of bacteriocins, resulting in immunity to bacteriocins [7,8]. Sprules and colleagues suggested that the hydrophobic pocket within the C-terminal domain of the immunity proteins may be attracting the immunity proteins to the surface of the cell membrane [9]. The mannose phosphotransferase system (man-PTS), which is a major sugar uptake system in Gram-positive and Gram-negative bacteria, consists of EI, Hpr and EII enzymes [10]. EII is composed of four distinct components: IIA, IIB, IIC and IID. IIA and IIB are normally represented by a single cytoplasmic protein (IIAB) and the membrane-located proteins IIC and IID together form a functional receptor for pediocin-like bacteriocins [11]. A recent study has shown that an extracellular loop of the IIC protein determined specificity for class IIa bacteriocins [12]. Immunity proteins form a strong complex with the bacteriocins and receptor, therefore protecting bacteriocin-producer cells [11]. The detailed mechanism of recognition of the membranelocated components IIC and IID by the immunity protein still remains unclear. In the present study, we demonstrated that both IIC and IID components play an important role in the formation of the bacteriocin-receptor-immunity protein complex using a series of hybrid receptors.

Bacterial strains, plasmids and growth conditions
The list of bacterial strains and plasmids used in this study are shown in Table 1. Lactobacillus spp. and Leuconostoc mesenteroides 05-43 were grown under anaerobic conditions at 37°C in de Man-Rogosa-Sharpe (MRS). Lactococcus lactis NZ9000 was propagated at 30°C in M17 broth supplemented with 0.5% w/v D-glucose. Escherichia coli were cultured in Lysogeny broth (LB) medium with shaking at 37°C. Pediocin PA-1 was produced by Lactobacillus plantarum strain LB-B1, which was originally and previously isolated from a traditionally fermented dairy product [13]. When required, relevant antibiotics (Sigma-Aldrich) were added at the following final concentrations: 50 μg/ml of kanamycin (Km), 10 μg/ml chloramphenicol (Cm) or 500 μg/ml of erythromycin (Em) for E. coli and 5 μg/ml Em or 10 μg/ml Cm for L. plantarum, respectively.

DNA manipulation techniques
Mini-prep plasmid isolations from both E. coli and L. plantarum were performed using the E. Z.N.A TM Plasmid Mini Kit Ι according to the manufacturer's instructions (OMEGA Bio-tek Inc., Doraville, GA, USA). Total DNA from Lactobacillus spp., Leuconostoc spp., and Lactococcus spp., were isolated using the genomic DNA extraction kit (Tiangen, Beijing, CN). Lysis of the bacterial cells was operated by adding lysozyme to TES buffer (50 mM Tris-Cl, 1 mM EDTA, 25% sucrose; pH 8.0) to a final concentration of 30 mg/ml, and the mixtures were incubated at 37°C for 1 h [18]. Plasmids were electroporated into E. coli and L. plantarum as described previously [19,20]. PCR products were amplified by using Ex Taq polymerase (Takara, Dalian, CN). Restriction endonuclease digestions were performed according to the supplier's instructions (Takara, Dalian, CN). DNA ligations were performed using the DNA Ligation kit (Tiangen, Beijing, CN). DNA sequences were determined with the Bigdye Terminator cycle sequencing kit (Sangon, Beijing, CN).

Construction of WQ0815 man-PTS EII mutant
The site-directed integration system described previously by Russell and Klaenhammer [17] was used to inactivate man-PTS EII in the genome of L. plantarum strain WQ0815. Genes encoding IIAB, IIC and IID components of EII are located within the same gene cluster, so the insertional inactivation of gene IIAB will also inactivate the downstream genes IIC and IID. A 725-bp internal region of the man-PTS IIAB gene of L. plantarum WQ0815 was chosen as a homologous sequence to construct WQ0815 man-PTS EII mutant. It was amplified using the forward primer HEAF (5'-CATGCCATGGCGGAATTTGCATATATATAAGTGA G-3') and the reverse primer HEAR (5'-CGCGGATCCGTCCGCAGTTCGTCTTTAG-3'), which were designed according to the gene IIAB (Accession no. CP002222.1). Restriction sites used for subsequent clones are underlined: NcoI and BamHI for the forward and reverse primers, respectively. These two sites were used to clone the amplified product into the integration plasmid pORI28 [14]. The recombinant plasmid, designated as pORIHEA, was transformed into L. plantarum WQ0815 containing pTRK669. Plasmid pTRK669 with chloramphenicol resistant (Cm r ) gene is a temperature-sensitive helper plasmid that provides RepA in trans for the replication of pORI28. A temperature increase from 37°C to 42°C resulted in the integration of plasmid pORIHEA into the WQ0815 genome and also the loss of pTRK669 and its associated chloramphenicol resistance phenotype. Plasmid pORIHEA was integrated into IIAB gene region through homologous recombination in the chromosome of L. plantarum WQ0815. The resulting mutant (erythromycin resistant) was designated L. plantarum W903. Then, PCR with the forward primer EmrF (5'-TTTTAGCAAACCCGTATTCCAC-3') and the reverse primer MptAR (5'-CCAAAATACCTTCCATACC-3') was performed to confirm the integration of pORIHEA at the correct genome locus. The primer EmrF was designed according to the DNA sequence of erythromycin resistance (Em r ) genes (Accession no. KM017875.1) and the primer MptAR was designed according to the DNA sequence of man-PTS gene (Accession no. CP002222.1). Then in order to induce expression of the genes of interest in W903, the nisRK genes from L. lactis NZ9000 were cloned into this plasmid using XbaI and HindIII restriction sites, creating a new plasmid pNZ8300. Next, lpIICD from L. plantarum WQ0815, lmIICD from L. mesenteroides 05-43 and laIICD from L. acidophilus 05-172 were respectively cloned into pNZ8300 between NcoI and XhoI sites to produce the recombinant plasmids, designated pNZlpCD, pNZlmCD and pNZlaCD, respectively. SphI and XhoI sites were used to clone the lsIICD genes from L. salivarius REN into pNZ8300, resulting in the recombinant plasmid pNZlsCD. These vectors were then transformed into L. plantarum W903, the sensitivity of these four recombinant strains to pediocin PA-1 was determined by the well-diffusion assay. In order to further examine whether the expressed man-PTS EII components could serve as receptors recognized by the immunity protein PedB, the immunity protein gene pedB was inserted to the downstream of lpIICD, lmIICD, lsIICD and laIICD genes in pNZ8300, respectively. The pedB gene was obtained by PCR from the chromosomal DNA of L. plantarum LB-B1, using the primers Im-F (5'-CCGCTCGAGGGCATCAATAAAGGGGTG-3') and Im-R (5'-TGCTCTAGACTA TTGGCTAGGCCACGTATT G-3'), which were designed according to the gene pedB (Accession no. AJ242489.1). Restriction sites used for subsequent cloning are underlined: XhoI and XbaI for the forward and reverse primers, respectively. The digested PCR products were respectively inserted into pNZlpCD, pNZlmCD, pNZlsCD and pNZlaCD to produce the recombinant plasmids, designated pNZlpCD-B, pNZlmCD-B, pNZlsCD-B and pNZlaCD-B, respectively. Then all the recombinant plasmids were transformed into L. plantarum W903, the sensitivity of all recombinant strains to pediocin PA-1 was determined by the well-diffusion assay. All the primer sequences for amplifying genes encoding IIC and IID components are given in Table 2.

Construction of hybrid receptor
In order to determine the part(s) or domains of IIC and/or IID that are responsible for specific recognition by immunity protein PedB, we constructed hybrid receptors by combining LpIIC or LpIID recognized by PedB with the heterologous IID or IIC compounds unrecognized by PedB, respectively. In this study, six different combinations of genes of lpIIC with lmIID, lmIIC with lpIID, lpIIC with lsIID, lsIIC with lpIID, lpIIC with laIID, and laIIC with lpIID were generated using a two-step PCR approach [12]. In this procedure, two separate DNA fragments were amplified in the first step by using one outer primer and one inner primer for each fragment. Overlapped sequences were introduced using the inner primers and the two fragments were fused in a second PCR using the outer primers. Hybrid receptor genes were cloned into pNZ8300 between NcoI and XhoI sites, or SphI and XhoI sites to produce the recombinant plasmids, designated pNZlpClmD, pNZlmClpD, pNZlpClsD, pNZlsClpD, pNZlpClaD and pNZlaClpD, respectively. Then the recombinant plasmids were transformed into L. plantarum W903. The sensitivity of the recombinant strains to pediocin PA-1 was determined by well-diffusion assay. Only hybrid receptors recognized by pediocin PA-1 were used in this study, because self-protection by the immunity protein only takes place after the formation of the bacteriocin receptor complex [11]. Furthermore, the PCR product of pedB gene was respectively cloned into pNZlpClmD, pNZlmClpD, pNZlpClsD, pNZlsClpD, pNZlpClaD and pNZlaClpD between XhoI and XbaI sites to produce the recombinant plasmids, designated pNZlpClmD-B, pNZlmClpD-B, pNZlpClsD-B, pNZlsClpD-B, pNZlpClaD-B and pNZlaClpD-B, respectively. These recombinant plasmids were also transformed into L. plantarum W903. The sensitivity of these six recombinant strains to pediocin PA-1 was determined by the well-diffusion assay. All recombinant plasmids were verified by DNA sequencing analyses. The primers and DNA templates used to construct hybrid receptors are detailed in Table 3.

Bacteriocin activity assays
Pediocin PA-1 was prepared using ammonium sulfate precipitation (70% saturation) from the culture supernatant of L. plantarum LB-B1. The precipitate was dissolved in a 10 mM  phosphate buffer (pH 6.0) followed by filtration by a 0.2 μm pore size filter. Bacteriocin activity was expressed in units, and one bacteriocin unit (U) was defined as the amount of bacteriocin required to reduce the growth of the indicator strain L. plantarum WQ0815 by 50% under the tested conditions of the assay [21]. Bacteriocin activity was assayed by the well-diffusion method. When the recombinant strains were used as the indicator strains, the well-diffusion assay was modified by adding nisin (200 ng/ml) in the soft agar with 10 6 CFU of the indicator strains [22]. For the bacteriocin activity assay, 128 U bacteriocin was added into each metal punch on the plates.

Inactivation of man-PTS EII genes in L. plantarum WQ0815 by homologous recombination
In order to eliminate the interference of the endogenous receptor, a L. plantarum WQ0815 man-PTS EII gene insertion mutant was constructed in this study. The mutant was constructed as shown in Fig 1A. To confirm the integration of pORIHEA at the correct genome locus, PCR was performed using the primers EmrF from sequence of Em r gene and MptAR from man-PTS gene. The PCR result showed that an expected 1.8-kb fragment could be observed, when chromosomal DNA from the mutant W903 was used as template (S1 Fig). Sequence analysis showed that this PCR product contained the expected fragments of the Em r gene and man-PTS IIAB gene. When chromosomal DNA from the wild-type strain WQ0815 was used as template in the PCR, no product was generated. This result indicated that the Em r gene has been integrated into the chromosome of mutant W903 by homologous recombination. Furthermore, the sensitivity of this mutant to pediocin PA-1 was tested by the well-diffusion assay. As shown in Fig 1B, an inhibition zone was observed on the plate of the wild-type strain WQ0815. However, the mutant W903 was resistant to pediocin PA-1, suggesting that receptor EII has been inactivated in this mutant. The PCR result, combined with the well-diffusion assay results, demonstrated that the single crossover homologous recombination event was successful.

The immunity protein PedB recognizes the specific receptor
In order to study recognition specificities between these four different receptors and the immunity protein PedB, all recombinant plasmids with the different receptors were first transformed into the host L. plantarum W903, the well-diffusion assay was employed to investigate the sensitivity of the recombinant strains to pediocin PA-1. The results showed that only the control strain W918 (W903 with pNZ8300) was still resistant to pediocin PA-1 (S2 Fig), whereas all the recombinant strains W919 with lpCD, W921 with lmCD, W927 with lsCD and W929 with laCD became sensitive to pediocin PA-1 (Fig 2), suggesting that LpIICD, LmIICD, LsIICD and LaIICD could be recognized by pediocin PA-1 in the host W903. Furthermore, the well-diffusion assays showed that only the recombinant strain W920 with lpCD-B was resistant to pediocin PA-1, while the recombinant strains W922 with lmCD-B, W928 with lsCD-B and W930 with laCD-B were still sensitive to pediocin PA-1 (Fig 2). The results indicated that the immunity protein PedB could only recognize the complex formed by protein IICD from L. plantarum WQ0815 with pediocin PA-1 and could not recognize the complex formed by proteins IICD from L. mesenteroides 05-43, L. salivarius REN or L. acidophilus 05-172 with pediocin PA-1.

Both IIC and IID components play an important role in specific recognition by immunity protein PedB
The well-diffusion assays showed that all the recombinant strains W931, W933, W935, W937, W939 and W941 produced sensitivity to pediocin PA-1, suggesting that all six hybrid-type receptor genes were successfully expressed in W903 (Fig 3). These hybrid receptors form a complex with pediocin PA-1, allowing us to determine whether the immunity protein could provide immunity or not to pediocin PA-1. Well-diffusion assays showed that all the recombinant strains expressing the hybrid receptor genes and pedB gene in W932, W934, W936, W938, W940 and W942 still remained sensitive to pediocin PA-1, which indicated that IIC component of L. plantarum WQ0815 could not be replaced by IIC components of L. mesenteroides 05-43, L. salivarius REN or L. acidophilus 05-172 in the formation of the immunity protein-receptor-bacteriocin complex. The IID component of L. plantarum WQ0815 could also not be replaced with the other three kinds of corresponding IID components. These results demonstrated that both IIC and IID components play important roles in specific recognition by the immunity protein PedB. Taken altogether, we concluded that the immunity protein PedB directly interacts with the IIC protein, IID protein and pediocin PA-1 to form a complex, therefore preventing membrane permeabilization and cell death.

Discussion
Pediocin-like bacteriocins have a strong inhibitory effect on Listeria, which illustrates their potential applications as antimicrobials in the food and feed industry. For the sensitive strains, both membrane-located proteins IIC and IID of man-PTS were confirmed to be necessary for the receptor function [11]. When heterologously expressing IIC or IID components, these may interact with the endogenous IID or IIC protein in L. plantarum and together form a hybrid receptor complex that resulted in sensitivity to pediocin-like bacteriocins [12]. In order to eliminate the interference of endogenous receptor, L. plantarum WQ0815 man-PTS EII gene insertion mutant was constructed by site-directed integration system.
In a previous work, the leucocin A immunity gene was heterologously expressed in Enterococcus faecalis and the resulting strain displayed immunity to enterocin A, pediocin PA-1 and leucocin A, whereas in Lactobacillus sakei and Carnobacterium piscicola, this gene could only provide immunity to leucocin A [23], indicating that strain-specific factors may influence the function of immunity proteins. In this study, we co-expressed immunity protein PedB with receptor IICD from L. plantarum WQ0815, L. mesenteroides 05-43, L. salivarius REN or L. acidophilus 05-172 in the host W903. Our results showed that only the receptor from L. plantarum WQ0815 could recognize the immunity protein, suggesting that the receptor contributed to strain-specific immune protein functionality. In order to investigate whether there are other host-specific factors involved in immunity, in our previous work, man-PTS IICD genes of L. plantarum WQ0815 and L. mesenteroides 05-43 were expressed in product with chromosomal DNA from W903 as a template. The arrow indicates the 1.8-kb amplified product.