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Bioactive Constituents, Metabolites, and Functions

Risks Related to High-Dosage Recombinant Antimicrobial Peptide Microcin J25 in Mice Model: Intestinal Microbiota, Intestinal Barrier Function and Immune Regulation Haitao Yu, Lijun Shang, Xiangfang Zeng, Ning Li, Hongbin Liu, Shuang Cai, Shuo Huang, Gang Wang, Yuming Wang, Qinglong Song, and Shiyan Qiao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03405 • Publication Date (Web): 09 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

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Journal of Agricultural and Food Chemistry

Risks Related to High-Dosage Recombinant Antimicrobial Peptide Microcin J25 in Mice Model: Intestinal Microbiota, Intestinal Barrier Function and Immune Regulation Haitao Yu,†, ‡ Lijun Shang,†, ‡ Xiangfang Zeng,†, ‡ Ning Li,†, ‡ Hongbin Liu,†, ‡ Shuang Cai,†, ‡ Shuo Huang,†, ‡ Gang Wang,†, ‡ Yuming Wang,

†, ‡

Qinglong Song

‡, ||

and Shiyan

Qiao†, ‡,*   †State Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural University, Beijing 100193, P.R. China   ‡Beijing Bio-feed additives Key Laboratory, Beijing 100193, P.R. China ||Bejing Longkefangzhou Bio-Egineering Technology Co.,Ltd, Beijing 100193, P.R. China

*S.Q., (Tel: +86 10-62733588; Fax: +86 10-62733688. E-mail: [email protected])

ACS Paragon Plus Environment 1


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ABSTRACT: Antimicrobial peptide (AMP) can be a promising alternative in various

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domains. However, the further risk information is required. In this study, mice were

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orally administrated different dosages of recombinant AMP microcin J25 (4.55, 9.1 and

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18.2 mg/kg; MccJ25) for 1 week, which its toxicity risk impacts were examined. We

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evidenced that middle-dosage administration mice had a lower inflammation, better body

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weight and ameliorated mucosal morphology, accompanied by reduced intestinal

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permeability and tighter intestinal barrier. Fecal microbiota composition analysis in

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middle or low-dosage mice revealed Bifidobacterium count was increased and coliform

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bacteria count was decreased, and increased in short-chain fatty acid levels.

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Unexpectedly, there was a risk that high-dosage mice increased intestinal permeability

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and imbalance of intestinal bacteria. Taken together, these data indicated a safe threshold

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for usage of MccJ25 in clinical practice. Such studies can effectively enhance the safety

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of various aspects like food preservative and drug.  

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KEYWORDS: recombinant Microcin J25, toxicity risk, intestinal inflammation, gut

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microbiota, intestinal permeability, BALB/c mic


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INTRODUCTION

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Chemical antimicrobial weapons are found throughout the entire living kingdom,

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from animals, plants to microorganism.1 Lactobacillus secrete bacteriocins, microproteins

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from Enterobacteria, and these bacteria basically produce more antibiotics than

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Eukaryota in number. On a certain scale, microcins are similar to the survival

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characteristics of Gram-positive bacteria, for example, they can perform antimicrobial

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activities (minimum inhibitory concentrations, MICs) within the nanomolar range of the

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pathogens they near, and the effects is overwhelming.2 Microcins are a class of small

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ribosomally-synthesized antimicrobial peptides (AMPs) produced by enterobacteria.3

25

Ribosomally-synthesized natural antibiotics contribute to innate immunity in higher

26

organisms and to competitive advantage in microbial communities.4

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A considerable number of studies on biochemical and structural characteristics have

28

been carried out on the scale of microcins, which are also reflected in the relevant

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mechanisms of action and the categories of producing strains.5,6 For example, microcin

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J25 (MccJ25) is a member of the lasso peptides class. It is a, plasmid-encoded, small

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ribosomally-synthesized AMP, which is separated from a fecal strain of E. coli

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containing 21 amino acid residues.7 MccJ25 essentially targets Salmonella species and E.

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coli, with MICs in the nanomolar range (between 2 and 50 nM).8 On the other hand,

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biological activity of MccJ25 is not affected in complex biological matrices.8 Thereby,

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based on its high antibacterial capability, and the huge stability of the appealing lasso

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structure, MccJ25 has attracted considerable interest for further applications.

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Compared with chemical synthesize composite, biogenic expression was invariably

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performed to produce AMPs around the world, E. coli is the worldwide used main



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bacteria for a large amount of expression of AMPs.9,10 Therefore, engineered MccJ25

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high-efficiency expression vector is existing in our laboratory utilizing recombinant DNA

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(Fig. S1). Recombinant AMP MccJ25 has been displayed strong antibacterial activity

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against Gram-negative bacteria (Table S1 and Fig. S3). In our previous study, dietary

43

supplementation with recombinant MccJ25 as feed additive have beneficial function not

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only decrease E. coli in the gut but also maintain micro-ecosystem homeostasis, and

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reduction in intestinal permeability and provokes anti-inflammatory responses.11

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Kaznessis et, al. also has been shown that engineered probiotic E. coli Nissle 1917, to

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large-scale produce the antimicrobial peptide, Microcin J25, which can clear the

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Salmonella rapidly in the ceca of birds.10

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Hence, MccJ25 is a promising alternative candidate as drug in clinical practice. A

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previous study has been shown mice treated with 3 mg/mouse MccJ25 by intraperitoneal

51

injection presented a marked antimicrobial activity in vivo, and conferred protection

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against infections caused by Salmonella Newport in mice model.8 However, the peptide

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lacked dosage toxicity risk in vivo. As drug application, the route of administration has

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something in common with humans over the utilization12,13 and toxicological studies

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including animals are a major component of safety assessment.14 In vitro recombinant

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MccJ25 has no cytotoxicity towards IPEC-J2 cells by CCK-8 and lactate dehydrogenase

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assays, furthermore, recombinant MccJ25 protects against intestinal disruption and

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proinflammatory response caused by ETEC K88, nominated the employment of

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recombinant MccJ25 as a novel therapeutic or prophylactic drug to lessen pathogen

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infection in human, food or animals.15 But one of the requirements for the application of

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recombinant AMP in foods and drug would be the evaluation of its in vivo toxicity risk.


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Hence, the study aims to determine the dosage effects of the recombinant AMP MccJ25

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in healthy mice model via evaluation of intestinal microbiota, intestinal permeability and

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immune regulation.

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MATERIALS AND METHODS

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Preparation of Recombinant MccJ25. The recombinant MccJ25 method was

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performed in our laboratory with a highly efficient expression vector as described in Yu

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et al. (2018), with some modifications: DNA reorganizing technology and

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codon-optimized genes coding that implements functions are the way to realize pMJ25

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expression (Fig. S1)15. Then, the plasmid, pMJ25, was converted into indicated bacteria

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E. coli BL21. The E. coli BL21 was cultured in sucrose-compound based medium. After

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hatch, recombinant indicated bacteria cell supernatant was collected by centrifuge. The

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recombinant MccJ25 was stored in the form of lyophilized powder at -20 °C. The

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antimicrobial activities of recombinant MccJ25 (Table S1 and Fig. S3) and approach of

75

peptide purification were added to the Supplementary Information section (Fig S2).

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Animals and Design of in vivo Experiments. The Institutional Animal Care and

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Use Committee of China Agricultural University issued rules that laboratory

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Animals-Guideline of Welfare and Ethics of China (ICS 65.020.30) which guided the

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process of this study. The protocol was approved by the same institution.

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Female BALB/c mice (6-7 weeks) used in this study were purchased from HFK

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Bioscience Co., Ltd. (Beijing, China). All mice were kept in a laminar flow cabinet and

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individually housed in the same temperature- and humidity- automatic dominated room

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on a 12 h light/dark cycle. Throughout the experimental period, feed and water are

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available. Before the mice experiment, 60 mice were randomly assigned to 4



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experimental groups (15 mice each treatment group in 20 cages), after the 3-d

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equilibration period, among which 4 groups were daily given a gavage of 0.3 mL sterile

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saline (control group) or 4.55 mg/kg, 9.1 mg/kg, and 18.2 mg/kg (mg per kg body weight

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[BW]) of MccJ25 in a total volume of 0.3 mL sterile saline, respectively. The dosages of

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MccJ25 were calculated based on our previous study.11 The treatment was conducted 7 d

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until they were euthanized. During the treatment period, all mice can obtain sterilized

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conventional feed and distilled water at any time.

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Samples Collection and Measurements. Body weight, rectal temperature and

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survival rate were monitored for 7 d. After 1-week treatment, fresh stool samples from 6

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mice per treatment (2 mice per cage) were collected in sterile tubes and served as scaling

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fecal bacterial counts and short chain fatty acids (SCFAs) analysis. After obtaining the

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fresh stool samples, which are immediately placed on ice (1-2 h) and transported to the

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laboratory. On the same day, further studies will be carried out in the laboratory. The

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fresh stool samples collected for short SCFAs analysis were freeze-dried at -80 °C.

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After 1-week treatment, the mice were euthanized, the jejunum and colon tissues

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were gathered and stored in liquid nitrogen presently, and then placed at -80 °C for

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subsequent analysis. Blood samples were picked up, and serum was acquired after

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centrifugation (Biofuge22R; Heraeus, Hanau, Germany) and then kept at -80 °C until

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analysis.

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Measurement of D-lactate and Diamine Oxidase in Serum. Serum specimens

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were thawed and mixed well at the largest scale before measuring. Serum levels of

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D-lactate and diamine oxidase (DAO) were determined, using commercially available

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mouse ELISA kits (Beijing Siliangchangyuan Biotechnology Co. Ltd. Beijing, P.R.


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China) by the manufacturer’s protocol. By a microplate reader, applying MPM 6.1

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software (Bio-Rad Laboratories, Hercules, CA) can provide the timey data.  

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Determination of Cytokines Concentration in Tissues. Tissues specimens were

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unfrozen and thoroughly mixed immediately before measuring. The concentrations of

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cytokines IL-6 (interleukin-6), tumor necrosis factor-α (TNF-α), and IL-10 in jejunum

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and colon were determined using commercially available mouse ELISA kits (Nanjing

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Jiancheng Bioengineering Institute, Jiangsu, China) based on standard procedures

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described by the manufacture. Total protein originated from the jejunum and colon with a

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lysis buffer (KeyGEN BioTECH, Nanjing, China), and the proportion of protein in the

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supernatant was measured utilizing a BCA protein assay kit (KeyGEN BioTECH),

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complying with the manufacturer’s instructions. By a microplate reader, applying MPM

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6.1 software (Bio-Rad Laboratories, Hercules, CA) can provide the timely data.

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Real-time PCR for Gene Expression Analysis. StepOnePlus real-time PCR system

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(Applied Biosystems) method used was as previously described in Liu et al. (2017), with

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some modification: The transcript levels of IL-6, IL-10 and TNF-α were determined by,

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which was displayed with SYBR Green master. Total RNA originated from jejunum and

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colon tissues utilizing TRIzol reagent (Invitrogen, Carlsbad, CA) and cryogenic lapping

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instrument (JXFSTPRP-CL, Shanghai, China). The purity and yield of the RNA were

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evaluated using NanoDrop 2000 (Thermo Fisher Scientific). RNA (1 μg) was used to

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generate cDNA in a total volume of 200 μl. The primers for the real-time PCR are shown

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in Table 1. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was built as internal

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reference in this study. The relative mRNA expression of the aimed genes was

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determined using the 2-∆∆Ct method.16



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Western Blot Analysis. Tissues for analysis of abundance of proteins by using

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Western blot as described above.17 Membranes were cultured with a primary antibody

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[GAPDH, β-actin, IL-10, TNF-α, claudin-1 and occludin (Santa Cruz Biotechnology,

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USA)] at 4 °C overnight and then flushed 3 times with TBST for 15 min. The membranes

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were then cultured with a horse radish peroxidase (HRP)-conjugated secondary antibody

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(Applygen Technology, Inc., Beijing, China) at room temperature for 1 h. The signal was

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measured with the Western Blot Luminance Reagent (Applygen, Beijing, China) through

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an ImageQuant LAS 4000 mini system (GE Healthcare Bio-sciences AB, Inc., Sweden),

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and gel-imaging system with Image Quant TL software (GE Healthcare Life Science)

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was used for generating statistics.

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Histological and Histopathology Analysis. The middle jejunum and middle colon

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tissues were harvested and enclosed in 4% paraformaldehyde. It used xylene to seal

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5-millimeter thick intestines and color it with H&E. By the DM3000 microscope (Leica

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Microsystems, Wetzlar, Germany), we could gain the images. Image-Pro software can

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measure the villous height (V) and crypt depth (C) of the jejunum as previously

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described.16 From the normal level (0) to the most severe level (4), the pathological

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changes of the tissue can be divided into four types: normal, edema, inflammation, and

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epithelial damage utilizing a scoring system reported as described above.18

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Fecal Microflor Composition. Each fresh fecal sample (1 g) was distributed in 9 ml

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sterile saline liquor well-proportioned, and 100 μl of proper dilutions of the homogenates

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were plated onto selective media (Beijing Land Bridge Technology Co., China) to count

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colonies. The groups of fecal bacteria investigated and the selective mediums adopted

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were as follows: 1) Coliforms, MacConkey’s agar plates. Some colonies obtained


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growing on MacConkey’s agar plates were took into account the enterobacteria-like

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bacteria should be geared to the family Enterobacteriaceae. 2) Clostridium spp. in

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tryptose sulphite cycloserine agar (TSCA) 3) Lactobacilli, MRS agar; 4) Bifidobateria,

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Tryptone-phytone Yeast (TPY) agar; 5) Total anaerobic bacteria, Tryptic soy (TS) agar.

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The plates for VRB agar were cultured for 24 h at 37 ℃ under aerobic conditions. On the

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contrary, TSCA, MRS agar, TPY agar and TS agar plates were incubated under anaerobic

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conditions for 48 h at 37 ℃. Results were presented as log10 CFU/g feces or digesta, and

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all data were performed in triplicate.  

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Measurement of SCFAs in Fecal. The extraction method used was as described in

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Yu et al. (2017), with some modifications: The fecal contents was weighed (01-0.15 g).

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Then, the fecal samples were mixed by ultrasonic bath for 30 min. The mixture was

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centrifuged to obtain the suspension, which was diluted with water. A 25 μL extraction

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sample was analyzed for the SCFAs, including acetate, propionate and butyrate by a high

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performance ion chromatography (ICS-3000, Dionex, USA). These SCFAs were

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separated from an AS11 analytical column (250 × 4 mm). The gradient conditions of

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AG11 guard column was similar to Yu et al. (2017). The gradient was performed with

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potassium hydroxide.

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Statistical Analyses. Statistical analysis was performed using one-way ANOVA

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procedure of SAS system (version 9.2, SAS Institute, Inc., Cary, NC). The significant

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difference was measured by Turkey’s multiple comparisons test. All indicators were

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studied in every repeat. Statistical results were represented by the mean and standard

175

 error, P value < 0.05 was considered to be statistically significant.  

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RESULTS



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Body Weight and Clinical Symptoms. The first step in our study was to test if

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recombinant MccJ25 (Fig. S1) had the antimicrobial activities against Gram-negative

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bacteria, such as the E. coli, Salmonella and Shigella. As we expected, recombinant

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MccJ25 exerted strong antimicrobial activity against E. coli, multidrug resistant bacteria

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E. coli AZ1, Salmonella pullorum and Shigell flexneri (Table S1 and Fig. S3). Then, with

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oral administration of 4.55 and 9.1 mg/kg MccJ25 for 7 d, they improved the final body

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weight with a non-different survival rate. However, mice-treated 18.2 mg/kg MccJ25

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significantly decreased the body weight at d 7 (P < 0.01, Fig. 1A and C). In addition,

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there was no variance of rectal temperature among control, 4.55 and 9.1 mg/kg MccJ25

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groups (Fig. 1B). On d 7 (P < 0.01), oral administration of 18.2 mg/kg MccJ25

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significantly reduced the mice rectal temperature, respectively, while no mortality was

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observed (Fig. 1B and C).

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Intestinal Morphology and Histopathology Evaluation. To reveal the reason for

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the improved body weight and test the risk of recombinant MccJ25 in tissue of jejunum

191

(Fig. 2A) and colon (Fig. 2C), mucosal morphology and histopathology were observed

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and determined. Only 9.1 mg/kg MccJ25-treated mice had a marked influence on the

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villous height (V) and crypt depth (C) and the V/C in jejunum compared with control

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group (P < 0.05) (Fig. 2C, D and E). When treating the mice with 18.2 mg/kg MccJ25,

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the V and C were lower (P < 0.05) than the 9.1 mg/kg MccJ25 one and were not

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distinguishable from that of control group (Fig. 2B and C), while mice-treated 18.2

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mg/kg MccJ25 significantly reduced the V/C in jejunum compared with control group (P

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< 0.05) (Fig. 2D). Notably, compared with control mice, recombinant MccJ25-treated

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mice showed no apparent jejunum mucosal morphology damage, but caused mucosal


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morphology damage in colon (Fig. 2F). Systematic Inflammation in Jejunum and Colon. To further evaluate if

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recombinant MccJ25 induced inflammatory responses risk caused by regulating pro-

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inflammatory secretion and expression. We examined the pro-inflammatory cytokines

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TNF-α, IL-6 and anti-inflammatory cytokines IL-10 in jejunum and colon. Interestingly,

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the secretion levels of cytokines in jejunum measured by ELISA revealed no differences

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among all treatments (Table 2). However, in colon tissues (Table 2), 18.2 mg/kg MccJ25

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treated mice significantly increased the pro-inflammatory cytokines TNF-α or IL-6

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secretion levels, as well as decreased the IL1-0 concentration compared to control group

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(P < 0.01). Concentration of TNF-α was markedly decreased (P < 0.01) after oral

210

administration of 9.1 mg/kg MccJ25 compared with control group, but the difference was

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not observed in IL-6 (P > 0.05). There was a significant difference (P < 0.01) in IL-10

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concentration in 9.1 mg/kg MccJ25 treatment compared to other treatment groups.

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To test the expression of mRNA of cytokines in colon tissues (Fig. 3A), we did a

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similar assay using RT-PCR. The results were consistent with the ELISA data. Based on

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the ELISA and RT-PCR results, the Western blot assay was carried out to further

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testifying the effect of administration of MccJ25 orally on TNF-α and IL-10 in colon

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tissues (Fig. 3B and C). As we expected, 18.2 mg/kg MccJ25-treated mice showed a

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significantly decreased the protein level of IL-10 (P < 0.05) and increased the TNF-α (P

219

< 0.01) protein level in colon tissues. Compared with the control treatment, oral

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administration of 9.1 mg/kg MccJ25 significantly increased IL-10 protein level (P
0.05).  

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Intestinal Permeability. Based on the above experiments, we also analyzed the effect



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of MccJ25 on intestinal permeability in serum. The

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(DAO) were assayed (Table 3). Compared with control groups, 18.2 mg/kg MccJ25

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treatments significantly increased the serum D-Lactate concentration (P < 0.01). Although

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the decreased level of the 4.55 and 9.1 mg/kg MccJ25 groups was not greater than the

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control, its decreasing was also significant (P < 0.05) on the basis of 18.2 mg/kg MccJ25

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group. These results suggested that and 9.1 mg/kg MccJ25 can improve epithelial barrier

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integrity.

D-Lactate

and diamine oxidase

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The Expression of Claudin-1 and Occludin in Jejunum and Colon. In order to

231

assess the MccJ25 cure to the signal pathway of intestinal barrier, the expression of

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mucosal barrier protein, including occludin, claudin-1 was tested through western blot

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(Fig. 4A and B). The expression of claudin-1 in jejunal mucosa was significantly

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enhanced in the 9.1 mg/kg MccJ25 group (P < 0.05) compared with the other

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groups (Fig. 4C and D). However, the claudin-1 expression in the 18.2 mg/kg was not

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weakened compared with that of the control and 4.55 mg/kg MccJ25 groups (P >

237

0.05). Interestingly, the expression of occludin in jejunal mucosa was tremendously

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decreased in the 18.2 mg/kg MccJ25 group (P < 0.05) compared with the other

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treatments. While in colonic mucosa, the expression of occludin was marked increased in

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the 9.1 mg/kg MccJ25 group (P < 0.05) as against the other groups (Fig. 4C and D).

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Furthermore, 18.2 mg/kg MccJ25-treated mice significantly decreased the abundance of

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occludin (P < 0.05). The abundance of claudin-1 in colonic mucosa had no difference in

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per group (P > 0.05).

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Microbial Composition and SCFAs Levels in Feces. Based on above studies, we

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further studied the mechanism of oral administration of MccJ25 on modified intestinal


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mucosal morphology, decreased intestinal permeability and inflammatory responses, the

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intestinal bacterial composition and SCFAs in fecal were determined (Fig. 5). There were

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no obvious differences in total bacterial numbers and Lactobacillus in feces among all

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treatments (P > 0.05). Compared with control group, the coliform bacteria and

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Clostridium spp. were significantly decreased in 9.1 mg/kg MccJ25 group, while the

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Bifidobactrium was marked increased (P < 0.05). Surprisingly, 18.2 mg/kg MccJ25 group

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had a greater coliform bacteria and Clostridium spp. numbers or a lower Bifidobactrium

253

number than control group (P < 0.05). Furthermore, oral administration of 9.1 mg/kg

254

MccJ25 significantly increased acetate, propionate and butyrate concentrations compared

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with the control groups (P < 0.05). 18.2 mg/kg MccJ25-treated mice had a lower butyrate

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concentration than control, 4.55 and 9.1 mg/kg MccJ25 groups. However, no significant

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differences were observed in acetate and propionate concentrations among all treatments

258

(P > 0.05).

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DISCUSSION  

260

In light of the increase in toxicity risks of natural, synthetic or biosynthetic

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antimicrobial peptide (AMP), 12,19 there is a need to exam the AMP for their application

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in various industrial domains, such as drug, food and animal. Microcin J25 (MccJ25) is a

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particularly compelling member of microcins for study as a novel antibiotic due to its

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unique structure and potent antibacterial activity.3,8,20 In addition, these peptides are

265

ribosomally-encoded, they can be genetically engineered to obtain potent biological

266

activities.9 In the present study, recombinant MccJ25 was genetically engineered to

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acquire potent biological activities based on natural MccJ25 as previous described with

268

minor modification.11 The expression vector pMJ25 obtained from engineered genes



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corresponding presumably to the gene cluster (Fig. S1). After engineering, recombinant

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MccJ25 had been shown exert strong antibacterial activity (Table S1 and Fig. S3). As we

271

expected, the antimicrobial activity in this study in an agreement with the pervious study

272

of Lopez, et. al.8 Considering the application of recombinant MccJ25 in different

273

industrial domains, such as food, clinical medicine and animals, as well as less

274

information is available regarding the optimum dosing regimens for AMP MccJ25.

275

Hence, the present work was undertaken to test the risk impacts of recombinant MccJ25

276

in the healthy mice by a 7-day oral administration.

277

Body weight, survival rate and rectal temperature are the ideal effective clinical

278

indicators for risks assessment in animal models.17,21,22 In this study, after 1 wk of

279

treatment, we demonstrated that oral administration of 4.55 and 9.1 mg/kg recombinant

280

MccJ25 effectively improved the body weight with a non-different survival rate and

281

rectal temperature. Previous study has shown that oral administration antimicrobial

282

peptide (AMP) CWA (Cathelicidin-WA) attenuated EHEC-induced clinical symptoms

283

and body weight loss in mice.23 In addition, some reports have indicated that mice-treated

284

AMP porcine β defensin 2 (pBD2), Cathelicidin-BF and sublancin by intraperitoneal

285

injection also ameliorated the clinical symptoms and body weight loss.17,24,25 Although

286

the 18.2 mg/kg recombinant MccJ25 did not cause the mice death, it caused the body

287

weight and rectal temperature significantly decreased, which maybe due to the damage

288

causing to the intestinal villus morphology and permeability.

289

The villus plays a critical role in nutrient absorption and transport. This capacity of

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the villi is closely related to the height of the villi. Specifically, the higher the height of

291

the villi, the wider the contact area, and the stronger the corresponding ability.26 In this


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study, not only the growth height of jejunum villus, but also the corresponding enlarged

293

depth were detected in the 9.1 mg/kg recombinant MccJ25 team when compared to the

294

control curing. Significant change in villi suggested that MccJ25 effectively enlarged the

295

area of the small intestine that absorbs nutrients, and its digestive and absorption capacity

296

increased by orders of magnitude. Consistent with the present study, previous studies

297

have found that dietary supplemented recombinant MccJ25 and sublancin significantly

298

improved the nutrient digestibility of weaning pigs and villus morphology of broilers,

299

respectively.15,17 However, adding 18.2 mg/kg recombinant MccJ25, microvillus height

300

and depth had no significant different compared with control group, while V/C was

301

significantly decreased when compared to the other treatment groups. This happens

302

because the rate of intestinal cell updated slows down, and the number of mature cells

303

decreased. Notably, the results of this study showed that the epithelial disruption was less

304

to be statistically significantly in mice at the jejunum histological level after curing with

305

18.2 mg/kg recombinant MccJ25, while there was inflammatory morphology of colon

306

segments with 18.2 mg/kg recombinant MccJ25, which may be due to the damage cause

307

to increase the intestinal permeability and inflammatory responses. To further paralleled

308

to our observation, the intestinal permeability and barrier function were determined. The

309

increased in D-lactate and diamine oxidase (DAO) in serum is associated with intestinal

310

permeability.27-29 With damage to intestinal barrier integrity, serum D-lactate and DAO

311

concentrations increase.30.31 As we expected, when applied to mice orally, 9.1 mg/kg

312

recombinant MccJ25 remarkable decreased

313

control group. 18.2 mg/kg recombinant MccJ25 marked increased the D-lactate and DAO

314

concentrations in serum.

D-lactate

levels in serum compared with



Page 16 of 40

315

Moreover, the epithelial barrier performance is hugely dominated by intercellular

316

tight junctions (TJs). The TJs are taking charge of limiting para-cellular movement of

317

compounds across the intestinal mucosa.32 In this study, 9.1 mg/kg recombinant MccJ25

318

significantly improved the protein expression of claudin-1 in jejunum and occludin in

319

colon compared with control group. Increased expression of TJs indicates that it can

320

provide a closer connection, not only can effectively increase the permeability of para

321

cells, enhance the ability of intercellular blockade, and thus limit the scope of solute

322

wandering in epithelial cell sheets.33-35 Amounts of investigations showed that the AMPs

323

could improve the intestinal structure and gut morphology. What’s more, the AMPs also

324

enhanced the close junction and improved the intestinal carrier performances, and

325

resulted in a balance in the disorder systems.11,15,23-25 Consistent with the results of serum

326

D-lactate

327

integrity in jejunum and colon via a decrease in occludin protein expression.

and DAO, 18.2 mg/kg recombinant MccJ25 damaged the intestinal barrier

328

As we know, increasing the permeability in TJs can lead to infection and

329

inflammation in the gut.18,36,37 In fact, we can derive the ability of AMPs to effectively

330

fight against inflammation from data from studies involving human and animals. In

331

addition, the normal functioning of anti-inflammatory activity is positively correlated

332

with the maintenance of intestinal integrity and normal intestinal epithelial function.11,15

333

Thus recombinant MccJ25 might enrich the gut barrier with the mechanism of its

334

anti-inflammatory function. In this study, we found that the proinflammatory cytokines

335

and interleukine-10 (IL-10) levels were no statistically significant in jejunum of all

336

treatment groups mice after oral administration of 4.55 and 9.1 mg/kg recombinant

337

MccJ25. Notably, the administration of 9.1 mg/kg recombinant MccJ25 suppressed the


Page 17 of 40


338

inflammatory responses by decreased production and expression of tumor necrosis

339

factor-α (TNF-α) and increased the production and expression of IL-10 in colon tissues.

340

The low dosages of recombinant MccJ25 might have a positive effect on barrier integrity

341

by supporting the expression of TJs, then decreasing the rigidity of gut inflammation and

342

full activation of the innate immune system. In addition, the increased in inflammatory

343

performance may resulting in the increased expression of inflammatory cytokines

344

including TNF-α and IL-6 caused by 18.2 mg/kg recombinant MccJ25.

345

Epithelial barrier damage and immune-mediated disorders are usually related to

346

disruptions in the microbial composition of the host’s intestine.38 There is a link between

347

the gut microbiota and human health, and this relation has attracted more and more

348

attention. The role of intestinal microbiota in host is irreplaceable by other

349

microorganisms. They can assist to absorb nutrients, metabolize drugs, ensure the

350

stability and structural integrity of the intestinal mucosal barrier, and adjust

351

immunomodulation.39 Microcins are believed to play a role in maintaining equilibrium

352

within the intestinal microbiota ecosystem, conducing to the management of probable

353

takeover by competing enterobacteria.18,9 Although the recombinant MccJ25 exerts

354

strong antibacterial activities against E. coli, Salmonella and Shigella (Table S1 and Fig.

355

S3), the different effects produced by different dosages of recombinant MccJ25 on the

356

intestinal barrier may be associated with the various situation in microbiota composition.

357

As investigated, dietary supplementation with recombinant MccJ25 or synthetic

358

AMP can enable microbiota living in the bowel of weaned pigs.11,40 In this study, we

359

found that 9.1 mg/kg recombinant MccJ25 significantly increased the Bifidobactrium

360

numbers, increased SCFA levels and decreased coliform bacteria in feces compared with



Page 18 of 40

361

control group. However, 18.2 mg/kg recombinant MccJ25 decreased the Bifidobactrium

362

number and the concentration of butyrate, and increased coliform bacteria and

363

Clostridium spp. counts in feces. Our previous study also indicates that dietary

364

supplementation with recombinant MccJ25 as feed additive in pigs can decrease the

365

coliform bacteria copy and increase the Bifidobactrium copy. In addition, some of studies

366

have been shown that nature MccJ25 or engineered MccJ25 administration can

367

effectively decrease the Salmonella pathogen numbers in mice and turkey models,

368

respectively. In this study, considering its influence on clinical symptoms, inflammation,

369

the intestinal barrier and microbiota composition, 4.55 and 9.1 mg/kg BW recombinant

370

MccJ25 did not cause negative impacts in mice model, especially, oral administration of

371

9.1 mg/kg BW recombinant MccJ25 had a remarkable positive effects on gut

372

micro-ecology.

373

In conclusion, our findings suggest that the toxicity risks of recombinant MccJ25 is

374

related to dosages. Middle-dosage 9.1 mg/kg recombinant MccJ25 can be more

375

effectively at enhancing intestinal resistance to external environment and improving

376

microbial composition and metabolic abilities in the intestines of BALB/c mice. The

377

high-dosage 18.2 mg/kg recombinant MccJ25 can cause the toxicity risk and deny the

378

positive effects, at least in part by disturbing the established microbial ecosystem and by

379

interfering with immune responses. The above data help to optimize the development of

380

drug regimens. And such a scheme can effectively confirm the potential of the

381

recombinant AMP MccJ25 severed as a substitution in various industrial domains,

382

including food, humans and animals.


Page 19 of 40


383

AUTHOR INFORMATION  

384

AUTHOR CONTRIBUTIONS  

385

Conceived and designed the experiments: S.Q. and X. Z. Performed the experiments:  H.

386

Y and L. S. Analyzed the data: H. Y H. L. and N. L. Contributed

387

reagents/materials/analysis tools: S. C., S. H. G. W. and Y. W. Wrote the paper: H. Y. All

388

authors read and approved the final manuscript.

389

FUNDING  

390

This work was supported by National Key Research and Development Program of China

391

(Grant number 2016YFD0501308), Agro-scientific Research in the Public Interest (Grant

392

number 201403047) and Research and application of key technologies for

393

enterobactercin

394

Z171100001317017) 

395

NOTES  

396

The authors declare no competing financial interest.  

397

ABBREVIATIONS USED  

398

MccJ25, Microcin J25; AMPs, antimicrobial peptides; MIC, minimum inhibitory

399

concentrations; ETEC, enterotoxigenic E. coli; SCFAs, short-chain fatty acids; TNF-α,

400

tumor necrosis factor-α; IL-10, interleukine-10; TJs, tight junctions; DAO, diamine

401

oxidase; VRB: violet red bile agar; TSCA, tryptose sulphite cycloserine agar; TPY,

402

tryptone-phytone yeast; TS, Tryptic soy; HRP, horse radish peroxidase; V, villous height;

new

veterinary

drug

product

development

(Grant

number

19

ACS Paragon Plus Environment


403

Page 20 of 40

C, crypt depth  

404

20


Page 21 of 40


405

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406

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27



Page 28 of 40

Table 1. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Primers Sequences Used in this Paper. Genes GAPDH

TNF-α IL-6

IL-10

Forward Reverse Forward Reverse Forward Reverse Forward Reverse

Sequence (5'-3') GAGAAACCTGCCAAGTATGATGA C TAGCCGTATTCATTGTCATACCAG CCACGCTCTTCTGTCTACTG ACTTGGTGGTTTGCTACGAC GAGTCACAGAAGGAGTGGCTAAGG A CGCACTAGGTTTGCCGAGTAGATCT GGACCAGCTGGACAACATACTGCT A CCGATAAGGCTTGGCAACCCAAGT

Size (bp) 212

NCBI Gene ID NM_017008.3

169

NM_010851.2

106

NM_031168.1

80

NM_010548.2

28


Page 29 of 40


Table 2. The Effects of Recombinant MccJ25 on Intestinal Proinflammatory and Anti-Inflammatory Cytokine Levels in BALB/c Mice. Item

Control

　 Jejunum TNF-α, pg/mg protein IL-6, pg/mg protein IL-10, pg/mg protein Colon TNF-α, pg/mg protein IL-6, pg/mg protein IL-10, pg/mg protein 1Values

Oral administration MccJ25, mg/kg BW 　

　

of SEM1

P-value

4.55

9.1

18.2

41.3 43.4 101.6

40.6 42.36 101.5

40.4 40.2 103.2

42.4 44.4 98.7

1.47 2.00 4.07

0.29 0.22 0.74

150.4b 63.4b

149.0bc 66.5ab

144.3c 61.5b

156.2a 72.1a

1.82 2.86

0.01 < 0.01

103.5b

106.5b

123.1a

4.77

< 0.01

92.19c

are means ± SEM, n = 6. Different italic superscript lowercase letters within each group mean significantly different (P

Risks Related to High-Dosage Recombinant ... - ACS Publications

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