Accepted Manuscript Construction of a new shuttle vector for DNA delivery into mammalian cells using noninvasive Lactococcus lactis Bhrugu Yagnik, Harish Padh, Dr. Priti Desai, Scientist-B PII:
S1286-4579(15)00254-3
DOI:
10.1016/j.micinf.2015.11.006
Reference:
MICINF 4352
To appear in:
Microbes and Infection
Received Date: 9 September 2015 Revised Date:
31 October 2015
Accepted Date: 22 November 2015
Please cite this article as: B. Yagnik, H. Padh, Priti Desai, Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis, Microbes and Infection (2015), doi: 10.1016/j.micinf.2015.11.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Construction of a new shuttle vector for DNA delivery into mammalian cells using non-
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invasive Lactococcus lactis
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Bhrugu Yagnik a, Harish Padh b, Priti Desai *, a
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a
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Research Development (PERD) Centre, Ahmedabad-380054, Gujarat, India
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b
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Department of Cell and Molecular Biology, B. V. Patel Pharmaceutical Education and
Sardar Patel University, Vallabh Vidhyanagar-388120, Gujarat, India
Bhrugu Yagnik
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E-mail: [email protected]
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Harish Padh
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E-mail: [email protected]
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Corresponding author*
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Dr. Priti Desai
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Scientist-B
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Department of Cell and Molecular Biology
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B. V. Patel PERD Centre, S.G. Highway,
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Thaltej, Ahmedabad-380054,
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Gujarat
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India
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Tel.: +91-79-27439375/ 27416409
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Fax: +91-79-2745049
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Email – [email protected]
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[email protected]
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ACCEPTED MANUSCRIPT Abstract
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Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for
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delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive
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bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA)
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or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the
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safety status of invasive L. lactis is questionable. In the present report, we have shown that
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non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY
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to mammalian cells without any chemical enhancers. The salient features of the vector are; I)
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Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria
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(LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green
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Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS) IV) Immunostimulatory
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CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-
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K1 and Caco-2 cells demonstrates its functionality. Non-invasive r-L. lactis was found
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efficient in delivering pPERDBY to Caco-2 cells. The in vitro data presented in this article
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supports the hypothesis that in the absence of invasive proteins or relevant chemical
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treatment, L. lactis was found efficient in delivering DNA to mammalian cells.
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Key words: DNA vaccine; EGFP; Lactococcus lactis; Non-invasive; Plasmid; Vaccine
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delivery.
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1 Introduction
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In comparison with proteins, immunization with DNA is favourable and is gaining increasing
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acceptability. For DNA vaccination, the plasmid DNA is favoured for the reason that it can
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be easily constructed and amplified in appropriate host. Therefore, use of plasmid DNA for
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immunization represents one of the recent advancement in development of newer vaccine
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technology. The potential of DNA vaccines against few infectious diseases has been
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established [1]. DNA based vaccination has several advantages over the conventional
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methods. DNA vaccination leads to vaccine encoded antigen expression by host cell leading
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to presentation of antigen with their native epitopes. Therefore, DNA based vaccination
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generally lead to activation of both humoral and cell mediated immune pathways [1,2]. In
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addition, presence of CpG dinucleotide clusters in plasmid DNA will work as a natural
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adjuvant further aiding in stimulating immune system [3,4].
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Use of attenuated pathogens such as Shigella flexneri, Yersinia enterocolitica, Listeria
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monocytogenes or Salmonella thiphymurium was common in earlier attempts in development
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of DNA vaccines. Attenuated bacteria in such cases invade professional and non-professional
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phagocytes and deliver plasmid DNA, which results in cellular expression of antigen and
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presentation to immune system, invoking strong immune response against antigen [5].
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However, the major risk in this approach would be when attenuated pathogens revert back to
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their virulent form [6].
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Against this background, Lactococcus lactis (L. lactis) is considered safe and an attractive
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alternative as it offers the advantages of immunostimulatory property and extremely safe
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profile. The intrinsic advantage and extensive usage of L. lactis therefore lies in the
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knowledge that they are non-colonizing, non-invasive and non-pathogenic even when given
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overt opportunity as would be the case in an on-going disease. They have also been
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veterinary clinical significance [7,8]. Further, great advancement in L. lactis genetic
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manipulation tools played an important role in use of L. lactis as a readily amenable delivery
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vector [9]. L. lactis is generally regarded as safe (GRAS) microorganism as it is part of
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number of fermented food products [10]. This non-commensal, Gram positive microorganism
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has been widely explored for oral delivery of antigens, antibodies and a variety of therapeutic
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proteins [11,12].
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Use of L. lactis as a DNA delivery vehicle is an attractive and an emerging field in vaccine
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technology. Non-invasive and invasive recombinant L. lactis expressing either Internalin A
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(InlA) or mutant Internalin A (mInlA) of Listeria monocytogenes or Fibronectin Binding
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Protein A (FnBPA) of Staphylococcus aureus have been explored for their potential use as a
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gene transfer vehicle in vitro as well as in vivo [13–16].
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Currently available L. lactis DNA delivery systems are either based on the use of invasive L.
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lactis or cell wall weakening agents like glycine or penicillin [6,17,18]. The safety status of
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L. lactis expressing invasive proteins is not established. However, it is believed that
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expression of such invasive genes in non-commensal, non-invasive bacteria may possess
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potential hazards by disturbing natural gastro-intestinal flora, immune system and systemic
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processes [19]. It is also believed that cell wall weakening chemical agents used to enhance
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invasiveness may compromise its survival and reduce efficiency of DNA delivery in vivo.
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Moreover, larger size of plasmids used for vaccination may reduce transformation efficiency
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[6,19].
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To overcome such limitations of existing DNA delivery systems, here, we have evaluated
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non-invasive L. lactis as DNA delivery vehicle using newly constructed Escherichia coli (E.
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coli)-Lactic acid bacteria (LAB) shuttle vector, pPERDBY. New plasmid pPERDBY was
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plasmid less than 5 kb. The plasmid DNA was constructed using the elements of pSEC:Nuc
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and pEGFP-N1. The final plasmid would have the size less than 5 kb and following salient
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features; I) An eukaryotic cassette, II) Reporter gene Enhanced Green Fluorescent Protein
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(EGFP), III) Origin of replication compatible with a broad range of bacteria, IV)
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Immunostimulatory CpG motifs.
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The constructed plasmid pPERDBY would be validated and its functionality would be tested
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by transfection into mammalian cell lines like CHO-K1 and Caco-2. This article represents
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the design, construction, validation and delivery of pPERDBY using non-invasive L. lactis to
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Caco-2 cells.
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2 Materials and methods
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2.1 Bacterial strains, plasmids and growth conditions
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Bacterial strains and plasmids used in this study are listed in Table-1. Escherichia coli (E.
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coli) DH5α was grown in Luria-Bertani (LB) medium and incubated at 37 °C with vigorous
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shaking. L. lactis NZ9000 was grown in M17 medium containing 0.5 % glucose (GM17).
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Antibiotics were added at the indicated concentrations as necessary; Chloramphenicol, 10
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µg/mL for E. coli and L. lactis and kanamycin, 30 µg/mL for E. coli.
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2.2 DNA manipulation
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DNA manipulations were performed as described previously [21] with the minor
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modifications. For plasmid isolation from L. lactis, TES buffer (50 mM Tris-HCl [pH 8], 1
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mM EDTA, 25% sucrose) containing lysozyme (10 mg/mL) was added for 15 min at 37 °C
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for protoplast preparation. Enzymes were used as recommended by manufacturer (Thermo
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Scientific, USA). Electroporation of L. lactis was performed as described earlier [22]. L.
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24 hour incubation at 30 °C.
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2.3 pPERDBY vector design and construction
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Plasmid pPERDBY contains two major parts; 1) An eukaryotic expression cassette derived
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from plasmid pEGFP-N1 and 2) Replication origin for E. coli (RepC), Replication origin for
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L. lactis (RepA) and chloramphenicol resistance gene (Cmr) from plasmid pSEC:Nuc [23].
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Eukaryotic expression cassette composed of cytomegalovirus promoter (PCMV), a multiple
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cloning site (Nhe I, Eco47 III, Bgl II, Xho I, Hind III, Apa I, Bsp120 I, Kpn I, Asp718 I,
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BamH I, Age I, Xma I, Sac II, Sma I, Sal I, Sac I, Acc I, Ecl136 II, EcoR I and Pst I) fused
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with enhanced green fluorescent protein gene (MCS-EGFP), and SV40 polyadenylation
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(polyA) tail, was amplified with polymerase chain reaction (PCR) using high fidelity
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polymerase with proof reading activity (Pfu DNA Polymerase, Thermo Scientific, USA).
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Oligonucleotides pEGB-For2 (5’ CGCGCATGTGATAACCGTATTACCGCCATG 3’) and
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pEGB-Rev2 (5’ GCCTTAATTAACACTCAACCCTATCTCGGTCTATT 3’) introduced
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restriction site of Xce I and Pac I (underlined) respectively, in the ~1735 bp amplicon. The
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resulting PCR amplicon was further digested with restriction enzymes and purified using
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GeneJet gel extraction kit (Thermo Scientific, IL, USA) (Supplementary Fig. 1). RepA, RepC
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and Cmr were obtained by digesting pSEC:Nuc with Pac I and Xce I, resulting in fragment of
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~3253 bp, which was also purified using GeneJet gel extraction Kit (Thermo Scientific, IL,
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USA) (Supplementary Fig. 2).
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These digested parts of both the plasmids were further ligated and used to transform E. coli
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DH5α and L. lactis NZ9000. The resulting plasmid, pPERDBY has rolling circle replication
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(RCR) origin. Its replicon is derived from plasmid pWV01. Integrity of pPERDBY sequence
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was confirmed by DNA sequencing (Applied Biosystems 3730xl DNA Analyzer platform,
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ACCEPTED MANUSCRIPT Labreq Bioscientific, India). Fig. 1 and Table 1 summarizes the detailed description and
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information of pPERDBY construction.
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“Fig. 1, Table 1”
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2.4 Identification of CpG dinucleotides
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For identification of potent CpG islands in pPERDBY, bioinformatics tools like EMBOSS
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New CpG reporter (EMBL-EBI), EMBOSS CpG plot (Version 6.6.0), Sequence
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Manipulation Suite (SMS), and DataBase of CpG islands and Analytical Tool (DBCAT)
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were explored. Gardiner – Garden and Frommer (1987) method was followed for
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identification of CpG islands [24].
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2.5 Transfection of CHO-K1 cells and Caco-2 cells with pPERDBY
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Plasmid pPERDBY was assayed for EGFP expression by transfection into CHO-K1 and
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Caco-2 cells. CHO-K1 and Caco-2 cells were maintained in Dulbecco modified Eagle
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medium (DMEM) supplemented with 10% FBS. 100 U penicillin and 100 g streptomycin
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were added to prevent bacterial and fungal growth. 70 to 80 % confluent cells were
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transfected with pPERDBY, pEGFP-N1 (positive control) and pSEC:Nuc (negative control)
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using Lipofectamine LTX Plus transfection reagent (Invitrogen) according to supplier’s
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recommendation. The EGFP expressing cells were visualised at 24, 48 and 72 hours after
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transfection with an epifluorescent microscope (Olympus Model IX51). Transfection assays
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were performed in triplicate.
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2.6 Delivery of pPERDBY by non-invasive L. lactis to Caco-2 cells
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To evaluate the delivery of pPERDBY, r- L. lactis harbouring pPERDBY were co-cultured
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with Caco-2 as described previously [18] with the following modifications. Briefly, Caco-2
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cells were cultured in P12 wells plates containing 0.5 × 106 cells per well in DMEM
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supplemented with 10% FBS. r-L. lactis::pPERDBY and wild type L. lactis (negative
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ACCEPTED MANUSCRIPT control) (OD600 = 0.9-1.0) were added to Caco-2 cells so that the multiplicity of infection was
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about 103 bacteria/cell. After incubation period of 1 hour, non-internalised cells were
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removed by washing thrice with phosphate buffer saline (pH 7.4). Caco-2 cells were
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incubated for 2 hours with medium containing gentamicin (20 mg/L) to kill non-internalised
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bacteria. EGFP expressing cells were visualised at 24 and 48 hours after gentamicin treatment
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using epifluorescent microscope (Olympus Model IX51). For quantification of fluorescent
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cells, flow cytometry was performed after 24 hours of co-culture on Fluorescent Activated
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Cell Sorter, BD FACS Calibur (FACS, Becton Dickson, France).
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2.7 RNA isolation and Reverse Transcriptase-PCR (RT-PCR)
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24 hours after gentamicin treatment, co-cultured Caco-2 cells were removed from the P12
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well plate. Total RNA from these cells were isolated using RNA Sure mini kit (Nucleopore,
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Genetix, India) according to manufacturer’s protocol. RT-PCR was performed using 1 µg of
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total RNA with Maxima cDNA synthesis kit (Thermo Scientific, USA). EGFP transcripts
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were detected with egfp specific primers (Forward 5’ TTCAAGGACGACGGCAACTAC 3’
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and Reverse 5’ CTCTTCGCGCTAGTGTACCAG 3’).
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2.8 Quantification of internalised bacteria
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To find the number of bacteria internalized after co-culture with wild type L. lactis and r-L.
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lactis::pPERDBY, gentamicin treated Caco-2 cells were subjected to 1 mL of PBS containing
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0.2% Triton X-100 for 30 min. Serial dilution of the cell lysate was plated onto GM17 agar
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and were kept at 30 °C for 16-20 hours. PBS treated Caco-2 cells were used as a negative
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control. Assays were performed in triplicates.
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2.9
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Statistical analyses were performed using GraphPad Prism 5. For CFU study, statistical
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significance between the groups was calculated using One Way ANOVA test, followed by
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Statistical analysis
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ACCEPTED MANUSCRIPT the “Bonferroni” post-test. Statistical significance between mean values of percentage EGFP
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positive cells was assessed using two-tailed unpaired Student’s t test. Results of all statistical
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analyses were considered significant at value of p ≤ 0.05.
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3 Results
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3.1 pPERDBY construction and characteristics
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E. coli-L. lactis shuttle vector was constructed using the segments of pSEC:Nuc and pEGFP-
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N1 and ligated as outlined in materials and methods section. As depicted in Fig. 1, the newly
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constructed,
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cytomegalovirus promoter (pCMV), a multiple cloning site fused with enhanced green
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fluorescent protein gene (MCS-EGFP), and SV40 early mRNA polyadenylation signal,
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required for gene expression by eukaryotic host cells. The presence of EGFP act as a reporter
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gene and MCS facilitates convenient cloning.
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Presence of pPERDBY plasmid in recombinant E. coli and L. lactis was confirmed by
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restriction digestion analysis and PCR (Supplementary Fig. 3 and 4). To further validate the
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construction of vector pPERDBY, the plasmid was sequenced and sequence is now deposited
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in GenBank (Accession no. KR072508).
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CpG islands naturally present in pPERDBY were identified using bioinformatics tools and
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the results are summarised in Supplementary Fig. 5. The results indicate that pPERDBY has
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major two CpG islands comprised of multiple immunostimulatory CpG motifs.
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3.2 Transfection assay of pPERDBY into CHO-K1 and Caco-2 cells
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In order to examine the functionality of pPERDBY into CHO-K1 and Caco-2, cells were
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examined 24, 48 and 72 hours of transfection with pPERDBY as outlined in materials and
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methods. As shown in Supplementary Fig. 6, fluorescence from EGFP expression was
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transfection indicating not only transfection but also functional integrity of the system.
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Fluorescence from EGFP was not evident in the cells transfected with pSEC:Nuc, which
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served as a negative control. The results clearly demonstrate the significance of various
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features of newly constructed pPERDBY and indicates its potential for DNA vaccine vector.
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“Fig. 2”
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3.3 Delivery of pPERDBY to Caco-2 cells using r-L. lactis harbouring pPERDBY
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3.3.1
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Data mentioned in section 3.2 using fluorescence marker suggested that pPERDBY was
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successfully transfected to CHO-K1 and Caco-2 cells. To substantiate DNA delivery and
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expression of recombinant genes, total RNA was isolated from Caco-2 cells which were co-
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cultured with r-L. lactis::pPERDBY and egfp specific transcripts were detected by Reverse
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Transcriptase-PCR (RT-PCR). As shown in Supplementary Fig. 7, a 350 bp amplicon
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supports the expression of EGFP in Caco-2 cells. Caco-2 cells chemically transfected with
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pPERDBY was used as a positive control.
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3.3.2
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Caco-2 cells were co-cultured with L. lactis NZ9000 and r-L. lactis::pPERDBY for 1 hour.
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Subsequently, the cells were observed for EGFP expression at 24 and 48 hours. As shown in
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Fig. 3, fluorescence resulting from expression of EGFP was visible in the cells co-cultured
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with r-L. lactis::pPERDBY, whereas, Caco-2 cells co-cultured with L. lactis NZ9000 served
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as a negative control. The data clearly suggest the potential of non-invasive L. lactis for
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delivery of reporter plasmid pPERDBY to epithelial cells. (Fig. 3).
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“Fig. 3”
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EGFP expression at RNA level
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Quantitation of internalised bacteria
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DNA delivery by L. lactis to mammalian cells involves entry of L. lactis inside the cell by
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phagocytosis and release of plasmid DNA [6]. In order to ascertain the internalisation of non-
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invasive L. lactis, the number of bacteria internalised by Caco-2 cells were compared to PBS
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treated Caco-2 cells. Significantly large number of bacteria internalized inside the cells were
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found (Fig. 4).
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3.3.4
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Fractions of Caco-2 cells expressing EGFP protein after co-culturing with r-L.
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lactis::pPERDBY were quantitated by FACS analysis. As shown in Fig. 5, significantly large
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number of percentage of Caco-2 cells were found to be EGFP fluorescent positive as compare
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to control (p = 0.0054).
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“Fig. 4, Fig. 5”
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4 Discussion
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A number of pathogens having invasive properties such as Shigella, Salmonella, Yersinia and
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Listeria have been exploited for their use in DNA delivery to mammalian cells. [1]. However,
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the risk of reverting to virulent phenotype cannot be completely mitigated and there remains a
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need to search for safer alternatives. L. lactis was exploited as a safer alternative compared
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with invasive pathogens [9]. The L. lactis which considered non-invasive has been converted
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to invasive with expression of certain invasive proteins or by chemical treatment to increase
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its delivery efficiency [15,6]. We believe that L. lactis converted invasive by one of the
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methods mentioned above would compromise the safety profile and efficiency of L. lactis.
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Therefore, in the present study, we have explored the potential of non-invasive L. lactis as a
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DNA carrier using a newly constructed reporter plasmid pPERDBY.
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ACCEPTED MANUSCRIPT The plasmid offers several attractive features for its use as DNA vaccine candidate. The
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plasmid is capable of replicating in E. coli as well as in several species of Lactic Acid
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Bacteria (LAB) i.e. Bacillus subtilis, Lactococcus lactis, Borrelia burgdorferi, and numerous
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Lactobacilli (namely reuteri, fementum, casei, acidophilus, pentosus and helveticus), which
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makes it convenient to construct and explore their delivery through suitable strains of LAB.
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The plasmid also contains egfp gene along with Multiple Cloning Site (MCS), permitting
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convenient cloning of gene of interest in-frame with reporter gene. Compared with other
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alternatives, pPERDBY is only 4.9 kb, one of the smallest and thereby facilitating better
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transformation efficiency [20]. In addition, presence of CpG motifs in pPERDBY accords an
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additional adjuvant activity and also play important role as immunostimulatory. The data
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presented in this manuscript provides the proof of concept that pPERDBY is a functional
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plasmid which can be successfully transfected into CHO-K1 and Caco-2 cells. Moreover,
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using non-invasive r-L. lactis, pPERDBY can be delivered to Caco-2 cells.
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To increase the DNA delivery efficiency of L. lactis, numerous attempts have been made.
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Various invasive proteins such as Internalin A (InlA) of L. monocytogenes or Fibronectin
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binding protein A (FnBPA) of S. aureus were expressed in L. lactis rendering it invasive
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[15,16,18]. Though, non-invasive L. lactis is Generally Regarded As Safe (GRAS); to what
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extent recombinant L. lactis expressing such invasive proteins of pathogens is safe is
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questionable.
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Another approach to increase the delivery efficiency was assessed by Tao et al., in 2011
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wherein they showed that the treatment of L. lactis with glycine and other cell wall
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weakening agents resulted in increased uptake of L. lactis by Caco-2 cells. However, plasmid
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DNA delivery by L. lactis and expression followed by its uptake in Caco-2 cells are not
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shown [6]. Furthermore, the cell wall integrity of L. lactis is critical for its viability and
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survival in acidic environment of stomach as well as in the presence of bile. [26,27].
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step for successful delivery of plasmid DNA to mammalian cells [5]. Hence, such cell wall
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weakening treatments may compromise L. lactis not only for its survival, but also its ability
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to deliver DNA, which in turn reduces its potential as a DNA vaccine carrier in vivo.
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Considering the safety and efficiency aspects, here we have shown the successful delivery of
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newly constructed E. coli – LAB shuttle vector, pPERDBY to mammalian cells using non-
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invasive L. lactis. In addition, L. lactis NZ9000 is reported to induce pro-inflammatory
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effects [28], potentiating the advantage of using L. lactis NZ9000 as a live vaccine vector.
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The flow cytometry analysis performed in our study, revealed that non-invasive recombinant
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L. lactis is capable to deliver functional gene coding for EGFP in infected Caco-2 cells,
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which indicates the strength of non-invasive L. lactis as a DNA delivery vehicle.
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Non-invasive r-L. lactis::pPERDBY can also be used for efficient and safer delivery and
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monitoring of DNA vaccines, interference RNA and/or bioactive peptide [6]. With the
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advancement of technology such as UVP iBOX Scientia Small Animal Imaging System, it is
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now even possible to monitor vaccine uptake in vivo [29]. Further, attempts can be made to
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increase its adhesion capacity by expressing proteins like S-Layer protein of Lactobacillus
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brevis [30], instead of chemical treatment.
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Our hypothesis for DNA delivery through non-invasive L. lactis in vitro is; adhesion of L.
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lactis to epithelial cells [31] followed by its entry inside the cells [6], lysis of internalized
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bacteria inside the vacuole/phagosome, release of plasmid DNA and entry of plasmid inside
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the nucleus resulting in expression of gene of interest [1].
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In conclusion, a newly constructed shuttle vector pPERDBY with EGFP as reporter gene was
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constructed which allowed cloning and monitoring the expression of gene of interest.
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Moreover, using non-invasive L. lactis, delivery of pPERDBY to Caco-2 cells was
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ACCEPTED MANUSCRIPT confirmed. Hence, our study contributes in the field of new DNA delivery vectors using
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harmless non-invasive L. lactis, and its application in DNA immunization specifically for
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attainting mucosal immunity.
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Acknowledgment
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We gratefully acknowledge Dr. Luis Bermudez-Humaran, INRA, France for the gift of
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backbone vector pSEC:Nuc and L. lactis NZ9000 and Dr. Elke Ziska, Max Plank Institute of
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Infection Biology, Berlin, Germany for gift of pEGFP-N1 plasmid. Bhrugu Yagnik is
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recipient of Lady Tata Memorial Trust’s Fellowship. This work was financially supported by
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the grant of Indian Council of Medical Research (ICMR). This research has been facilitated
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by the infrastructure and resources provided by B.V. Patel PERD Centre.
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Conflict of Interest
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The authors declare no conflict of interest with respect to authorship, funding and publication
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of this article.
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References
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[26] Cotter PD, Hill C. Surviving the Acid Test : Responses of Gram-Positive Bacteria to Low pH 2003;67:429–53.
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[27] Drouault S, Corthier G, Ehrlich SD, Renault P. Survival, physiology, and lysis of Lactococcus lactis in the digestive tract. Appl Environ Microbiol 1999;65:4881–6.
[28] Yam KK, Pouliot P, N’diaye MM, Fournier S, Olivier M, Cousineau B. Innate inflammatory responses to the Gram-positive bacterium Lactococcus lactis. Vaccine
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Lactobacillus brevis S-Layer Protein in Lactococcus lactis Provides Nonadhesive
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lactococci with the Ability To Adhere to Intestinal Epithelial Cells. Appl Environ
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Microbiol 2003;69:2230–6.
[31] Kimoto H, Kurisaki J, Tsuji NM, Ohmomo S, Okamoto T. Lactococci as probiotic
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strains: adhesion to human enterocyte-like Caco-2 cells and tolerance to low pH and
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bile. Lett Appl Microbiol 1999;29:313–6.
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(A) Unique restriction enzyme sites PacI and XceI in pSEC:Nuc plasmid flank the region of
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origin of replication for E. coli (RepC), L. lactis (RepA) and Chloramphenicol resistance
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gene (Cmr) (B) Eukaryotic expression cassette composed of cytomegalovirus promoter
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(PCMV), multiple cloning site (MCS) fused with enhanced green fluorescence protein gene
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(EGFP) and simian virus 40 (SV40) polyadenylation tail (Poly A) present in pEGFP-N1
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plasmid (C) Eukaryotic expression cassette amplified from pEGFP-N1 plasmid flanking XceI
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and PacI restriction sites (D) Ligation of PacI and XceI digested product of pSEC:Nuc and
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amplified product of pEGFP-N1 plasmid resulting in a new DNA vaccine reporter plasmid,
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pPERDBY
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Figure 2: Epifluorescent micrographs of EGFP expressing CHO-K1 and Caco-2 cells after 72
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hours of transfection (100x magnification)
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CHO-K1 and Caco-2 cells were transfected with pEGFP-N1 (Positive control), pSEC:Nuc
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(Negative control) and pPERDBY plasmid; (A) CHO-K1 cells transfected with pEGFP-N1
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and pPERDBY show fluorescence whereas no fluorescence was observed in pSEC:Nuc
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transfected cells; (B) Similar profile with Caco-2 cells was observed.
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Figure 3: EGFP expression analysis in Caco-2 cells after delivery of pPERDBY using L.
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lactis (200x magnification)
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Epifluorescent micrographs of EGFP expressing Caco-2 cells co-cultured with wt. L. lactis
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(NZ9000) as a negative control, r-L. lactis::pPERDBY and control Caco-2 cells; (A) and (B)
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No fluorescence observed with control and wt. L. lactis; (C) and (D) Fluorescence was visible
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in Caco-2 cells after 24 and 48 hours of co-culture with r-L. lactis::pPERDBY.
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Figure 4: Internalised wt. L. lactis and r-L. lactis::pPERDBY after co-culture with Caco-2
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cells
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with Caco-2 cells. PBS treated Caco-2 cells were used as negative control. Results are mean
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± standard deviation from triplicates. Error bars represent standard deviation.
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Figure 5: In vitro gene transfer after 24 hours of co-culture of Caco-2 cells with r-L.
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lactis::pPERDBY and wt. L. lactis
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EGFP expression was assessed in Caco-2 cells after 24 hours of co-culture through FACS
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analysis. (A) Caco-2 cells co-cultured with wt. type L. lactis NZ9000; (B) with r-L.
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lactis::pPERDBY; Results presented are of one experiment representative of three performed
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independently; (C) Comparison of percentage of EGFP expressing Caco-2 cells. Results are
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means ± standard deviation from triplicates. Error bars represent standard deviation. **, %
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EGFP expressing cells were statistically comparable (the Students t test, P< 0.05)
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Table 1: Strains and plasmids used in the study
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ACCEPTED MANUSCRIPT Table 1: Strains and plasmids used in the study Strains and Plasmids
Characteristics
Reference
F-φ80dlacZ∆M15 ∆(lacZYA- argF)U169 endA1 recA1
Lab Source
E. coli DH5α
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Strains
hsdR17(rk- mk+) deoR thi-1 supE44 λ- gyrA96 relA1 L. lactis NZ9000
MG1363 (nisRK genes into chromosome), plasmid free
[12]
Cm, NZ9000 harboring pPERDBY
This work
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r-L. lactis::pPERDBY Plasmids
pSEC:Nuc
Kan, PCMV fused to egfp gene
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Cm, usp45-nuc fusion expressed under PnisA,
Gift from Dr. Elke Ziska [23]
RepA/RepC origin of replication
Cm, PCMV fused to egfp gene, E. coli-LAB shuttle vector
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pPERDBY
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*Chloramphenicol (Cm) was used at 10 µg/ml for E. coli and L. lactis. Kanamycin (Kan) at
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30 µg/ml was used for E. coli.
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S1286-4579(15)00254-3
DOI:
10.1016/j.micinf.2015.11.006
Reference:
MICINF 4352
To appear in:
Microbes and Infection
Received Date: 9 September 2015 Revised Date:
31 October 2015
Accepted Date: 22 November 2015
Please cite this article as: B. Yagnik, H. Padh, Priti Desai, Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis, Microbes and Infection (2015), doi: 10.1016/j.micinf.2015.11.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Construction of a new shuttle vector for DNA delivery into mammalian cells using non-
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invasive Lactococcus lactis
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Bhrugu Yagnik a, Harish Padh b, Priti Desai *, a
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a
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Research Development (PERD) Centre, Ahmedabad-380054, Gujarat, India
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b
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Department of Cell and Molecular Biology, B. V. Patel Pharmaceutical Education and
Sardar Patel University, Vallabh Vidhyanagar-388120, Gujarat, India
Bhrugu Yagnik
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E-mail: [email protected]
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Harish Padh
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E-mail: [email protected]
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Corresponding author*
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Dr. Priti Desai
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Scientist-B
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Department of Cell and Molecular Biology
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B. V. Patel PERD Centre, S.G. Highway,
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Thaltej, Ahmedabad-380054,
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Gujarat
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India
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Tel.: +91-79-27439375/ 27416409
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Fax: +91-79-2745049
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Email – [email protected]
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[email protected]
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ACCEPTED MANUSCRIPT Abstract
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Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for
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delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive
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bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA)
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or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the
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safety status of invasive L. lactis is questionable. In the present report, we have shown that
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non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY
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to mammalian cells without any chemical enhancers. The salient features of the vector are; I)
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Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria
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(LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green
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Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS) IV) Immunostimulatory
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CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-
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K1 and Caco-2 cells demonstrates its functionality. Non-invasive r-L. lactis was found
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efficient in delivering pPERDBY to Caco-2 cells. The in vitro data presented in this article
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supports the hypothesis that in the absence of invasive proteins or relevant chemical
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treatment, L. lactis was found efficient in delivering DNA to mammalian cells.
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Key words: DNA vaccine; EGFP; Lactococcus lactis; Non-invasive; Plasmid; Vaccine
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delivery.
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1 Introduction
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In comparison with proteins, immunization with DNA is favourable and is gaining increasing
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acceptability. For DNA vaccination, the plasmid DNA is favoured for the reason that it can
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be easily constructed and amplified in appropriate host. Therefore, use of plasmid DNA for
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immunization represents one of the recent advancement in development of newer vaccine
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technology. The potential of DNA vaccines against few infectious diseases has been
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established [1]. DNA based vaccination has several advantages over the conventional
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methods. DNA vaccination leads to vaccine encoded antigen expression by host cell leading
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to presentation of antigen with their native epitopes. Therefore, DNA based vaccination
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generally lead to activation of both humoral and cell mediated immune pathways [1,2]. In
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addition, presence of CpG dinucleotide clusters in plasmid DNA will work as a natural
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adjuvant further aiding in stimulating immune system [3,4].
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Use of attenuated pathogens such as Shigella flexneri, Yersinia enterocolitica, Listeria
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monocytogenes or Salmonella thiphymurium was common in earlier attempts in development
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of DNA vaccines. Attenuated bacteria in such cases invade professional and non-professional
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phagocytes and deliver plasmid DNA, which results in cellular expression of antigen and
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presentation to immune system, invoking strong immune response against antigen [5].
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However, the major risk in this approach would be when attenuated pathogens revert back to
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their virulent form [6].
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Against this background, Lactococcus lactis (L. lactis) is considered safe and an attractive
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alternative as it offers the advantages of immunostimulatory property and extremely safe
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profile. The intrinsic advantage and extensive usage of L. lactis therefore lies in the
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knowledge that they are non-colonizing, non-invasive and non-pathogenic even when given
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overt opportunity as would be the case in an on-going disease. They have also been
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veterinary clinical significance [7,8]. Further, great advancement in L. lactis genetic
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manipulation tools played an important role in use of L. lactis as a readily amenable delivery
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vector [9]. L. lactis is generally regarded as safe (GRAS) microorganism as it is part of
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number of fermented food products [10]. This non-commensal, Gram positive microorganism
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has been widely explored for oral delivery of antigens, antibodies and a variety of therapeutic
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proteins [11,12].
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Use of L. lactis as a DNA delivery vehicle is an attractive and an emerging field in vaccine
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technology. Non-invasive and invasive recombinant L. lactis expressing either Internalin A
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(InlA) or mutant Internalin A (mInlA) of Listeria monocytogenes or Fibronectin Binding
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Protein A (FnBPA) of Staphylococcus aureus have been explored for their potential use as a
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gene transfer vehicle in vitro as well as in vivo [13–16].
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Currently available L. lactis DNA delivery systems are either based on the use of invasive L.
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lactis or cell wall weakening agents like glycine or penicillin [6,17,18]. The safety status of
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L. lactis expressing invasive proteins is not established. However, it is believed that
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expression of such invasive genes in non-commensal, non-invasive bacteria may possess
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potential hazards by disturbing natural gastro-intestinal flora, immune system and systemic
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processes [19]. It is also believed that cell wall weakening chemical agents used to enhance
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invasiveness may compromise its survival and reduce efficiency of DNA delivery in vivo.
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Moreover, larger size of plasmids used for vaccination may reduce transformation efficiency
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[6,19].
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To overcome such limitations of existing DNA delivery systems, here, we have evaluated
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non-invasive L. lactis as DNA delivery vehicle using newly constructed Escherichia coli (E.
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coli)-Lactic acid bacteria (LAB) shuttle vector, pPERDBY. New plasmid pPERDBY was
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plasmid less than 5 kb. The plasmid DNA was constructed using the elements of pSEC:Nuc
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and pEGFP-N1. The final plasmid would have the size less than 5 kb and following salient
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features; I) An eukaryotic cassette, II) Reporter gene Enhanced Green Fluorescent Protein
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(EGFP), III) Origin of replication compatible with a broad range of bacteria, IV)
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Immunostimulatory CpG motifs.
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The constructed plasmid pPERDBY would be validated and its functionality would be tested
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by transfection into mammalian cell lines like CHO-K1 and Caco-2. This article represents
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the design, construction, validation and delivery of pPERDBY using non-invasive L. lactis to
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Caco-2 cells.
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2 Materials and methods
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2.1 Bacterial strains, plasmids and growth conditions
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Bacterial strains and plasmids used in this study are listed in Table-1. Escherichia coli (E.
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coli) DH5α was grown in Luria-Bertani (LB) medium and incubated at 37 °C with vigorous
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shaking. L. lactis NZ9000 was grown in M17 medium containing 0.5 % glucose (GM17).
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Antibiotics were added at the indicated concentrations as necessary; Chloramphenicol, 10
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µg/mL for E. coli and L. lactis and kanamycin, 30 µg/mL for E. coli.
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2.2 DNA manipulation
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DNA manipulations were performed as described previously [21] with the minor
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modifications. For plasmid isolation from L. lactis, TES buffer (50 mM Tris-HCl [pH 8], 1
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mM EDTA, 25% sucrose) containing lysozyme (10 mg/mL) was added for 15 min at 37 °C
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for protoplast preparation. Enzymes were used as recommended by manufacturer (Thermo
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Scientific, USA). Electroporation of L. lactis was performed as described earlier [22]. L.
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24 hour incubation at 30 °C.
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2.3 pPERDBY vector design and construction
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Plasmid pPERDBY contains two major parts; 1) An eukaryotic expression cassette derived
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from plasmid pEGFP-N1 and 2) Replication origin for E. coli (RepC), Replication origin for
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L. lactis (RepA) and chloramphenicol resistance gene (Cmr) from plasmid pSEC:Nuc [23].
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Eukaryotic expression cassette composed of cytomegalovirus promoter (PCMV), a multiple
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cloning site (Nhe I, Eco47 III, Bgl II, Xho I, Hind III, Apa I, Bsp120 I, Kpn I, Asp718 I,
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BamH I, Age I, Xma I, Sac II, Sma I, Sal I, Sac I, Acc I, Ecl136 II, EcoR I and Pst I) fused
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with enhanced green fluorescent protein gene (MCS-EGFP), and SV40 polyadenylation
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(polyA) tail, was amplified with polymerase chain reaction (PCR) using high fidelity
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polymerase with proof reading activity (Pfu DNA Polymerase, Thermo Scientific, USA).
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Oligonucleotides pEGB-For2 (5’ CGCGCATGTGATAACCGTATTACCGCCATG 3’) and
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pEGB-Rev2 (5’ GCCTTAATTAACACTCAACCCTATCTCGGTCTATT 3’) introduced
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restriction site of Xce I and Pac I (underlined) respectively, in the ~1735 bp amplicon. The
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resulting PCR amplicon was further digested with restriction enzymes and purified using
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GeneJet gel extraction kit (Thermo Scientific, IL, USA) (Supplementary Fig. 1). RepA, RepC
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and Cmr were obtained by digesting pSEC:Nuc with Pac I and Xce I, resulting in fragment of
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~3253 bp, which was also purified using GeneJet gel extraction Kit (Thermo Scientific, IL,
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USA) (Supplementary Fig. 2).
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These digested parts of both the plasmids were further ligated and used to transform E. coli
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DH5α and L. lactis NZ9000. The resulting plasmid, pPERDBY has rolling circle replication
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(RCR) origin. Its replicon is derived from plasmid pWV01. Integrity of pPERDBY sequence
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was confirmed by DNA sequencing (Applied Biosystems 3730xl DNA Analyzer platform,
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information of pPERDBY construction.
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“Fig. 1, Table 1”
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2.4 Identification of CpG dinucleotides
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For identification of potent CpG islands in pPERDBY, bioinformatics tools like EMBOSS
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New CpG reporter (EMBL-EBI), EMBOSS CpG plot (Version 6.6.0), Sequence
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Manipulation Suite (SMS), and DataBase of CpG islands and Analytical Tool (DBCAT)
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were explored. Gardiner – Garden and Frommer (1987) method was followed for
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identification of CpG islands [24].
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2.5 Transfection of CHO-K1 cells and Caco-2 cells with pPERDBY
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Plasmid pPERDBY was assayed for EGFP expression by transfection into CHO-K1 and
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Caco-2 cells. CHO-K1 and Caco-2 cells were maintained in Dulbecco modified Eagle
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medium (DMEM) supplemented with 10% FBS. 100 U penicillin and 100 g streptomycin
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were added to prevent bacterial and fungal growth. 70 to 80 % confluent cells were
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transfected with pPERDBY, pEGFP-N1 (positive control) and pSEC:Nuc (negative control)
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using Lipofectamine LTX Plus transfection reagent (Invitrogen) according to supplier’s
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recommendation. The EGFP expressing cells were visualised at 24, 48 and 72 hours after
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transfection with an epifluorescent microscope (Olympus Model IX51). Transfection assays
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were performed in triplicate.
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2.6 Delivery of pPERDBY by non-invasive L. lactis to Caco-2 cells
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To evaluate the delivery of pPERDBY, r- L. lactis harbouring pPERDBY were co-cultured
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with Caco-2 as described previously [18] with the following modifications. Briefly, Caco-2
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cells were cultured in P12 wells plates containing 0.5 × 106 cells per well in DMEM
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supplemented with 10% FBS. r-L. lactis::pPERDBY and wild type L. lactis (negative
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about 103 bacteria/cell. After incubation period of 1 hour, non-internalised cells were
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removed by washing thrice with phosphate buffer saline (pH 7.4). Caco-2 cells were
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incubated for 2 hours with medium containing gentamicin (20 mg/L) to kill non-internalised
167
bacteria. EGFP expressing cells were visualised at 24 and 48 hours after gentamicin treatment
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using epifluorescent microscope (Olympus Model IX51). For quantification of fluorescent
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cells, flow cytometry was performed after 24 hours of co-culture on Fluorescent Activated
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Cell Sorter, BD FACS Calibur (FACS, Becton Dickson, France).
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2.7 RNA isolation and Reverse Transcriptase-PCR (RT-PCR)
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24 hours after gentamicin treatment, co-cultured Caco-2 cells were removed from the P12
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well plate. Total RNA from these cells were isolated using RNA Sure mini kit (Nucleopore,
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Genetix, India) according to manufacturer’s protocol. RT-PCR was performed using 1 µg of
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total RNA with Maxima cDNA synthesis kit (Thermo Scientific, USA). EGFP transcripts
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were detected with egfp specific primers (Forward 5’ TTCAAGGACGACGGCAACTAC 3’
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and Reverse 5’ CTCTTCGCGCTAGTGTACCAG 3’).
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2.8 Quantification of internalised bacteria
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To find the number of bacteria internalized after co-culture with wild type L. lactis and r-L.
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lactis::pPERDBY, gentamicin treated Caco-2 cells were subjected to 1 mL of PBS containing
181
0.2% Triton X-100 for 30 min. Serial dilution of the cell lysate was plated onto GM17 agar
182
and were kept at 30 °C for 16-20 hours. PBS treated Caco-2 cells were used as a negative
183
control. Assays were performed in triplicates.
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2.9
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Statistical analyses were performed using GraphPad Prism 5. For CFU study, statistical
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significance between the groups was calculated using One Way ANOVA test, followed by
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Statistical analysis
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positive cells was assessed using two-tailed unpaired Student’s t test. Results of all statistical
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analyses were considered significant at value of p ≤ 0.05.
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3 Results
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3.1 pPERDBY construction and characteristics
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E. coli-L. lactis shuttle vector was constructed using the segments of pSEC:Nuc and pEGFP-
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N1 and ligated as outlined in materials and methods section. As depicted in Fig. 1, the newly
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constructed,
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cytomegalovirus promoter (pCMV), a multiple cloning site fused with enhanced green
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fluorescent protein gene (MCS-EGFP), and SV40 early mRNA polyadenylation signal,
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required for gene expression by eukaryotic host cells. The presence of EGFP act as a reporter
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gene and MCS facilitates convenient cloning.
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Presence of pPERDBY plasmid in recombinant E. coli and L. lactis was confirmed by
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restriction digestion analysis and PCR (Supplementary Fig. 3 and 4). To further validate the
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construction of vector pPERDBY, the plasmid was sequenced and sequence is now deposited
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in GenBank (Accession no. KR072508).
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CpG islands naturally present in pPERDBY were identified using bioinformatics tools and
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the results are summarised in Supplementary Fig. 5. The results indicate that pPERDBY has
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major two CpG islands comprised of multiple immunostimulatory CpG motifs.
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3.2 Transfection assay of pPERDBY into CHO-K1 and Caco-2 cells
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In order to examine the functionality of pPERDBY into CHO-K1 and Caco-2, cells were
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examined 24, 48 and 72 hours of transfection with pPERDBY as outlined in materials and
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methods. As shown in Supplementary Fig. 6, fluorescence from EGFP expression was
of
eukaryotic
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transfection indicating not only transfection but also functional integrity of the system.
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Fluorescence from EGFP was not evident in the cells transfected with pSEC:Nuc, which
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served as a negative control. The results clearly demonstrate the significance of various
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features of newly constructed pPERDBY and indicates its potential for DNA vaccine vector.
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“Fig. 2”
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3.3 Delivery of pPERDBY to Caco-2 cells using r-L. lactis harbouring pPERDBY
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3.3.1
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Data mentioned in section 3.2 using fluorescence marker suggested that pPERDBY was
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successfully transfected to CHO-K1 and Caco-2 cells. To substantiate DNA delivery and
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expression of recombinant genes, total RNA was isolated from Caco-2 cells which were co-
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cultured with r-L. lactis::pPERDBY and egfp specific transcripts were detected by Reverse
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Transcriptase-PCR (RT-PCR). As shown in Supplementary Fig. 7, a 350 bp amplicon
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supports the expression of EGFP in Caco-2 cells. Caco-2 cells chemically transfected with
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pPERDBY was used as a positive control.
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3.3.2
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Caco-2 cells were co-cultured with L. lactis NZ9000 and r-L. lactis::pPERDBY for 1 hour.
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Subsequently, the cells were observed for EGFP expression at 24 and 48 hours. As shown in
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Fig. 3, fluorescence resulting from expression of EGFP was visible in the cells co-cultured
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with r-L. lactis::pPERDBY, whereas, Caco-2 cells co-cultured with L. lactis NZ9000 served
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as a negative control. The data clearly suggest the potential of non-invasive L. lactis for
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delivery of reporter plasmid pPERDBY to epithelial cells. (Fig. 3).
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Quantitation of internalised bacteria
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DNA delivery by L. lactis to mammalian cells involves entry of L. lactis inside the cell by
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phagocytosis and release of plasmid DNA [6]. In order to ascertain the internalisation of non-
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invasive L. lactis, the number of bacteria internalised by Caco-2 cells were compared to PBS
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treated Caco-2 cells. Significantly large number of bacteria internalized inside the cells were
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found (Fig. 4).
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3.3.4
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Fractions of Caco-2 cells expressing EGFP protein after co-culturing with r-L.
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lactis::pPERDBY were quantitated by FACS analysis. As shown in Fig. 5, significantly large
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number of percentage of Caco-2 cells were found to be EGFP fluorescent positive as compare
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to control (p = 0.0054).
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“Fig. 4, Fig. 5”
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4 Discussion
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A number of pathogens having invasive properties such as Shigella, Salmonella, Yersinia and
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Listeria have been exploited for their use in DNA delivery to mammalian cells. [1]. However,
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the risk of reverting to virulent phenotype cannot be completely mitigated and there remains a
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need to search for safer alternatives. L. lactis was exploited as a safer alternative compared
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with invasive pathogens [9]. The L. lactis which considered non-invasive has been converted
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to invasive with expression of certain invasive proteins or by chemical treatment to increase
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its delivery efficiency [15,6]. We believe that L. lactis converted invasive by one of the
253
methods mentioned above would compromise the safety profile and efficiency of L. lactis.
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Therefore, in the present study, we have explored the potential of non-invasive L. lactis as a
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DNA carrier using a newly constructed reporter plasmid pPERDBY.
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plasmid is capable of replicating in E. coli as well as in several species of Lactic Acid
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Bacteria (LAB) i.e. Bacillus subtilis, Lactococcus lactis, Borrelia burgdorferi, and numerous
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Lactobacilli (namely reuteri, fementum, casei, acidophilus, pentosus and helveticus), which
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makes it convenient to construct and explore their delivery through suitable strains of LAB.
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The plasmid also contains egfp gene along with Multiple Cloning Site (MCS), permitting
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convenient cloning of gene of interest in-frame with reporter gene. Compared with other
263
alternatives, pPERDBY is only 4.9 kb, one of the smallest and thereby facilitating better
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transformation efficiency [20]. In addition, presence of CpG motifs in pPERDBY accords an
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additional adjuvant activity and also play important role as immunostimulatory. The data
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presented in this manuscript provides the proof of concept that pPERDBY is a functional
267
plasmid which can be successfully transfected into CHO-K1 and Caco-2 cells. Moreover,
268
using non-invasive r-L. lactis, pPERDBY can be delivered to Caco-2 cells.
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To increase the DNA delivery efficiency of L. lactis, numerous attempts have been made.
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Various invasive proteins such as Internalin A (InlA) of L. monocytogenes or Fibronectin
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binding protein A (FnBPA) of S. aureus were expressed in L. lactis rendering it invasive
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[15,16,18]. Though, non-invasive L. lactis is Generally Regarded As Safe (GRAS); to what
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extent recombinant L. lactis expressing such invasive proteins of pathogens is safe is
274
questionable.
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Another approach to increase the delivery efficiency was assessed by Tao et al., in 2011
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wherein they showed that the treatment of L. lactis with glycine and other cell wall
277
weakening agents resulted in increased uptake of L. lactis by Caco-2 cells. However, plasmid
278
DNA delivery by L. lactis and expression followed by its uptake in Caco-2 cells are not
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shown [6]. Furthermore, the cell wall integrity of L. lactis is critical for its viability and
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survival in acidic environment of stomach as well as in the presence of bile. [26,27].
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step for successful delivery of plasmid DNA to mammalian cells [5]. Hence, such cell wall
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weakening treatments may compromise L. lactis not only for its survival, but also its ability
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to deliver DNA, which in turn reduces its potential as a DNA vaccine carrier in vivo.
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Considering the safety and efficiency aspects, here we have shown the successful delivery of
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newly constructed E. coli – LAB shuttle vector, pPERDBY to mammalian cells using non-
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invasive L. lactis. In addition, L. lactis NZ9000 is reported to induce pro-inflammatory
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effects [28], potentiating the advantage of using L. lactis NZ9000 as a live vaccine vector.
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The flow cytometry analysis performed in our study, revealed that non-invasive recombinant
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L. lactis is capable to deliver functional gene coding for EGFP in infected Caco-2 cells,
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which indicates the strength of non-invasive L. lactis as a DNA delivery vehicle.
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Non-invasive r-L. lactis::pPERDBY can also be used for efficient and safer delivery and
293
monitoring of DNA vaccines, interference RNA and/or bioactive peptide [6]. With the
294
advancement of technology such as UVP iBOX Scientia Small Animal Imaging System, it is
295
now even possible to monitor vaccine uptake in vivo [29]. Further, attempts can be made to
296
increase its adhesion capacity by expressing proteins like S-Layer protein of Lactobacillus
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brevis [30], instead of chemical treatment.
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Our hypothesis for DNA delivery through non-invasive L. lactis in vitro is; adhesion of L.
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lactis to epithelial cells [31] followed by its entry inside the cells [6], lysis of internalized
300
bacteria inside the vacuole/phagosome, release of plasmid DNA and entry of plasmid inside
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the nucleus resulting in expression of gene of interest [1].
302
In conclusion, a newly constructed shuttle vector pPERDBY with EGFP as reporter gene was
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constructed which allowed cloning and monitoring the expression of gene of interest.
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Moreover, using non-invasive L. lactis, delivery of pPERDBY to Caco-2 cells was
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harmless non-invasive L. lactis, and its application in DNA immunization specifically for
307
attainting mucosal immunity.
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Acknowledgment
309
We gratefully acknowledge Dr. Luis Bermudez-Humaran, INRA, France for the gift of
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backbone vector pSEC:Nuc and L. lactis NZ9000 and Dr. Elke Ziska, Max Plank Institute of
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Infection Biology, Berlin, Germany for gift of pEGFP-N1 plasmid. Bhrugu Yagnik is
312
recipient of Lady Tata Memorial Trust’s Fellowship. This work was financially supported by
313
the grant of Indian Council of Medical Research (ICMR). This research has been facilitated
314
by the infrastructure and resources provided by B.V. Patel PERD Centre.
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Conflict of Interest
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The authors declare no conflict of interest with respect to authorship, funding and publication
317
of this article.
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References
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(A) Unique restriction enzyme sites PacI and XceI in pSEC:Nuc plasmid flank the region of
406
origin of replication for E. coli (RepC), L. lactis (RepA) and Chloramphenicol resistance
407
gene (Cmr) (B) Eukaryotic expression cassette composed of cytomegalovirus promoter
408
(PCMV), multiple cloning site (MCS) fused with enhanced green fluorescence protein gene
409
(EGFP) and simian virus 40 (SV40) polyadenylation tail (Poly A) present in pEGFP-N1
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plasmid (C) Eukaryotic expression cassette amplified from pEGFP-N1 plasmid flanking XceI
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and PacI restriction sites (D) Ligation of PacI and XceI digested product of pSEC:Nuc and
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amplified product of pEGFP-N1 plasmid resulting in a new DNA vaccine reporter plasmid,
413
pPERDBY
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Figure 2: Epifluorescent micrographs of EGFP expressing CHO-K1 and Caco-2 cells after 72
415
hours of transfection (100x magnification)
416
CHO-K1 and Caco-2 cells were transfected with pEGFP-N1 (Positive control), pSEC:Nuc
417
(Negative control) and pPERDBY plasmid; (A) CHO-K1 cells transfected with pEGFP-N1
418
and pPERDBY show fluorescence whereas no fluorescence was observed in pSEC:Nuc
419
transfected cells; (B) Similar profile with Caco-2 cells was observed.
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Figure 3: EGFP expression analysis in Caco-2 cells after delivery of pPERDBY using L.
421
lactis (200x magnification)
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Epifluorescent micrographs of EGFP expressing Caco-2 cells co-cultured with wt. L. lactis
423
(NZ9000) as a negative control, r-L. lactis::pPERDBY and control Caco-2 cells; (A) and (B)
424
No fluorescence observed with control and wt. L. lactis; (C) and (D) Fluorescence was visible
425
in Caco-2 cells after 24 and 48 hours of co-culture with r-L. lactis::pPERDBY.
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Figure 4: Internalised wt. L. lactis and r-L. lactis::pPERDBY after co-culture with Caco-2
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cells
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429
with Caco-2 cells. PBS treated Caco-2 cells were used as negative control. Results are mean
430
± standard deviation from triplicates. Error bars represent standard deviation.
431
Figure 5: In vitro gene transfer after 24 hours of co-culture of Caco-2 cells with r-L.
432
lactis::pPERDBY and wt. L. lactis
433
EGFP expression was assessed in Caco-2 cells after 24 hours of co-culture through FACS
434
analysis. (A) Caco-2 cells co-cultured with wt. type L. lactis NZ9000; (B) with r-L.
435
lactis::pPERDBY; Results presented are of one experiment representative of three performed
436
independently; (C) Comparison of percentage of EGFP expressing Caco-2 cells. Results are
437
means ± standard deviation from triplicates. Error bars represent standard deviation. **, %
438
EGFP expressing cells were statistically comparable (the Students t test, P< 0.05)
439
Table 1: Strains and plasmids used in the study
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ACCEPTED MANUSCRIPT Table 1: Strains and plasmids used in the study Strains and Plasmids
Characteristics
Reference
F-φ80dlacZ∆M15 ∆(lacZYA- argF)U169 endA1 recA1
Lab Source
E. coli DH5α
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hsdR17(rk- mk+) deoR thi-1 supE44 λ- gyrA96 relA1 L. lactis NZ9000
MG1363 (nisRK genes into chromosome), plasmid free
[12]
Cm, NZ9000 harboring pPERDBY
This work
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pSEC:Nuc
Kan, PCMV fused to egfp gene
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Cm, usp45-nuc fusion expressed under PnisA,
Gift from Dr. Elke Ziska [23]
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*Chloramphenicol (Cm) was used at 10 µg/ml for E. coli and L. lactis. Kanamycin (Kan) at
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