Antimicrobial peptide MPX alleviates the lethal attack of Escherichia coli in mice Текст научной статьи по специальности «Биологические науки»

Ukrainian Journal of

Veterinary and Agricultural Sciences!

http://uivas.com.ua

Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv

original article | UDC 619:615.28 | doi: 10.32718/ujvas4-3.03

Volume 4 Number 3

Antimicrobial peptide MPX alleviates the lethal attack of Escherichia coli in mice

Xueqin Zhao

Sumy National Agrarian University, Herasima Kondratieva Str., 160, Sumy, 40021, Ukraine

College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang, 453003, China

Article info Received 02.07.2021 Received in revised form

05.08.2021 Accepted 06.08.2021

Correspondence author Xueqin Zhao

E-mail: 1124227899@qq.com

2021 Zhao X. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Contents

1. Introduction................. .. 16

2. Materials and methods .... .. 17

3. Results and discussion .... .. 17

4. Conclusions................ .. 20

.. 20

Abstract

Escherichia coli is an important zoonotic pathogen causing intestinal diseases. In recent years, due to the unreasonable use of antibiotics, the drug resistance of bacteria has been increasing, and the proportion of multi-drug resistant strains has also been rising, which directly threatens the health of animals and humans. The antimicrobial peptide MPX was isolated from wasp venom and had better antibacterial activity against Gram-positive and Gramnegative bacteria. Studies have found that MPX had better bactericidal activity against E. coli in vitro. However, whether MPX also has better bactericidal activity in mice is still unknown. This study found that E. coli infected mice lost appetite, diarrhea, and grouping together, while MPX treatment significantly alleviated these symptoms. The autopsy results found that the intestinal congestion, bleeding, thinning of the intestinal wall, yellow viscous fluid in the intestinal cavity, congestion of the lungs, necrosis in the liver, congestion, and bleeding of the spleen, and MPX treatment effectively relieved the above symptoms. The qRT-PCR results found that MPX could increase the mRNA expression of the antibacterial protein TFF3 in the jejunum and colon and reduce the expression of the antibacterial protein Remlp and REG3y in the jejunum and colon. H&E staining results further found that MPX could alleviate the pathological damage of mouse intestines and organs caused by E. coli infection. The above results show that MPX has good bactericidal activity against E. coli infection in mice, providing an essential reference for screening drugs for the clinical treatment of E. coli infection.

Keywords: antimicrobial peptide MPX, Escherichia coli, mice.

Citation:

Zhao, X. (2021). Antimicrobial peptide MPX alleviates the lethal attack of Escherichia coli in mice. Ukrainian Journal of Veterinary and Agricultural Sciences, 4(3), 16-21.

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1. Introduction

Escherichia coli is a gram-negative bacterium and common zoonotic pathogen, which causes many human epidemics. In the United States, more than 100,000 people are infected with EHEC 0157:H7 every year (Malavolta et al., 2019). Studies have reported that EHEC infection in pig intestinal tract contents and feces in central China is high (Bae et al., 2021). The harm of E. coli is not only manifested in causing animal diseases and bringing substantial economic losses to the breeding industry and animal husbandry, but also a reservoir of drug resistance genes for other pathogenic bacteria, and the drug resistance genes carried by the food chain passed to Humans (Liang et al., 2021). 80 % of E. coli are multi-drug resistant strains, with strong resistance to aminoglycosides, sulfonamides, tetracyclines, and chloramphenicol (Buxser, 2021). E. coli is exceptionally harmful and challenging to control. Therefore, there is an urgent

need to find new antibacterial drugs against E. coli infection, and not easy to develop drug resistance.

Antimicrobial peptides are small-molecule peptides that can resist the invasion of pathogenic microorganisms into the body. They are an essential part of the innate immune system. Their small molecular weight, good water solubility, and resistance to resistance are considered the best alternative to antibiotics and have become a research hotspot in recent years (Santos et al., 2021). Antimicrobial peptides have various biological functions such as antibacterial, antivirus, anti-parasitic, anti-inflammatory, anti-cancer, improving animal performance and immunity (Xie et al., 2020; Al Adwani et al., 2021; Piyadasa et al., 2021; Gong et al., 2021). MPX was extracted from wasp venom consisting of 14 amino acids and had four positive charges, which had good bactericidal activity against both Gram-positive and Gram-negative bacteria (Zhao et al., 2021). Previous studies of our group found that MPX had good bactericidal activity against E. coli in vitro. Whether MPX also had good bacte-

ricidal activity in vivo is still unknown. This study aims to explore further the effect of MPX against E. coli infection in vivo.

2. Materials and methods

tion kit (Takala, Japan) was used to reverse RNA into cDNA. The primer sequences as shown in Table 1.

Table 1

The primer sequences for qRT-PCR

Ethics Statement. BALB/c mice (18-22 g, female) were purchased from Zhengzhou University (Henan Province, China). All animal experiments were approved by the Animal Ethics Committee and were performed following the guidelines of the Animal Welfare and Research Ethics Committee.

Peptide Synthesis. Antimicrobial peptide MPX (H-INWKGIAAMAKKLL-NH2) was synthesized and purified by Ji er sheng Hua (Shanghai, China) at a purity greater than 98 %, and antimicrobial peptide MPX was very soluble in ddH2O.

Clinical symptoms and observation of necropsy lesions. BALB/c mice were randomly divided into 4 groups, namely control group, E. coli, E. coli + MPX, E. coli + en-rofloxacin. The dose of E. coli infected BALB/c mice was 4.5x107 CFU/mice, MPX (20 mg/kg), and Enro (20 mg/kg) were treated by intraperitoneal injection after infection with E. coli for 2 h, and treatment was continued for 3 days. Observed the clinical manifestations and necropsy of the mice after E. coli infection, took out the mouse lungs, liver, spleen, and intestines with scissors and toothless forceps, observed the pathological changes of the mouse intestines and organs, and took pictures.

qRT-PCR. Total RNA extraction kit (Solarbio, China) extracted total RNA from mouse jejunum and colon. The jejunum and colon powder were slowly added to 1.5 mL EP, 200 ^L chloroform was added to each well, and shaken on a shaker for 15 s, centrifuged at 12000 rpm, 4 °C for 10 min, added 500 ^L isopropanol and mix well, centrifuged at 12000 rpm, 4 °C for 10 min, discard the supernatant, adding 1 mL to each tube 75 centrifuge in% ethanol, 12000 rpm, 4 °C for 5 min, added 20-30 ^L of DEPC water and mix well, then measure the RNA concentration. A reverse transcrip-

Genes

Sequence

F:5'-CCCGACACTGGGCTATGAAC-3' R:5'-GGTACCACAGTGATTGCCTGA-3' F:5' -CTGATAGTCCCAGGGAACGC-3' R:5'-GTCTGCCAGAAGACGTGACA-3' F:5' -CCTGGTTGCTGGGTCCTCTG-3' R:5'-GCCACGGTTGTTACACTGCTC-3' F:5' -GAGAAACCTGCCAAGTATGATGAC-3' R:5'-TAGCCGTATTCATTGTCATACCAG-3'

Reg3y Relmß TFF3 GAPDH

H&E staining. After wiping clean with alcohol cotton, the mouse organs and intestines were fixed with 4 % para-formaldehyde, paraffin-embedded, sectioned, and H&E stained to observe the pathology of the mouse duodenum, ileum, colon and liver, spleen, and lungs. Change, refer to the specific operation steps (He et al., 2015).

Statistical Analysis. GraphPad Prism 5 data processing software to carry out and difference analysis of experimental results (One-Way ANOVA), P < 0.05 means significant difference (marked in the text *P < 0.05; **P < 0.01;

*** P < 0.001; #P < 0.05; ##P < 0.01; < 0.001).

3. Results and discussion

Results

MPX alleviates the clinical manifestations of mice.

Observation of clinical symptoms after infection of E. coli in mice was shown in Figure 1A and B: mice infected with E. coli alone showed loss of appetite, rapid heartbeat, body tremor, loose hair, bunching up, arched back, anal prolapse, feces clinical manifestations such as irregularities, while MPX treatment significantly alleviated the adverse reactions caused by E. coli infection. Mice increased appetite, smooth coat, and the effect was better than enrofloxacin treatment. The control group did not show any adverse reactions.

Control

E.coi i

ssSt

E.CO//+MPX

E.coli+ Enro

Fig. 1. Observation of clinical symptoms of E. coli infection with BALB/c mice (A, B)

MPX alleviates the pathological changes of mice by

necropsy. The results of the necropsy were shown in Figure

2, the intestines of mice in the control group were normal, with thick and flexible intestinal walls, and no pathological changes were seen in the liver, spleen, and lungs. Mice infected with E. coli had intestinal congestion, hemorrhage, intestinal wall thinning, and easy to rupture, the intestinal

cavity was filled with a yellow viscous liquid, the jejunum was severely congested, and the lungs, liver, and spleen were congested and bleeding. While MPX could effectively alleviate the intestinal inflammatory response and organ pathological damage caused by E. coli infection, its effect was equivalent to that of the antibiotic Enro.

Intestinal Lung Liver Spleen

Control

E. coli

E.coli+ MPX

E.coli+ Enro

Fig. 2. Autopsy results of mouse intestines and organs after E. coli infection.

MPX increases the expression of intestinal antimicrobial peptide protein. The mRNA expression of intestinal antibacterial related proteins REG3y, Remlp, and TFF3 by qRT-PCR. In the jejunum (Figure 3A), compared with the control group, the TFF3 gene expression level in the jejunum of the E. coli group was increased (P< 0.05);. In contrast, the TFF3 gene expression in the jejunum of E. coli+MPX was significantly lower than in the E. coli group (P < 0.05), with no significant difference from the control group. Compared with the control group, the mRNA expres-

sion level of Remlp in the jejunum tissue of E. coli infected mice was significantly increased (P < 0.001). MPX significantly reduced the mRNA expression level of Remlp, which was equivalent to the effect of Enro. In contrast, the expression level of Remlp in the mouse colon was not significantly different in another group. In addition, E. coli infection leads to increased REG3y expression in mouse jejunum and colon, and MPX could significantly reduce REG3y mRNA expression caused by E. coli infection.

Fig. 3. The mRNA expression of the antibacterial protein in the mouse intestine. A: The mRNA expression of TFF3 in mouse jejunum and colon; B: The mRNA expression of Remlp in mouse jejunum and colon; C: The mRNA expression of REG3y in

mouse jejunum and colon

MPX relieves pathological intestine damage. Further H&E staining was used to observe the pathological changes of the duodenum, ileum, and colon after E. coli infection. As shown in Figure 4, the duodenum, ileum, and colon of mice infected with E. coli showed intestinal villi shedding, breaking and falling into the intestinal lumen, catarrhal enteritis, degeneration, necrosis, shedding of intestinal mucosal epithelial cells, congestion of the lamina pro-pria and a large

number of neutrophil infiltration, showing the pathology of necrotizing enteritis and fibrinous necrotizing enteritis Changes (Figure 4A, B, C). At the same time, the pathological changes of each bowel segment were significantly alleviated after treatment with MPX. The intestinal villi of the control were neatly arranged without the above-mentioned pathological changes.

a** J Cflrim O. gtijgfj

o jfm - O Î %

MJ.VI MPX îCina-hj) m ¡P L.twYMVIPX &

KiL-Enra 2örf*9! O. jt^JHí i Wimm iSD*^: B o 1««

A

Fig. 4. H&E staining of intestines after E. coli infection in mice. A: H&E staining of duodenum after E. coli infection in mice; B: H&E staining of ileum after E. coli infection in mice; C: H&E staining of the colon after E. coli infection in mice

MPX relieves pathological damage of organs in mice.

E. coli-infected mice developed acute interstitial pneumonia, widened alveolar septum, ruptured alveoli, neutrophil infiltration, and mild lung disease, showing local pulmonary congestion and a small amount of red blood cell and inflammatory cell infiltration (Figure 5A). Symptoms of hem-orrhagic splenitis, congestion, local necrosis, small splenic corpuscles appear in the spleen, a large number of neutro-

phil infiltration in the splenic sinus (Figure 5B), degeneration and necrosis of hepatocytes, and acute necrosis in the liver, disintegration of liver cells, congestion, liver congestion, dilation of liver sinusoids, infiltration of red blood cells and neutrophils (Figure 5C). The above symptoms were significantly alleviated after treatment with MPX, indicating that MPX can protect mice against the damage of E. coli to the organs.

lllllll ¡Si rr.-Ä

— »It mm lg¡ mu

L -nWrjPX aurgfífl) Slip kwtewñi 111

Cull>4*ni» A lg!! S su

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Kü 91

Fig. 5. H&E staining of the organs infected with E. coli in mice. A: H&E staining of lung infected with E. coli in mice; B: H&E staining of spleen infected with E. coli in mice; C: H&E staining of liver infected with E. coli in mice

Discussion

In this study, E. coli was used to establish the BALB/c mouse infection model and MPX treatment to evaluate the effect against E. coli infection in mice. The clinical symptoms, intestinal and organ necropsy, pathological changes, and the mRNA expression of the antibacterial protein in mice were evaluated. The results showed that MPX could alleviate the clinical symptoms of mice caused by E. coli infection, relieve the pathological changes of the intestines and organs, and increase the mRNA expression of the antimicrobial protein TFF3. This study evaluated the effect of MPX against E. coli in vivo, laying a foundation for the study of MPX in mice, providing a reference of drugs for treating E. coli infection.

MPX can alleviate the intestinal damage caused by E. coli infection in mice. The intestine is the largest digestion and absorption organ of animals and the most important immune organ of the body. Zhang et al. found that adding antimicrobial peptide plectasin to chicken diets could improve chicken performance, immune function, and intestinal health and increase intestinal villi length (Zhang et al., 2021). Roque-Borda CA et al. found that the antimicrobial peptide Ctx(Ile)-Ha could effectively alleviate pathological intestinal damage (Roque-Borda et al., 2021). Shang et al.

found that the antimicrobial peptide Microcin J25 could alleviate DSS-induced intestinal Inflammation and improve intestinal morphology (Shang et al., 2021). Xiong et al. found that oral antimicrobial peptide-defensin-1 (DEFB1) could improve intestinal function and enhance intestinal barrier function (Xiong et al., 2021). The results found that MPX could effectively reduce the intestinal damage caused by E. coli infection in mice.

The intestine is in direct contact with the external environment and colonizes a large number of microorganisms. Antimicrobial proteins secreted by intestinal epithelial cells play an essential role in maintaining the homeostasis of the intestinal epithelium and normal microbial flora (Gallo & Hooper, 2012; Wlodarska et al., 2010). REG3y is mainly expressed in the small intestine tissues of mice and humans. In addition, REG3y is also conditionally expressed when pathogen infection or Inflammation occurs in the large intestine tissues (Christa et al., 1996). The study showed that REG3y was almost not expressed in the intestinal tract of sterile mice, and the expression of REG3y was significantly increased after the normal flora was colonized (Cash et al., 2006). The expression of RemLp is mainly regulated by Th2 cytokines, which play an essential role in the process of innate immunity and host defense (Hosoya et al., 2017).

TFF3 is produced by mucous secreting cells, which play an essential role in the intestinal mucus layer and mucosal repair function (Ge et al., 2015). This study found that MPX can increase the mRNA expression of the antimicrobial protein TFF3 in the jejunum and colon and reduce the expression of the antimicrobial protein Remlp and REG3y in the jejunum and colon.

4. Conclusions

MPX can resist the lethal attack of E. coli in mice, alleviate the pathological changes of mice intestines and organs, and increase or decrease the mRNA expression of antimicrobial proteins in the jejunum or colon to varying degrees, providing significant reference value for clinical drug screening of E. coli infection.

Author's contributions

Xueqin Zhao participated in the study design, carried out data analyses, participated and performed measurements, laboratory testing's and wrote this manuscript.

Acknowledgments

This work was supported by the Young Talent Lifting Project in Henan Province (2020HYTP041); the Key scientific research projects of colleges and universities in Henan Province (21A230004); the National Key Research and Development Program of China (2019YFC605700); Open Project of State Key Laboratory of Marine Resources Utilization in South China Sea (Hainan University, MRUKF2021004) ; the Youth Backbone Teacher Project of Colleges and Universities of Henan Province (2020GGJS162); the Innovative Research Team (in Science and Technology) in University of Henan Province (20IRTSTHN025); Climbing Project of Henan Institute of Science and Technology (2018JY02).

Conflict of interest

The author does not report any financial or personal connections with other persons or organizations, which might negatively affect the contents of this publication, and claim authorship rights to this publication.

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