Milk from transgenic goat expressing human lysozyme for recovery and treatment of gastrointestinal pathogens

Accepted Manuscript Milk from transgenic goat expressing human lysozyme for recovery and treatment of gastrointestinal pathogens

Igor de Sá Carneiro, José Nilson Rodrigues de Menezes, Julyana Almeida Maia, André Marrocos Miranda, Victor Bruno Soares de Oliveira, James D. Murray, Elizabeth A. Maga, Marcelo Bertolini, Luciana Relly Bertolini PII: DOI: Reference:

S0928-0987(17)30623-1 doi:10.1016/j.ejps.2017.11.005 PHASCI 4294

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

25 April 2017 20 October 2017 4 November 2017

Please cite this article as: Igor de Sá Carneiro, José Nilson Rodrigues de Menezes, Julyana Almeida Maia, André Marrocos Miranda, Victor Bruno Soares de Oliveira, James D. Murray, Elizabeth A. Maga, Marcelo Bertolini, Luciana Relly Bertolini , Milk from transgenic goat expressing human lysozyme for recovery and treatment of gastrointestinal pathogens. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), doi:10.1016/j.ejps.2017.11.005

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MILK FROM TRANSGENIC GOAT EXPRESSING HUMAN LYSOZYME FOR RECOVERY AND TREATMENT OF GASTROINTESTINAL PATHOGENS Corresponding author: Luciana Relly Bertolini (L. Bertolini) Phone number: +55 (51) 3320-3931

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e-mail address: [email protected]

Postal address: Biotechnology and Genetic Engineering Lab

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School of Pharmacy, Pontifical Catholic University of Rio Grande do Sul (PUCRS)

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Av. Ipiranga, 6681 - Porto Alegre, RS - Brazil - 90.619-900

Igor de Sá Carneiroa, José Nilson Rodrigues de Menezesb, Julyana Almeida

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Maiac; André Marrocos Mirandad; Victor Bruno Soares de Oliveirae; James D. Murrayf; Elizabeth A. Magag; Marcelo Bertolinih; Luciana Relly Bertolinia,i a

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Laboratory of Molecular Biology and Development, University of Fortaleza, Avenida Washington

Soares, 1321, Edson Queiroz, 60.811-905, Fortaleza, Ceará, Brazil. E-mail: [email protected] b

Laboratory of Molecular Biology and Development, University of Fortaleza, Avenida Washington

Soares, 1321, Edson Queiroz, 60.811-905, Fortaleza, Ceará, Brazil. E-mail: [email protected] Soares,

1321,

Edson

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Laboratory of Molecular Biology and Development, University of Fortaleza, Avenida Washington Queiroz,

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c

60.811-905,

Fortaleza,

Ceará,

Brazil.

E-mail:

[email protected] d

Laboratory of Molecular Biology and Development, University of Fortaleza, Avenida Washington

Soares,

1321,

Edson

Queiroz,

60.811-905,

Fortaleza,

Ceará,

Brazil.

E-mail:

e

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[email protected]

Laboratory of Molecular Biology and Development, University of Fortaleza, Avenida Washington 1321,

Edson

Queiroz,

60.811-905,

Fortaleza,

Ceará,

Brazil.

E-mail:

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Soares,

[email protected] f

Department of Animal Science and Department of Population Health and Reproduction, University of

California, Davis, CA 95616, USA. E-mail: [email protected] g

Department of Animal Science and Department of Population Health and Reproduction, University of

California, Davis, CA 95616, USA. E-mail: [email protected] h

School of Veterinary Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. E-

mail: [email protected] i

Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil. E-mail:

[email protected]

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ABSTRACT Lysozyme is an important non-specific immune protein in human milk, modulating the immune response against bacterial infections. The aim of this study was to characterize the milk of a transgenic goat expressing a recombinant human lysozyme (rhLZ) in the milk, also testing the in vitro antibacterial activity of the rhLZ milk against pathogens of the gastrointestinal tract. Milk samples collected from Tg and non-

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transgenic goats (nTg) from the 3rd to the 11th week of lactation were submitted to physicochemical analyses, rhLZ semi-quantification, and to rhLZ antimicrobial activity

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against Micrococcus luteus, Shiguella sonnei and Enterococcus faecalis. Viability and cell migration were studied in ileum epithelial cells (IEC-18) in absence or presence

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of E. faecalis, Staphylococcus aureus, Escherichia coli (EPEC) and S. sonnei. The expression of ZO-1 and IL-6 genes were evaluated in IEC-18 to evaluate the effect of

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rhLZ milk on intestinal barrier function and intestinal inflammation. Physicochemical parameters between goat Tg and nTg milk were similar and within normal values for

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human consumption, with hLZ concentrations being similar between Tg (224 µg/mL) and human (226 µg/mL) milk. The Tg milk had bactericidal activity against M. luteus, no bactericidal effect on S. sonnei, and relative to discrete sensitivity against E.

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feacalis than controls. Better migrating parameters were observed in cells in culture

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with nTg and Tg than controls. In the presence of pathogens, the Tg milk promoted improved migrating parameters than controls, except for S. sonnei, with lower cell numbers in the presence of nTg samples and E. faecalis and S. sonnei. No

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differences in ZO-1 relative expression patterns were observed in cultured cells, with increased expression in IL-6 in cells exposed to nTg milk than controls, with the Tg

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group being similar to all groups. In conclusion, goat milk containing rhLZ demonstrated valid evidence for its potential use as a nutraceutical for improvement of health and nutrition quality in humans.

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1. INTRODUCTION Breast milk is the ideal source of nutrition for infant development, providing nutrients and factors that promote health and fight infections. The benefits of human milk are attributed to the antimicrobial action of milk proteins such as Lysozyme (LZ). Such enzyme is part of the passive immunity and natural defence against Grampositive bacteria and, with cooperation of other factors present in milk, LZ has also

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demonstrated activity against Gram-negative bacteria, viruses, parasites and fungi (Rollins et al., 2016; Victora et al., 2016).

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Lysozyme is capable of catalyzing the hydrolysis of glycosidic bonds

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between 1-4 acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer of the bacterial cell walls. Its antimicrobial activity results from that cleavage, which causes leakage of the internal components of the cells and consequent destruction of

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bacteria (Norman et al., 2012). Lysozyme is a globular protein with 129 amino acid residues and 14.314 kDa, which, besides being present in human milk, can be found

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in different concentrations in various species and secretions such as tears, sweat and saliva. In human milk, the typical concentration is about 200-400 μg/mL, many fold higher than the content of LZ present in the milk of goats (0.25 μg/mL), cattle (0.13

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μg/mL) and pigs (0.065 μg/mL), for instance (Cerven et al., 2008; Maga et al., 2012;

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Ning et al., 2009; Pupek et al., 2003; Yang et al., 2011). Considering the benefits of LZ, several research groups have developed genetically engineered organisms for production of recombinant human lysozyme

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(rhLZ). The rhLZ expression by the transgenic brown rice has reached 0.6% of grain weight (Huang et al., 2002). Transgenic chickens produced an average of 29.90

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μg/mL in the egg white (Wang et al., 2016). The concentrations of rhLZ in the milk of transgenic mice varied from 250 to 710 μg/mL (Maga and Murray, 1995); transgenic cows and transgenic pigs expressed in their milk about 25.96 μg/mL (Yang et al., 2011) and 116 mg/mL (Lu et al., 2014) of rhLZ, respectively. Milk from transgenic dairy animals expressing rhLZ has potential to prevent and treat infant diarrhea and some other infectious diseases, also reducing in the burden of malnutrition (Brundige et al., 2008; Cerven et al., 2008; Maga et al., 2012). Goat rhLZ milk can indeed modulate gut microbial populations in a fashion similar to

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the human milk (Cooper et al., 2013; Maga et al., 2012). In previous studies, the fecal microbiota analysis of young pigs fed with pasteurized milk of rhLZ transgenic goats showed significant increase in the number of bacteria associated with gut health (Bifidobacteriaceae and Lactobacillaceae) and decreased number of bacteria associated with diseases (Escherichia coli, Mycobacteriaceae, Streptococcaceae and Campylobacterales; Maga et al., 2006b, 2012). Young pigs have also presented

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improved gut morphology and circulating metabolites (Cooper et al., 2011; Brundige et al., 2010), helping to ameliorate symptoms of diarrhoea (Cooper et al., 2013). To

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further investigate and explore the benefits of human lysozyme, a line of transgenic dairy goats expressing rhLZ in their milk was produced in Brazil. In this study, we

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aimed to validate the potential usefulness of the milk as from such transgenic line by evaluating the physicochemical characteristics of the milk, also identifying and

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quantifying rhLZ in milk, and its enzyme activity in vitro on intestinal epithelial cell (IEC-18) cultures and against gastrointestinal bacterial pathogens. This represents

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the first necessary step in testing the milk properties in systematic in vitro and in vivo studies for future potential use of the rhLZ goat milk as nutraceutic product in

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humans.

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2. MATERIAL AND METHODS 2.1 Ethics statement

This study was conducted in compliance with guidelines of the Brazilian Technical

Biosafety

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National

Committee

(Comissão

Técnica

Nacional

de

Biossegurança - CTNBio), in facilities accredited by the Biosafety Certification the

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Number 0294/10 and the Ethical Principles of Animal Experimentation described by Brazilian

College

of

Animal

Experimentation

(Colégio

Brasileiro

de

Experimentação Animal - COBEA, 1991). The described experiments were approved by the Research Ethics Committee of the University of Fortaleza (process number 003/2016). 2.2 Transgenic animals A line of transgenic goats expressing rhLZ in milk was generated by pronuclear microinjection of one-cell stage embryos with a gene construct (23 Kb)

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consisted of the human lysozyme cDNA (540 bp) under the bovine αS1-casein promoter and 3′ regulatory elements, according to Maga (2003). Milk from a female transgenic founder (Tg) and from two non-transgenic control goats (nTg1 and nTg2) matching the same breed, parity and stage of lactation was used throughout this study. 2.3 Milk sample collections

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Transgenic and non-transgenic goats were kept in separate stalls, and provided hay, mineral salt and water ad libitum, and daily exposure to sunlight. Milk

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samples from the Tg goat and from the nTg1 and nTg2 goats were collected once a week, starting at the third week of natural lactation, and for the subsequent eight

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weeks. The nTg2 goat was used only for comparisons in the physicochemical analyses. The collection procedure was performed manually and under aseptic

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conditions, with teats sanitized and dried prior to each collection, as recommended by biosafety standard protocols. As for the human control milk (hM), samples were 2.4 Physicochemical analyses

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obtained from unidentified donors, approximately at the fourth month of lactation. Upon each milk collection, during the eight-weeks period, 50 mL of fresh milk

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collected from the transgenic and non-transgenic goats were subjected to physical

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and chemical analyses in triplicate. Samples from two nTg were used for substantial equivalence, as previously defined by the OECD (Organization for Economic Cooperation and Development, 1998). The amount of fat, lactose, protein and non-fat

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solids, as well as the pH and the freezing point were verified in each milk sample for identification of potential alterations in milk composition between groups, using a milk

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analyser apparatus (Lactoscan S, Milkotronic Ltd., New Zagora, Bulgaria). The lactic acid content was measured by titrating the milk with 0.1 N Sodium hydroxide (Dornic solution) and the alkaline level was measured in the presence of a phenolphthalein indicator at pH 8.0 (FAO, 2017; Salva, 2011). To analyse the specific gravity of milk, the density was obtained following FAO recommendations (Food and Agriculture Organization, 2012) and to determine the proximate analysis for nutrition evaluation, the ash content, which represents the total mineral content, was obtained by total incineration of the samples through 24-h heating in an oven at 100°C, followed by 6 h

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in muffle furnace at 550°C (Nielsen, 2006). After each weekly physicochemical analyses, the remaining milk samples were cooled down and pasteurized at 64°C for 35 min. Samples were immediately cooled to -4°C, being stored at -80°C and thawed only at runtime analyses. 2.5 Lysozyme identification and quantification The rhLZ contents in milk samples were quantified by Western blot in

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triplicates. The nTg, Tg and hM were initially diluted 1:1 in Laemmli buffer and denatured at 95°C for 3 min, for separation by 15% SDS-PAGE in electrophoretic run

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(90 min at 100 V). Commercial recombinant human lysozyme (L1667, Sigma-Aldrich, St. Louis, MO, USA) diluted at 270 µg/mL was used as positive control and also to

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quantify the rhLZ in milk. Proteins were transferred to nitrocellulose membrane for 2 h at 100 V by wet transfer in the Mini Trans-Blot® Cell device (Bio-Rad, Hercules, CA,

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USA). Membranes were blocked for 1 h using TBS-T with 5% non-fat dry milk (8 g/L NaCl, 2.42 g/L Tris, 0.05% Tween-20 detergent). Subsequently, membranes were

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rinsed three times for 15 min in TBS-T, and human anti-lysozyme antibody (DAKO, Glostrup, Denmark) was diluted to 1:2,500 in the blocking solution for 16 h at room temperature (RT). Incubation was then performed with alkaline phosphatase-labelled

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anti-rabbit secondary antibody (Invitrogen®, Carlsbad, CA, USA) diluted to 1:20,000

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in TBS-T (at RT for 1 h). Three additional 15-min washes were carried out in TBS-T, with the membranes then covered with the NBT/BCIP (Sigma-Aldrich). After 24 h, semi-quantification of rhLZ expression in milk was determined by the analysis of

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digitalized and photographed gels, where the intensity of each specific western blot band diameter was measured using the Image J software 1.4 (NIH, Bethesda, MD,

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USA) comparing to known amounts of commercial rhLZ (S-rhLZ group). Results were converted into numeric values referring to each measured band. 2.6 Activity test

To assess the biological activity of lysozyme in the milk produced by the transgenic goat, we assayed its antimicrobial activity analysing the formation of bacterial inhibitory growth zones into culture plates. Samples of Tg, nTg, hM, S-rhLZ, and a solution identified as nTg+rhLZ (a preparation of the nTg sample added with 270 µg/mL of commercial rhLZ, meant to simulate the rhLZ concentration in human

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milk) were subjected to a spot-on-the-lawn activity assay, by incubating 30 µL of each sample in a punched hole of an agarose plate incorporated with 10% Micrococcus luteus (ATCC 00356). After the 48-h plate incubation at 37°C, photographic images were analysed and the inhibitory growth zone measurement was assessed for each group. The assay was based on the diffusion procedure adapted from Maga et al. (1995).

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2.7 Bactericidal assay

The ATCC (Rockville, MD, USA) strains used to investigate the bactericidal

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activity of lysozyme were Shiguella sonnei (ATCC 25931), Enterococcus faecalis (ATCC 29212), and M. luteus (ATCC 00356). Bacteria from each strain were

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cultivated at 37°C for 24 h and then diluted in 0.9% saline solution to a 0.5 concentration in the McFarland scale. Then, each bacterial suspension was diluted

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1,000-fold in Luria-Bertani (LB) broth. Milk samples from the Tg, nTg, S-rhLZ and nTg+rhLZ groups were also diluted 20-fold in LB broth, remaining at 37°C under 220

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x g for 60 min. Then, 15 µL were plated onto two LB-agar medium plates. After 24 h, the colony-forming units (CFU) were observed, counted and calculated. The experiment was performed in triplicates.

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2.8 Cell proliferation assay

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Cellular viability, migration and gene expression in presence of Tg and controls were determined by using rat intestinal epithelial cells (IEC-18 line, ATCC, Rockville, MD, USA) in culture, at passage 29, as previously described (Brito et al.,

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2005). In brief, cells were cultured at 37°C in 5% CO2 in DMEM (Dulbecco’s modified Eagle medium, GIBCO, Grand Island, NY, USA) containing 10% heat-inactivated

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fetal bovine serum, 50 IU/mL penicillin and 50 μg/mL streptomycin (GIBCO). Milk samples from each group were diluted 1:5, 1:20 and 1:40 in DMEM, with phosphatebuffered saline (PBS) used as negative control. 2.8.1 Cell viability To determine cell viability in the presence of different milk dilutions, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Sigma-Aldrich) was used to measure the mitochondrial reductase activity in living cells (Mosmann, 1983). In brief, IEC-18 were seeded in 96-well plates in a total concentration of 4 x

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104 cells/well in 100 µL of culture medium. After 24 h incubation, the media was removed and replaced by DMEM containing PBS or each sample dilution in triplicate (n=3). After 24, 48, and 72 h, cells were washed with PBS and then incubated for 4 h in 100 µL culture medium with 10 μL MTT solution (5 mg/mL). Plates were centrifuged at 3,000 rpm for 15 min, media containing MTT were removed by fast inversion, and formazan crystals were diluted with acidified isopropanol solution (0.04

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N HCl). Prior to reading, plates were stirred for 5 min and the absorbance was measured in a ELISA reader set at 575 nm.

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2.8.2 Migration assay

The ability of hrLZ-containing milk to affect the IEC-18 migration in the

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presence or absence of bacteria was determined as previously described (Carvalho et al., 2012). Briefly, cell migration was evaluated in the presence of rhLZ in the milk.

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IEC-18 were transferred to a 12-well plate at a concentration of 2.4 x 10 5 cells/well. After a 24 h growth period, cells were incubated with Mitomycin C (5 μg/mL; Sigma-

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Aldrich) for 30 min, the monolayer was scratched with the aid of a sterile blade, so that cells were dragged from the center to the right-side edge of the well. Culture medium containing Mitomycin C was discarded and replaced by fresh medium with

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PBS or milk dilutions (1:5, 1:20, and 1:40) from each group. After incubation for 24 h,

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wells were washed twice with PBS for observation of cell migration across the scratch line under an inverted microscope at 10X magnification. Cell migration analysis was based on the number of migrating cells, the migration distance and software.

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growth velocity, after assessment of photographic images by using the Image J

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2.8.3 Cell migration in the presence of pathogens The ability of the milk from transgenic or non-transgenic goats to modulate

cell migration was assessed in the presence of main intestinal pathogens associated with stomach diseases and diarrhea Escherichia coli EPEC (ATCC 3905), Shiguella sonnei (ATCC 25931), Enterococcus faecalis (ATCC 29212) and Staphylococcus aureus (ATCC 6535), based on van Vuuren et al. (2015). Milk samples were diluted at 1:20 in DMEM, with bacteria (2.5x105 CFU/mL) added to cell cultures. Following 24 h incubation, cells were washed with PBS and examined for CFU, as described

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above (Carvalho et al., 2012). 2.8.4 Gene expression As intestinal inflammation may be associated with intestinal barrier disruption, qRTPCR analysis was used to evaluate the expression of genes for the tight junction protein ZO-1 and the pro-inflammatory cytokine IL-6 in IEC-18 in culture, using βactin as housekeeping gene for normalization. Briefly, IEC-18 were seeded into 12-

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well plates until 60-70% confluence, when Tg, nTg, nTg+S-rhLZ and S-rhLZ samples were added in three biological replicates for each treatment, remaining in culture for

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additional 24 h. Cells were washed twice with PBS and harvested by trypsinization. Suspended cells were washed in PBS, and then stored at -80°C. Samples were

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thawed and total RNA was extracted using the RNeasy spin column purification kit (Qiagen, Valencia, CA, USA), following the manufacturer's instructions. RNA

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concentration was quantified and purity was checked by UV absorbance at 260 nm and 280 nm (NanoDrop 2000, Waltham, Massachusetts, USA). cDNA was

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synthesized via reverse transcription PCR, using 1 µg total RNA treated with DNase, oligo (dT) as primers, and Super Script III enzyme (Invitrogen). RT-qPCR reactions were performed in 20 µL containing 10 µL Power SYBR® Green PCR Master Mix 2x

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(Applied Biosystem, Foster City, CA, USA), 1 µL cDNA with 600 nM of gene-specific

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primers designed to assess ZO-1 and IL-6 transcript expression, and 6.6 µL ultrapure water. Primers were synthetized by Invitrogen (São Paulo, Brazil), with nucleotide sequences shown in Table A.1. Amplification consisted of 5 min at 94°C, followed by

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40 cycles of 30 s at 94°C, 30 s at the annealing temperature (60°C), and 30 s at 72°C, followed by 40 cycles of 0.5°C increments (10 s each) for the melting curve,

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starting at 75°C. Fluorescence was measured during the annealing step of each cycle. All amplifications were carried out using the thermocycler StepOne PlusTM (Applied Biosystems, Foster City, CA, USA). 2.9 Statistical analysis Data obtained from the bactericidal evaluation, cell viability and migration, cell number, migration velocity and distance for samples and pathogens were compared by the χ2 or Fisher’s tests. The normality of the quantitative data was analysed by the Kolmogorov-Smirnov test and, when required, values were

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normalized by logarithmic transformation on a base 10 and analyzed by ANOVA with paired comparisons by the Tukey’s test (GLM-Minitab, State College, PA, USA). A simple correlation (Pearson) test was used to assess relationships between variables. Graphic figures were produced by GraphPad Prism® software (Version 5.01). Gene expression data, representative of three independent biological replicates for each treatment, were subjected to analysis of variance, using Minitab®

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Statistical Software (Minitab Inc., State College, PA, USA), with means compared by the Tukey’s test. The level of significance was 5%.

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3. RESULTS 3.1 Physicochemical analysis

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Physicochemical analyses showed the mean milk parameter values for goat Tg milk samples to be similar to goat nTg milk throughout the eight consecutive

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weeks of collection, with all parameters falling well within the required values for human consumption, according to minimum quality standards by MAPA (Ministério da

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Agricultura e Pecuária, Brazil, 2000). However, in spite of the normal range values, milk from the transgenic goat founder had significantly lower fat and higher protein contents than milk from both non-transgenic control goats (Table A.2).

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3.2 Lysozyme identification and quantification

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The rhLZ was identified by Western blot in milk samples from Tg, hM and SrhLZ (Fig. A.1). After comparing each specific band intensity for each sample to the S-rhLZ group (270 µg/mL commercial rhLZ), the mean concentration values for

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human lysozyme in Tg milk was 224 µg/mL. Such mean LZ value was similar to that found in the hM control (Table A.3).

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3.3 Activity test in vitro The M. luteus growth inhibition zone was observed after exposure to Tg, S-

rhLZ, nTg+rhLZ and hM samples, demonstrating a growth restraining effect, whereas no inhibition zone was observed for the nTg milk sample (Fig. A.2), as expected. 3.4 Bactericidal assay in vitro The bactericidal activity of the Tg milk group against M. luteus was demonstrated by a 7- to 8-fold reduction in CFU number observed after incubation in comparison to the Negative Control and to the nTg group. Additionally, the S-rhLZ

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and nTg+rhLZ samples completely inhibited the M. luteus growth. Samples used against S. sonnei showed no bactericidal effect, whereas E. feacalis showed a relative sensitivity to nTg+rhLZ and a discrete sensitivity to Tg and S-rhLZ samples when compared to the Negative Control and to the nTg Groups (Table A.4). 3.5 In vitro cell viability The evaluation of the sample dilution effects (1:5, 1:20, and 1:40) on cell

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viability showed that the 1:20 and 1:40 dilutions of nTg, S-rhLZ and nTg+rhLZ groups significantly reduced the viability after 24 h of exposure when compared to the other

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groups. After 48 h of exposure, however, cells exposed to Tg samples at 1:5 and 1:20 dilutions had lower viability when compared to controls and the nTg group. After 72 h,

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the Tg and nTg dilutions were statistically similar between one another and between groups, with all dilutions used in the control group showing increased cell viability.

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The rhLZ group showed a significantly decrease in cell viability when compared to the others group after 24, 48 and 72 h of culture (Fig. A.3).

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3.6 In vitro cell migration assay

The migration features of IEC-18 were not negatively affected by milk or the presence of human lysozyme. An increase in number of migrating cells was observed

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at the 1:20 dilution for all groups in comparison to controls. An increase in migrating

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velocity was observed at all dilutions for the nTg, Tg and S-rhLZ groups, being similar to controls when rhLZ is added to nTg. The migration distance was also increased at the 1:20 and 1:40 dilutions for the nTg and Tg groups, whereas a reduction was seen

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for the nTg+rhLZ group at 1:5 and 1:40 dilutions. No differences were detected between dilutions within groups (Table A.5). The 1:20 dilution for the nTg and Tg

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groups had significantly better migrating parameters for cells in culture (p<0.05) than controls.

3.6.1 In vitro cell migration assay in the presence of pathogens In general, and compared to controls, the presence of rhLZ in culture was favourable for in vitro cell migrating velocity and distance in the absence or presence of E. faecalis, S. aureus and E. coli, with no positive effect observed for S. sonnei. In the absence of selected pathogens at 1:20 dilution, the Tg groups improved the number of migrating cells, the nTg, Tg and S-rhLZ groups improved migrating

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velocity, and all groups increased migrating distance in cells in culture when compared to controls. In the presence of pathogens, the Tg milk, S-rhLZ and nTgrhLZ groups at 1:20 dilution promoted an increase in cell number (E. coli for Tg and S-rhLZ, and S. aureus for S-rhLZ), growth migration velocity (E. faecalis, S. aureus and E. coli) and distance (E. faecalis, S. aureus and E. coli) when compared to the control group, except for S. sonnei (p<0.05). Conversely, nTg samples showed lower

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cell numbers in the presence of E. faecalis and S. sonnei. In the presence of S. sonnei, S-rhLZ and nTg+rhLZ samples were associated with lower number of cells,

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with lower migrating distance (p<0.05) in the presence of nTg+rhLZ samples (Table A.6).

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3.7 Gene expression

No differences were observed between groups for the relative expression

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patterns of ZO-1 in cultured cells. However, albeit the IL-6 expression pattern was statistically similar between the control (PBS) and the rhLZ groups, its expression

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was increased in the group of cells exposed to nTg milk samples (nTg and nTg+rhLZ), with the Tg group being similar to all groups (Table A.7).

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4. DISCUSSION

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Goat (Capra hircus) milk contributes with approximately 2.4% of the global milk production, representing an important source of proteins in poor communities (FAO, 2013). Goat milk has more protein and mineral contents than human milk

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(FAO, 2013), and the easy adaptation of goats to environments of extreme poverty renders this species attractive for the production of recombinant proteins and

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nutraceuticals for human consumption. Consequently, boosting goat milk with human lysozyme may be an interesting strategy to combat undernutrition and endemic diseases. In this study, in view of such potentiality, we report the production of a transgenic goat lineage for expression of rhLZ in the milk and the characterization of the milk produced by the founder animal, which is an important step in testing the milk properties for future potential use of the rhLZ goat milk as nutraceutic product. Most analyses and parameters evaluated in this study indicated features of great potential for such application.

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Food composition is an issue of interest in the food industry for product development, quality control, or regulatory purposes, to ensure quality and safety, and the physicochemical milk analysis is an important requirement for human consumption (Nielsen, 2006). The proximate composition of foods includes the contents of macronutrients, specifically moisture, ash, lipid, protein, and carbohydrate (Nielsen, 2006). Fresh milk contains the natural ability to resist to pH changes, owing

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to its "natural acidity"; however, the action of bacteria that normally develop in raw milk produces more or less lactic acid (FAO, 2017). Physical and chemical properties

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of fresh milk from the transgenic goat founder was analysed during eight weeks, being widely similar to those observed in non-transgenic goats, and in compliance

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with the quality standards required by law for human consumption in Brazil (MAPA, 2000). The significance of the differences in fat and protein composition observed in

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the milk from the transgenic goat, although within normal values, may be related to the animal effect. Future studies including more animals are necessary to unravel

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those findings.

The rhLZ mean content in the milk from the transgenic goat was about 224 µg/mL, which represents 56% of the typical lysozyme concentration found in human

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breast milk (400 µg/mL; Ning et al., 2009; Yang et al., 2011). The mean lysozyme

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concentration values in human milk varies, as it decreases from the colostrum (370 µg/mL) to the transitional milk (270 µg/mL), and to the mature milk between 15 and 28 days (240 µg/mL) of lactation. However, lysozyme levels increase in mature milk al., 2001).

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from days 29-56 (330 µg/mL) up to days 57-84 (890 µg/mL) of lactation (Montagne et

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The rhLZ present in milk from transgenic goat has been characterized in previous studies as capable of modulating microbial population (Maga et al. 2006a, b; Scharfen et al. 2007). Experiments using the pig model, where animals were fed with transgenic goats’ milk containing rhLZ at 270 µg/mL, have shown positive impact on the gastrointestinal morphology, serum metabolites, lymphocyte populations, and increased anti-inflammatory cytokine expression (Brundige et al. 2008, 2010; Cooper et al. 2011). In fact, after lactation, the transgenic goats kept a lower level of somatic cell counts in milk in comparison to non-transgenic goats. Such measure is used to

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monitor infection of the mammary gland, indicating a healthier udder. Such benefit is probably associated with the antimicrobial activity of lysozyme, resulting in improved milk safety and animal welfare (Carvalho et al., 2012; Maga et al., 2006a; 2006b). The analysis of the antimicrobial effect of the milk showed that Tg, S-rhLZ and nTg+rhLZ samples had antibacterial and bactericidal activity against M. luteus, a Gram-positive strain normally used as reference organism for studies on lysozyme

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activity (Diler et al., 2011). The other Gram-positive strain, E. faecalis, showed discreet sensitivity to the presence of Tg and S-rhLZ samples, whereas the CFU

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number decreased significantly in the nTg+rhLZ group, which contained rather higher rhLZ amounts. The high resistance of E. faecalis to the action of lysozyme is already

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well known, which allows survival of the pathogen in the mammalian host (Benachour et al., 2012; Varahan et al., 2013).

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Despite the effective bactericidal effect against Gram-positive strains, lysozyme was shown effective against Gram negative strains only when associated

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with other substances, such as lactoferrin (Cerven et al., 2008). Both proteins are present in mammalian milk, presenting synergistic antimicrobial properties. Lactoferrin binds to lipopolysaccharides on the outer bacterial membrane, thus

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contributing to membrane disruption and allowing lysozyme to have better access to

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the peptidoglycan layer in Gram negative bacteria (Leitch and Wilcox, 1999). Therefore, as expected for the Gram negative bacteria S. sonnei, no sensitivity was verified in the sole presence of lysozyme. Our results corroborate with other studies

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(Masschalck et al., 2001), in which the presence of lysozyme (at concentrations of 10 and 100 µg/mL) resulted in no variation or delay in growth curves for any of the six

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tested types of Gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri). The potential antimicrobial effect of lysozyme, with positive modulation of the microbial population, has been previously described by multiple studies. Moreover, the stability of LZ to heat treatments and acidic conditions ensures integrity and effectiveness along the gastrointestinal tract (Masschalck et al., 2001; Mcinnins et al., 2015), which makes the transgenic milk containing rhLZ a promising nutraceutical

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product, suitable for consumption as fresh raw or pasteurized milk. Furthermore, the rhLZ can be purified from milk for use as supplement to oral rehydration solutions. In fact, the use of transgenic goat milk as potential nutraceutic product or as supplement to oral rehydration therapy was reported by Carvalho et al. (2012), which demonstrated that the presence of nutrients in culture medium, in special proteins and fat of goat milk, can be beneficial to intestinal cell proliferation in vitro. Results in

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the cell assay obtained in this study showed alterations in cell viability in the presence of diluted samples. After 24 h, the 1:20 dilution of Tg milk sample was

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significantly similar to the control group and different from nTg, showing positive effect on cell migration.

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Besides the nutrient source, the antimicrobial ability of the transgenic milk was evaluated by comparing cell migration in the presence of 1:20 sample dilutions,

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24 h after the in vitro inoculation of pathogenic bacteria. Improved cell proliferation was observed in cultures exposed to samples from the Tg group along with E.

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faecalis, S. aureus (statistically similar to S-rhLZ and nTg+rhLZ), and S. sonnei. Although Tg milk samples showed no bactericidal effect against E. faecalis, the presence of milk per se was beneficial to cell proliferation. The lower cell migration

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effect observed after exposure to samples from the S-rhLZ and nTg+rhLZ groups

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may be due to higher LZ protein activity in the Tg milk than in the rhLZ-supplemented milk, as the lysozyme produced in vivo in the milk through the mammary gland has been previously shown to be more potent as antimicrobial agent than the milk

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supplementation with purified protein (Carvalho et al., 2012; Maga et al., 2006a). Tight junction proteins are mainly responsible to function as intestinal

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mucosa barrier against macromolecular diffusion (Zhang and Gou, 2009). The presence of samples in cell culture did not alter relative gene expression pattern for ZO-1 protein, suggesting no effect on intestinal epithelial permeability in cultured cells. Conversely, the expression pattern of pro-inflammatory cytokine IL-6 by IEC-18 was increased in the presence of milk samples (nTg, Tg and nTg+rhLZ), which suggests that transgenic or non-transgenic goat milk may elicit an intestinal inflammatory process under in vitro conditions (Atreya and Neurath, 2005). However, the in vivo analysis of gut regions (duodenum and ileum) of pigs that received an

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association of pasteurized transgenic goat milk and pasteurized non-transgenic cow milk, with a final concentration of 135 µg/mL rhLZ, showed no increase in the expression of IL-6 (Cooper et al., 2013). More studies are needed to evaluate the in vitro and in vivo effects of the Tg and nTG milk on the intestinal epithelium, on proand anti-inflammatory molecules, and on gut permeability. 5. CONCLUSIONS

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This study focused on the characterization of the goat milk from a transgenic line that expresses human lysozyme in the milk. The physicochemical properties of

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milk samples from transgenic and non-transgenic goats were similar and in compliance with the minimum quality requirements for human consumption (MAPA,

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2000). Active rhLZ is present in the milk of the transgenic goat line at amounts comparable to values found in human breast milk, also demonstrating in vitro

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antibacterial and bactericidal effects. The transgenic milk did not alter or affect the in vitro viability, proliferation and migration of intestinal epithelial cells (IEC-18) in

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culture. This study was carried out in the Brazilian Northeast region, where the number of infant deaths due to malnutrition and infectious diseases is high. The rhLZ produced in milk has the potential to be used in infant formulas or in natura, as

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nutraceutic product, with the nutritional and medical values comparable to human

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milk. Additional in vitro and in vivo studies using animal models are under way, which are absolutely necessary to be performed to ensure safety as well as the observation of beneficial effects with no detrimental or unintended consequences of the use of

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transgenic goat milk containing rhLZ.

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ACKNOWLEDGEMENT This study was funded by the Studies and Projects Funding Agency

(Financiadora de Estudos e Projetos – Finep) of the Ministry of Science and Technology of Brazil, under grant number 0460.08. CONFLICT OF INTEREST Igor de Sá Carneiro, José Nilson Rodrigues de Menezes, Julyana Almeida Maia, Victor Bruno Soares de Oliveira, James D. Murray, Elizabeth A. Maga, Marcelo Bertolini and Luciana Relly Bertolini state that there are no conflicts of interest.

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Brundige D.R., Maga E.A., Klasing K.C., Murray J.D. (2010). Consumption of pasteurized human lysozyme transgenic goats’ milk alters serum metabolite profile in

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Cerven D., DeGeorge G., Bethell D. (2008). 28-Day repeated dose oral toxicity of recombinant human apo-lactoferrin or recombinant human lysozyme in rats. Regulatory Toxicology and Pharmacology, https://doi:10.1016/j.yrtph.2008.03.007. Cooper C.A., Brundige D.R., Reh W.A., Maga E.A., Murray J.D. (2011) Lysozyme transgenic goats’ milk positively impacts intestinal cytokine expression and morphology. Transgenic Research, https://doi:10.1007/s11248-011-9489-7.

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Lu D., Li Q., Wu Z., Shang S., Liu S., Wen X., Li Z., Wu F., Li N. (2014). High-level recombinant human lysozyme expressed in milk of transgenic pigs can inhibit the growth of Escherichia coli in the duodenum and influence intestinal morphology of sucking pigs. Plos One, https://doi:10.1371/journal.pone.0089130. Maga E.A., Cullor J.S., Smith W., Anderson G.B., Murray J.D.

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Wang H., Wu H., Wang K., Cao Z., Yu K., Lian L., Lian Z. (2016). Expression of recombinant human lysozyme in transgenic chicken promotes the growth

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Yang B., Wang J., Tang B., Liu Y., Guo C. (2011). Characterization of bioactive

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Figures

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Fig. A.1. Representative Western blot for the identification of the hLZ in samples of non-transgenic goat milk (nTg), transgenic goat milk (Tg), human breast milk (hM)

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and S-rhLZ solution (270 µg/mL of commercial rhLZ from Sigma-Aldrich diluted in saline) used as standard control. L=protein ladder (ProSieve® Color Protein Maker -

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Lonza).

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Fig. A.2. In vitro antimicrobial activity (linear measurement, in mm, and surface area, in mm2, of the inhibition zone) of (nTg) non-transgenic goat milk (0,00 mm, 0,000

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cm2), (Tg) transgenic goat milk (1,29 mm, 0,502 cm 2), (hM) human breast milk (2,30 mm, 0,73 cm2), and commercial rhLZ diluted (S-rhLZ) in saline (2,39 mm, 0,830 cm2)

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or (nTg-rhLZ) in nTg milk (1,51 mm, 0,670 cm2) against Micrococcus luteus strain.

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Fig. A.3. In vitro cell viability in the presence of 1:5, 1:20 and 1:40 dilutions of nontransgenic goat milk (Tg), transgenic goat milk (nTg), human breast milk (hM), and commercial rhLZ diluted in saline (S-rhLZ) or in nTg milk (nTg+rhLZ) exposed for 24

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(A), 48 (B) and 72 h (C) in comparison to LZ-free control sample.

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different superscripts for each dilution differ (p<0.05).

a – d:

Columns with

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Tables Table A.1. Sequence of PCR primers, melting temperatures (°C) and GenBank accession number for gene products analysed in cells in culture exposed to nontransgenic goat milk (nTg), transgenic goat milk (Tg), human milk (hM), and commercial rhLZ diluted in saline (S-rhLZ) or in nTg milk (nTg+rhLZ).

temperature (°C)

accession

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GenBank

F GAGGCTTCAGAACGAGGCTATTT ZO-1

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R CATGTCGGAGAGTAGAGGTTCGA

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Primer sequence 5’-3’

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Gene

Melting

number

81.7

NM_001106266.1

77.1

NM_012589.2

78.2

NM_031144.3

F ACCACCCACAACAGACCAGT IL-6

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R ACAGTGCATCATTCGCTGTTC F CCCTGGCTCCTAGCACCAT β-Actin

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R GAGCCACCAATCCACACAGA

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Table A.2. Physicochemical analysis of milk samples from the transgenic founder goat (Tg) and two non-transgenic control goats (nTg and nTg2). Results represent mean values obtained for milk collected during eight consecutive weeks starting at the third week of lactation. nTg

nTg2

MAPA (2000)1

Lactic acid (%)

0.16a

0.20a

0.18a

0.13 – 0.18

Density at 15 °C (g/mL)

1.02a

1.01a

1.00a

1.02 – 1.03

Cryoscopic constant (°H)

-0.55a

-0.55a

-0.55a

-0.55 – -0,585

2.7b

2.4b

< 2.9

2.2a

Protein (%)

3.5a

2.9b

2.9b

> 2.8

Lactose (%)

4.5a

4.3a

4.4a

> 4.3

8.4a

8.2a

8.2a

> 8.2

6.5a

6.4a

6.5a

--*

0.7a

0.8a

0.9a

> 0,7

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Fat (%)

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Tg

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Standards by

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Non-fat solids (%)

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pH Ashes (%) a,b:

Numbers with different supercripts in the row differ (p<0.05) MAPA recommendations (Ministério da Agricultura, Pecuária e Abastecimento, Brazil). *Not described.

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1

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Table A.3. Quantification of recombinant human lysozyme (rhLZ) present in the milk from the transgenic founder (Tg), at the third week of lactation, in comparison to milk from non-transgenic goat (nTg), human breast milk (hM) and commercial rhLZ diluted into saline solution (S-rhLZ). Results represent the

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semi-quantitative conversion of the lysozyme band intensity assessed from the WB relative to the S-rhLZ group (270 µg/mL commercial rhLZ), analysed by the Image J

Western nTg

Tg, µg/mL

1

-

2

-

3

-

Mean

-

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software.

hM, µg/mL

S-rhLZ*, µg/mL

230

226

270

221

231

270

221

221

270

224

226

270

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*Saline solution with known amounts of commercial rhLZ (270 µg/mL rhLZ).

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Table A.4. Evaluation of the bactericidal activity of non-transgenic (nTg) and transgenic (Tg) milk samples, commercial rhLZ diluted in saline solution (S-rhLZ) or in nTG milk (nTg+rhLZ) samples against pathogenic bacteria in culture . nTg

Tg

S-rhLZ

nTg+rhLZ

M. luteus

28.0 ± 3.2a

32.3 ± 11.9a

4.0 ± 1.2b

0.0 ± 0.0c

0.0 ± 0.0c

S. sonnei

117.7 ± 18.8a

140.3 ± 11.5a

111.3 ± 5.7a

105.3 ± 10.3a

90.7 ± 5.6a

E. faecalis

165.3 ± 14.4a

142.0 ± 18.2a

113.7 ± 12.8ab

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Means in the row with different superscripts differ (p<0.05).

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Control

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CFU

110.7 ± 14.7ab

63.3 ± 15.0b

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Table A.5. Number of migrating cells, velocity and distance of cell migration in vitro in the presence of nTg, Tg, S-rhLZ and nTg-rhLZ samples diluted at 1:5, 1:20 and 1:40.

Migrating velocity

Migrating distance

11.3 ± 0.5a

471.6 ± 12.3a

Tg

S-rhLZ

nTg+rhLZ

1:5

23.3 ± 0.9aA

23.7 ± 1.2aA

23.7 ± 0.3aA

22.6 ± 1.2aA

1:20

27.3 ± 2.0bA

32.0 ± 1.5bA

27.7 ± 0.3bA

28.0 ± 2.1bA

1:40

23.3 ± 0.7aA

25.0 ± 1.0aA

23.7 ± 0.7aA

23.0 ± 0.6aA

1:5

22.3 ± 1.6bA

23.3 ± 0.0bA

18.9 ± 1.9bA

15.2 ± 1.0aA

1:20

22.2 ± 2.5bA

24.7 ± 2.6bA

19.0 ± 0.0bA

17.0 ± 1.2abA

1:40

21.7 ± 2.6bA

23.6 ± 2.6bA

19.32 ± 1.3bA

15.8 ± 1.5aA

1:5

518.0 ± 55.2aA

537.2 ± 32.5aA

449.1 ± 26.5abA

381.2 ± 33.6bA

1:20

550.4 ± 55.3bA

593.7 ± 62.7bA

473.0 ± 33.6aA

407.5 ± 28.6aA

505.6 ± 52.0bA

567.8 ± 61.8bA

438.3 ± 32.7abA

301.2 ± 33.6cA

1:40 a,b:

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22.0 ± 0.6a

nTg

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cells, n

Dilution

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Migrating

Control

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Migration

Means in the same row with different superscripts differ (p<0.05). Means in the same column with different superscripts, for each migration assay, differ (p<0.05).

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Table A.6. Number of migrating cells, velocity and distance of cell migration in vitro in the presence of nTg, Tg, S-rhLZ and nTg-rhLZ samples diluted at 1:20 and

Tg

S-rhLZ

nTg+rhLZ

Absent

22.0 ± 0.6a

27.3 ± 2.0ab

34.7 ± 41.2b

27.7 ± 0.3ab

28.0 ± 2.1ab

E. feacalis

21.7 ± 0.3ab

16.0 ± 0.0b

23.0 ± 3.0a

25.0 ± 1.2a

23.3 ± 1.2a

S. aureus

21.3 ± 0.3ab

18.0 ± 1.2a

26.3 ± 1.3bc

30.67 ± 2.7c

26.7 ± 2.3bc

E. coli

20.7 ± 0.3a

24.3 ± 2.4ab

28.3 ± 1.2bc

22.7 ± 1.5ab

29.7 ± 0.7c

S. sonnei

20.3 ± 0.3ab

16.7 ± 1.9b

23.3 ± 1.8a

14.0 ± 1.2b

12.7 ± 3.2b

Absent

11.3 ± 0.5a

23.2 ± 2.3b

24.0 ± 2.6b

21.4 ± 1.7b

17.0 ± 1.2ab

E. feacalis

11.1 ± 0.1a

9.3 ± 0.0a

17.2 ± 1.6b

19.3 ± 0.8b

17.4 ± 0.4b

S. aureus

11.3 ± 0.0a

15.2 ± 2.4ab

21.6 ± 0.5b

22.1 ± 1.5b

21.6 ± 1.5b

E. coli

11.0 ± 0.1a

16.0 ± 2.6ab

20.1 ± 5.1b

17.5 ± 0.8b

18.8 ± 1.0b

S. sonnei

11.2 ± 0.0ab

15.7 ± 1.5a

17.7 ± 2.1a

12.4 ±1.6ab

7.2 ± 1.0b

Absent

271.6 ± 12.3a

555.7 ± 55.0b

593.8 ± 62.7b

473.0 ± 33.6b

407.6 ± 28.6b

270.8 ± 0.5a

222.6 ± 0.0a

412.1 ± 39.4b

412.0 ± 39.4b

417.6 ± 10.1b

270.2 ± 0.2a

394.3 ± 66.0ab

507.6 ± 10.9b

530.0 ± 36.0b

518.2 ± 35.7b

E. feacalis S. aureus

CE

Migrating distance

RI

SC

E. coli

269.6 ± 0.2a

383.6 ± 62.1ab

410.0 ± 60.5b

418.87 ± 18.6b

450.8 ± 24.4b

S. sonnei

269.2 ± 0.2ab

402.0 ± 37.3a

425.6 ± 51.2a

297.6 ± 37.9ab

173.6 ± 23.2b

AC

a b:

PT

nTg

NU

Migrating velocity

Control

D

Migrating cells, n

Pathogens

PT E

Migration assay

MA

pathogenic bacteria.

Means in the same row with different superscripts differ (p<0.05).

ACCEPTED MANUSCRIPT 32

Table A.7. Relative expression levels for the ZO-1 and IL-6 genes in IEC-18 in culture after incubation in the presence of nTg, Tg, S-rhLZ and nTg-rhLZ samples.

Expression

nTg

Tg

ZO-1

1.2 ± 0.1

a

0.8 ± 0.1

IL-6

1.8 ± 0.8

b

12.9 ± 0.3

a

S-rhLZ

0.8 ± 0.2

a

7.6 ± 3.5

a

ab

1.1 ± 0.1 1.7 ± 0.3

CE

PT E

D

MA

NU

SC

RI

Means within the same row with different superscripts differ (p<0.05).

AC

a,b:

Control

PT

Gene

nTg+rhLZ

a

0.7 ± 0.1

a

b

10.3 ± 0.5

a

ACCEPTED MANUSCRIPT 33

AC

CE

PT E

D

MA

NU

SC

RI

PT

Graphical abstract