Curr Microbiol DOI 10.1007/s00284-014-0703-8

Attachment of Escherichia coli to Listeria monocytogenes for Pediocin-Mediated Killing Shanna Liu • Timo M. Takala • Justus Reunanen Ossian Saris • Per E. J. Saris

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Received: 18 June 2014 / Accepted: 19 August 2014 Ó Springer Science+Business Media New York 2014

Abstract Listeria phage endolysin cell wall-binding domain (CBD) from the Listeria phage A500 was fused with flagellar subunit FliC in Escherichia coli, aiming at binding of E. coli cells to Listeria cells, followed by enhanced killing of Listeria by pediocin production. FliC::CBD chimeric flagella were expressed and detected by Western blot. However, only few chimeric flagella could be isolated from the recombinant cells compared with sufficient amount of wild-type flagella obtained from the host cells. Interestingly, wild-type flagella extract showed capacity of binding Listeria cells. Pediocinsecreting E. coli cells with Listeria-binding flagella killed approximately 40 % of the Listeria cells, whereas cell-free spent growth medium with the same pediocin concentration only inhibited Listeria growth. These results suggested that binding the Listeria to bacteriocin-secreting cells improves killing.

Introduction Listeria monocytogenes is a food pathogen causing listeriosis, a disease affecting young, pregnant, elderly, and immune S. Liu College of Food Science and Bioengineering, Tianjin Agricultural University, 22 Jinjing Road, 300384 Tianjin, People’s Republic of China S. Liu  T. M. Takala  O. Saris  P. E. J. Saris (&) Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland e-mail: [email protected] J. Reunanen Department of Veterinary Biosciences, University of Helsinki, P.O. Box 66, 00014 Helsinki, Finland

compromised humans with high mortality rate [2]. One way to reduce human exposure to Listeria cells is to utilize bacteriocins or bacteriocin-producing cultures in foods or intestine [3, 8]. The antilisterial peptide pediocin produced by many lactic acid bacteria has been extensively studied and used for such purpose [10]. However, in food and intestine, the secreted bacteriocin is adsorbed on fat and other particles reducing the probability to reach the target cells [1]. In this study, we hypothesized that introduction of Listeria-binding ability to a pediocin-producing E. coli strain could promote the antilisterial effect of pediocin by bringing the bacteriocin-producing E. coli cells to close proximity with the listerial cells. The secretion of pediocin by Listeria-binding E. coli would increase the chance of pediocin to hit its target before adsorbing to food particles. To test this hypothesis, we chose the cell wall-binding domain of the Listeria phage A500 endolysin (CBD500) as the Listeria-binding protein [6] to be displayed on the surface of the pediocin-producing E. coli. The CBD500 was fused with the flagellar subunit FliC of E. coli [9]. The Listeria-binding E. coli strains were equipped with pediocin secretion cassettes, and tested for the Listeria killing capability. Antilisterial capacity was compared with that of spent culture medium containing same amount of pediocin.

Materials and Methods Bacteria, Plasmids, and Culture Conditions The Escherichia coli strains and plasmids used in this study are listed in Table 1. E. coli JT1 strain was cultured in Luria–Bertani (LB) medium at 37 °C. Strains with plasmids were grown in LB medium supplemented with 100 lg/ml ampicillin, when necessary. L. monocytogenes

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S. Liu et al.: Attachment of E. coli to Listeria monocytogenes Table 1 Escherichia coli strains and plasmids Strains or plasmids

Description

Source or reference

E. coli strains JT1

C 600 Hag::Tn10 fimA:cat

[11]

ECO770

JT1/pBluescript/fliCDH7

This work

ECO768

JT1/pLEB738

This work

ECO769

JT1/pLEB739

This work

pHPL500 pBluescript/ fliCDH7

Vector containing CBD500 gene Derivative of pBluescript

[6] [11]

pLEB738

Vector for FliC::CBD500 surface display, derivative of pBluescript/ fliCDH7

This work

pLEB691 pLEB739

Vector containing P45- SSusp45-papA P45- SSusp45-papA in pBluescript/ fliCDH7

[4] This work

Plasmids

WSLC 1019 (gift from Prof. Martin Loessner, ETH Zu¨rich, Switzerland) was cultivated in brain–heart infusion (BHI) medium (Oxoid) at 30 °C. Construction of FliC::CBD500 Chimera and Pediocin Production Plasmids For FliC::CBD500 chimeric flagella, CBD500 gene was amplified with primers FliCBD500F (50 -ACGGTAGA CCAAAACACTAATACAAATTC- 30 ) and FliCBD500R (50 -ATCGTCTACCTTATCGTCATCGTCTTTTAAGA AGTATTC- 30 ), and cloned into the AccI site of the pBluescript/fliCDH7 vector, generating pLEB738. Plasmid pLEB738 was transferred into E. coli JT1. Cells were cultured on plates for 48 h at 28 °C for expression of the chimeric flagella. Enterokinase digestion (final concentration 2 ng/ml, purchased from New England Biolabs) at room temperature for 4 h was used to verify the introduced enterokinase recognition site in the chimeric protein. The P45-SSusp45-papA fragment was amplified with primers P45F (50 -ATTGCGGCCGCGAATTCCGTTAGG GGCTTGAAC- 30 ) and PedR (50 -GACGTCGACTAGCAT TTATGATTACCTTG- 30 ), using previously constructed pediocin secretion plasmid pLEB691 as template. PCR product was ligated with pBluescript/fliCDH7 cut by SmaI. The ligation mixture was introduced into E. coli JT1 cells, resulting in the plasmid pLEB739 and the strain ECO769. SDS-PAGE and Western Blot Analysis The protein samples were analyzed on 12 % SDS-PAGE. Separated proteins were electroblotted onto polyvinylidene-

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fluoride (PVDF) membranes (0.45 lm) (Millipore) in transfer buffer (25 mM Tris, 192 mM glycine, 20 % methanol). The anti-H7 flagella antibody [11] was used for Western blot analysis (Invitrogen WesternBreeze chromogenic kit) according to manufacturer’s instructions. Flagella Isolation and Binding Assay To obtain flagella extracts, cells were harvested from plates, suspended in PBS (pH 7.0) and vortexed for 30 min before centrifuging. Supernatants were collected by centrifugation at 10,0009g for 10 min and stored at -20 °C for further analysis. Flagellar extractions were mixed with Listeria cells in PBST for 20 min at room temperature. Flagella-cell mixtures were washed three times followed by SDS-PAGE and Western blot analysis. Antilisterial Activity of Pediocin-Producing E. coli Pediocin production in E. coli ECO769 harboring papA gene was examined by the agar diffusion method. L. monocytogenes WSLC 1019 was grown in BHI medium overnight. Then the prepared BHI agar plate was overlaid with 10 ml soft agar inoculated with 50 ll overnight culture of L. monocytogenes WSLC 1019. E. coli cells were streaked onto BHI plates with L. monocytogenes lawn and incubated at 30 °C overnight. Listeria killing test was done by mixing either 200 ll washed E. coli cells (OD600 = 1) or supernatant of cultures with same amount of E. coli cells incubated for 3 h, with 0.2 % L. monocytogenes WSLC 1019 in 2 ml BHI medium. The mixtures were incubated for 3 h at 30 °C before plating on Listeria-selective Oxford agar (Scharlab) plates for counting of the surviving Listeria cells.

Results and Discussion Surface Display of Chimeric FliC::CBD500 Flagella in E. coli CBD500 (GenBank: X85009.1), originated from L. monocytogenes phage endolysin Ply500 [7], features high binding specificity to Listeria cell wall and designed to be displayed on the surface of E. coli cells. In our previous work [5], CBD500 was fused with the outer membrane anchor of Yersinia adhesin YadA and shown to be displayed on the E. coli cell surface. However, CBD500YadA fusion was toxic for E. coli, which was replaced by another fusion of CBD to the C-terminal part of membrane anchor OmpA. The localization of the CBD500 on the external surface of E. coli was verified by Western blot. The accessibility of the CBD domain on the cell envelope was shown to be compromised by whole-cell ELISA.

S. Liu et al.: Attachment of E. coli to Listeria monocytogenes

recognizing FliC, a protein band larger than FliC was detected in recombinant construct (Fig. 1a). This protein band was absent if cells were treated with enterokinase before Western blot analysis (Fig. 1a). Hence, expression of the Listeria-binding domain CBD500 as a protein chimera with the flagella subunit FliC did result in detectable production level. The protein chimera was located on the surface of the cells as the enterokinase treatment of the cells resulted in breakdown of the protein chimera. Binding of the Flagella to Listeria Cells

Fig. 1 Western analysis. a The FliC proteins expressed in E. coli ECO770 (=JT1/vector) and ECO768 (=JT1/FliC::CBD). M prestained molecular weight marker, 1 E. coli ECO770 cells, 2 E. coli ECO770 cells after enterokinase digestion, 3 E. coli ECO768 cells, 4 E. coli ECO768 cells after enterokinase digestion, 5 flagella extract of E. coli JT1 cells, 6 flagella extract of ECO770 cells, 7 flagella extract of E. coli ECO768 cells. b Binding test of flagella extract. 1 L. monocytogenes WSLC 1019 cells, 2 L. monocytogenes WSLC 1019 cells after mixing with flagella extract from ECO770 cells. The FliC protein band is marked by an asterisk and the FliC::CBD500 protein band with an arrow

Therefore, flagella display system was investigated for presenting the Listeria-binding CBD better on the surface of E. coli cells. Flagella are well exposed to the environment, and CBD500 was expressed as a chimeric protein with flagellar subunit FliC. In Western blot analysis with antibodies

To test if the FliC::CBD flagella could bind to Listeria cells, flagella extracts were isolated, mixed with Listeria cells, washed, and analyzed by Western blot. The wild-type flagella from ECO770 cells (JT1 cells with flagella expression vector) could be extracted and bound to Listeria cells (Fig. 1). However, the FliC::CBD flagella could not be extracted from the cells (Fig. 1a), and their binding to Listeria could not be determined. Clearly, normal flagella could not be formed with the FliC::CBD500 chimera. Surprisingly, the wild-type flagella of the E. coli host strain for the FliC::CBD500 expression could bind to Listeria cells. Such cells with Listeria-binding flagella were used in the Listeria killing assay with or without pediocin secretion. Pediocin Production and Antilisterial Effect of Flagellated E. coli Pediocin production was transferred to the flagellaexpressing cells. The antilisterial activity of ECO769 cells

Fig. 2 Pediocin expression in E. coli ECO769 (=JT1/papA) and effects on Listeria survival. a Detection of pediocin activity with L. monocytogenes WSLC 1019 as the indicator strain. 1 ECO770 (=JT1/vector) cells cultured on the BHI medium with L. monocytogenes WSLC 1019, 2 ECO769 cells cultured on the BHI medium with L. monocytogenes WSLC 1019. b L. monocytogenes WSLC 1019 killing test. Survival of Listeria after incubation (3 h) with E. coli strains or their spent growth media

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S. Liu et al.: Attachment of E. coli to Listeria monocytogenes

harboring papA gene was confirmed by agar diffusion method (Fig. 2a) and compared to that of ECO769 cell-free spent growth medium containing same amount of pediocin. The results showed that E. coli secreting pediocin and having Listeria-binding flagella could kill approximately 40 % of the Listeria cells in growth medium in 3 h, whereas the cell-free medium with the corresponding amount of pediocin could only inhibit cell growth but did not decrease the number of viable Listeria cells during the 3 h incubation (Fig. 2b). We interpret these results in favor of our original hypothesis that intimate contact between Listeria and pediocin-producing cells enhances the antilisterial effect of the bacteriocin by preventing its dilution in the environment and adsorption onto particles before taking effect to the target cells. Clearly, pediocin-secreting cells with Listeria-binding flagella killed Listeria more efficiently than the corresponding amount of pediocin in spent growth medium without producer cells. Especially, as with the pediocin in the spent growth medium the Listeria cells encountered directly, the pediocin concentration accumulated during the 3 h incubation and was present for the following 3 h incubation period in the maximal pediocin concentration. Pediocin in the spent growth killed Listeria at the beginning of the 3 h incubation period, but further incubation could not kill more Listeria cells, only retard growth. Whereas, when Listeria cells were incubated with the pediocin-producing E. coli cells, the pediocin concentration was low in the beginning and gradually increased to the end of the 3 h incubation period.

Conclusions The aim of this study was to test the hypothesis that bacteriocin-secreting cells kill target cells more efficiently if they can also bind to the target cells. We showed that this holds true for Listeria and E. coli secreting pediocin and having Listeria-binding flagella. The results have implications for bacteriocin applications showing that when possible, a bacteriocin-producing culture with binding capacities to the target cells should be used. A similar strategy could be adopted to construct a set of probiotic strains that would specifically bind to different pathogens and efficiently kill them by bacteriocins thereby protecting

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the host from intestinal infections. We are currently planning to transfer the above described technology combining the Listeria-binding flagella with pediocin production to probiotic E. coli strain resulting in a new innovative vehicle for protection against listeriosis. Acknowledgment This work was supported by China Scholarship Council, the University of Helsinki, Finland, and the Academy of Finland (project number 177321). Professor Martin Loessner is acknowledged for the kind gift of the Listeria strain and the CBD500 encoding DNA.

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