SspH2 as anti-inflammatory candidate effector and its contribution in Salmonella Enteritidis virulence

Journal Pre-proof SspH2 as anti-inflammatory candidate effector and its contribution in Salmonella Enteritidis virulence Makarem O.E. Shappo, Qiuchun Li, Zhijie Lin, Maozhi Hu, Jingwei Ren, Zhengzhong Xu, Zhiming Pan, Xinan Jiao PII:

S0882-4010(19)31712-7

DOI:

https://doi.org/10.1016/j.micpath.2020.104041

Reference:

YMPAT 104041

To appear in:

Microbial Pathogenesis

Received Date: 25 September 2019 Revised Date:

2 February 2020

Accepted Date: 3 February 2020

Please cite this article as: Shappo MOE, Li Q, Lin Z, Hu M, Ren J, Xu Z, Pan Z, Jiao X, SspH2 as anti-inflammatory candidate effector and its contribution in Salmonella Enteritidis virulence, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/j.micpath.2020.104041. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author Statement Makarem O.E. Shappo: Conceptualization, Methodology, Software, Writing-Original draft preparation Qiuchun Li: Data curation, Writing-Review & Editing, Project administration. Zhijie Lin: Visualization, Investigation. Maozhi Hu: Software, Validation. Jingwei Ren: Methodology, Validation. Zhengzhong Xu: Formal analysis. Zhiming Pan: Resources. Xinan Jiao: Funding acquisition, Supervision.

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SspH2 as anti-inflammatory candidate effector and its contribution in

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Salmonella Enteritidis virulence

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Makarem O. E. Shappo1,2, Qiuchun Li1,2,3, Zhijie Lin1,2,3, Maozhi Hu1,2, Jingwei Ren1,2,3,

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Zhengzhong Xu1,2,3, Zhiming Pan1,2,3 and Xinan Jiao1,2,3,*

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1

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Agri-food Safety and Quality, Ministry of Agriculture of China, Yangzhou University, China

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2

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Important Animal Infectious Diseases and Zoonoses, Yangzhou University, China

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3

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Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for

Jiangsu Key Lab of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of

Joint International Research Laboratory of Agriculture and Agri-Product Safety, Yangzhou

University, China

11 12

* Correspondence: Xinan Jiao, Tel: (0086) 514-87971136; Fax: (0086) 514-87311374; E-mail:

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[email protected]

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Abstract

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Salmonella enterica is a facultative intracellular pathogen deploying the type III secretion system

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(T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) to transfer effector proteins into host

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cells to modify its functions and accomplish intracellular replication. To study the effect of SspH2 on

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immune response induced by S. Enteritidis, we generated a deletion mutant of the effector gene

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sspH2 and a plasmid mediated complementary strains, in S. Enteritidis C50336. The results of LD50

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showed that SspH2 has no obvious effect on the virulence of S. Enteritidis. However, deletion of

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sspH2 decreased the invasion and intercellular colonization of the bacteria in Caco2 BBE cells.

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Using bacteriological counts from tissue homogenates the result of colonization in internal organs

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showed that in spleen and liver tissues, at 3rd and 4th day p.i there is a significance decreased number

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of C50336-∆sspH2 compared to C50336-WT and C50336-∆sspH2-psspH2, respectively. The

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qRT-PCR analysis results both in vivo and in vitro experiments clearly showed that the mutant strain

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C50336∆sspH2 significantly promoted expression of IL-1β, INF-γ, IL-12, and iNOS cytokines

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compared to the groups infected with the wild type or complementary strains, while the IL-8

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synthesis was decreased in the mutant strain infected group. All of these findings revealed that

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SspH2 promotes the colonization of S. Enteritidis in host cells, and it is an important

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anti-inflammatory biased effector in Salmonella.

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Keywords: Salmonella enterica serovar Enteritidis (S. Enteritidis); SspH2; anti-inflammatory

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response; virulence.

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1. Introduction

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Salmonella is a foodborne pathogen, which can cause human disease, including gastroenteritis and

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enteric or typhoid fever, through consumption of Salmonella-contaminated food and water [1].

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Salmonella enter into the host cell and reside in a membrane-bound vacuole, originated from

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macropinocytosis and spacious phagosome formation [2, 3]. With difference to the other

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phagosomes in which bacteria are degraded, the Salmonella-containing vacuole (SCV) forms an

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ideal nice for the bacteria to persist and replicate even after fusion with the lysosomal compartment

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[4]. Survival and replication within host phagocytic cells is essential to Salmonella pathogenesis in

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animals [1]. Salmonella has developed various virulence mechanisms to benefit their survival by

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manipulating host cell functions, especially the two type III secretion systems (T3SS1 and T3SS2),

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which are encoded by Salmonella pathogenicity islands SPI-1 and SPI-2. Through both T3SS, a

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series of bacterial effectors are delivered into host cells with the intention to reprogram eukaryotic

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cell functions [5-7]. The T3SS1 appears to be required for penetration into the epithelial cells or the

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intestinal mucosa, while the T3SS2 is necessary for survival of Salmonella in host cells and the

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systemic infection.

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For survival in host cells, Salmonella used SPI-2 to exert many effectors to modify host cell

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functions in multiple ways, such as ubiquitination. In eukaryotes, ubiquitination is involved in many

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processes, including cell cycle, immune response, etc [8-11]. SspH2 is an E3 ubiquitin ligase (NEL)

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effector identified in S. Typhimurium, and it can activate NOD1 to enhance the IL-8 secretion

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However, most of the work has been performed in S. Typhimurium, the pathogenesis of S. Enteritidis

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and the function of SspH2 in the pathogen is poorly understood.

[12].

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This study was designed to investigate the effects of SspH2 effector from SPI2 in host immune

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response and it is contribution on S. Enteritidis virulence. To better understand the pathogenesis

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effects of SspH2, we successfully constructed the recombinant strains C50336∆sspH2 and C50336∆

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sspH2- psspH2 based on S. Enteritidis C50336-WT strain. In the following parts we will outline the

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findings of each experiment to detect (i) the characterizations of sspH2 gene (ii) role of SspH2

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effector in bacteria survival inside the host cells and (iii) eliciting immune responses in host.

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2. Materials and methods

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2.1. Bacterial strains and growth conditions

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The S. Enteritidis reference strain C50336 was obtained from the Chinese National Institute for the

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Control of Pharmaceutical and Biological Products. Bacterial growth conditions were as follows: Luria

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Broth (LB) cultures (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) at 37°C with shaking /180rpm,

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LB medium was supplemented with ampicillin (Amp) (100 mg/L and chloramphenicol (Cm) (35

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mg/L) according to constructed strain conditions. The solid LB media were prepared by adding agar

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(15 g/L). Bacterial strains and plasmids are shown in Table 1.

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2.2. Construction of the sspH2 knockout strain and complementary strain

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Deletion of sspH2 gene from the chromosome of S. Enteritidis was performed using standard λ-Red

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recombinase system as previously described [13,14]. The primers were designed to amplify the

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resistance gene fragment and FRT of chloramphenicol in pKD3 plasmid (Table 2). Briefly, the

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chloramphenicol (cat) resistance cassette was obtained from pKD3 by PCR with primers (SspH2-Cat-F

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and SspH2-Cat-R) and the amplification of homologous arm fragment for Red homologous

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recombination includes 39 nt on both side of the sspH2 gene. The constructed bacteria

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C50336∆sspH2::cat were screened and identified on both CmR and AmpR LB plates. Knockout of

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sspH2 was verified by PCR and DNA sequencing, then C50336∆sspH2 as obtained through FLP

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recombinase expressed by pCP20. The full-length sspH2 gene with ORF was amplified from C50336

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using primers R-SspH2-F and R-SspH2-R (Table 2) and cloned into plasmid pBR322 using

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ClonExpressTM II One Step Cloning Kit (Vazyme Biotech Co.,Ltd.) according to the manufacturer’s

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instruction, and then the recombinant plasmid was transformed into C50336∆sspH2, the transformants

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were screened and identified on LB agar plates with ampicillin. The complementary strain was further

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verified by PCR and sequencing analysis.

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2.3. Growth curves of S. Enteritidis

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The growth characteristics of S. Enteritidis strains:

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C50336∆sspH2-psspH2 was performed as previously described [15,16]. Briefly, single colony of

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each bacteria strain was inoculated in 5 mL LB medium for cultivation with shaking at 37 °C

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overnight, 50 µl of the bacteria culture was then transferred into 5 ml LB medium and cultured with

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shaking at 37 °C. The OD600 was measured every hour for 16 h to obtain the growth curve.

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2.4. Determination of LD50

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S. Enteritidis strains (C50336-WT, C50336∆sspH2, C50336∆sspH2-psspH2) were cultured in LB

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medium at 37°C overnight without shaking OD600 was measured as ~1.00. Ten fold dilutions of the

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culture were then prepared in phosphate buffered saline (PBS) (pH 7.4) and used to infect 85

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BLAB/c mice (6-8 weeks in old, females), which were obtained from the Comparative Medicine

C50336-WT, C50336∆sspH2 and

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Center, Yangzhou University. Mice were divided into 3 groups each containing 25 mice/ S.

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Eneteritidis strain (5 mice/dose) and as control 10 mice were used which were treated with PBS. The

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Experiment was approved by the Animal Care and Ethics Committee of Yangzhou University. The

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dilutions were plated on XLT4 agar plates for identification and quantification of the culture as

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Salmonella. Mice were infected with 200 µl of diluted bacteria orally via a feeding needle. The

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treated mice were kept at separate cages under free pathogenic condition and were observed daily,

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and their mortality over the following 2 weeks was recorded, and the LD50 was calculated by the

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method of Reed and Muench [17].

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2.5. Invasion assay

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Bacterial invasion assay was performed as described with some modifications [18]. Briefly, bacteria

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(C50336-WT, C50336∆sspH2 and C50336∆sspH2-psspH2 were cultured overnight in LB media at

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37◦C without shaking. OD600 was measured (=1.0), after centrifugation the pellet (bacterial cells) was

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washed twice using PBS (pH=7.4), finally the pellet was dissolved in 500 µl PBS and OD600 was

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adjusted as 1.0. The Caco2 BBE cells were grown in T75 flasks and checked to cover >90% of the

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field of view, the cells were washed twice with PBS (pH 7.4) and the cells were liberated from the

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T75 flask with 3 ml trypsin-EDTA (0.25%) to complete confluency. The cells were then cultured in

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2 ml of complete DMEM media (FBS 10%) in 24-well culture plates [MOI] = 100:1) for 48h.

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µl of bacterial cells (109 CFU) was added to each well containing Caco2 BBE cells. After incubation

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(37°C) at 2, 4, 8 and 24 h timepoint, the non-adherent bacteria were washed by PBS (pH 7.4) for

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three times, and the cells were treated with 200 µl of 1% Triton X-100 in PBS. After removal of

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Triton X-100, the cells were harvested in 500 µl of PBS, serial dilutions were prepared by took 50 µl

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from concentrated vial in 450 µl PBS. 105 ,106 and 107 were used to plate the cells in LB plates

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incubated at 37°C overnight and colonies were counted (106 was the best for counting the colonies of

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bacteria). The result was analyzed using GraphPad prism 5.0 (32 bit).

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2.6. Cell infection assay

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The human colonic cell lines Caco-2 BBE cells were used for the infection assay. The Caco-2 BBE

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cells routinely were cultured in DMEM media (FBS 20%) supplemented with antibiotics to a final

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concentration of 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were incubated at 37 °C

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in a humidified 5% (v/v) CO2 atmosphere. The infection assay by Salmonella strains (C50336-WT,

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C50336∆sspH2 and C50336∆sspH2-psspH2) was performed as previously described [3]. The

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overnight culture of S. Enteritidis were collected by centrifugation at 8,000 g for 5 min, followed by

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washing twice with PBS (pH 7.4), and then resuspended at a concentration of 2 × 107 CFU (colony

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forming units) ml-1 in DMEM medium. The prepared bacterial suspensions were added directly into

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24-well plates with washed Caco-2 BBE cells with 125 µl per well, and the plates were incubated at

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37 °C for 1.5 h. The cultured Caco-2 BBE cells were then washed three times with PBS (pH7.4) and

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incubated with 100 µg/ml gentamicin (time point, 0 h) for 1 h to kill the extracellular bacteria. For

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the survival analysis of Salmonella in Caco-2 BBE cells, the infected cells were washed three times

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with PBS and subjected to cultivation with 10 µg/ml gentamicin. The cells were collected at 30 min,

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1, 2, 4, 8, 12 and 24 h post infection, and lysed in 1 ml of 0.1 % Triton X-100 to release the

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intracellular bacteria for calculation on LB plate.

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2.7. In vivo study of SspH2 virulence in S. Enteritidis

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Specific-pathogen-free (SPF) female mice (C57BL/6; 6-8weeks) were obtained from the

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Comparative Medicine Center of Yangzhou University. All animal experiments were approved by

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the Jiangsu Administrative Committee for Laboratory Animals and complied with the guidelines of

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Yangzhou University’s Institutional Animal Care and Use Committee. For in vivo study, the mice

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were housed in groups of five under standard conditions. Food and water were withdrawn 4 h before

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treatment with 0.75 mg of streptomycin (100µl/mouse). Afterward, animals were supplied with water

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and food. Twenty hours later, food and water were withdrawn again for 4 h before the mice were

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inoculated with 108 CFU (oral gavage) of the corresponding bacterial strain of serovar Enteritidis

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(200µl/mouse) or treated with sterile PBS (control). At the indicated times p.i. (2nd, 3rd or 4th day),

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the mice were sacrificed to collect the tissue samples, including the cecum, spleens, and livers for

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further analysis.

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2.8. The bacterial colonization in tissues

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The bacterial loads in mice tissues were determined when mice orally infected by 200 µl doses with 1x

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108 cfu/ml amounts of the C50336-WT, C50336∆sspH2, C50336∆sspH2-psspH2 strains. At various

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times post infection (2nd, 3rd or 4th days), three mice were sacrificed from each group and tissues

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(spleen, liver and cecum) were collected and weighted. The portion of each organ was weighted and

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prepared as tissue homogenates in 1 ml PBS per organ. A series of dilutions of the homogenates were

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plated onto MacConkey agar plates and incubated at 37°C for 12-18 h. The bacterial CFUs per tissue

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were calculated and compared to reveal the colonization of the bacteria in different tissues.

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2.9. Quantitative real-time PCR (qRT-PCR)

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About 30 mg of spleen, liver or cecum was snap-frozen in liquid nitrogen, and stored at -80°C for

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total RNA extraction. RNeasy® Plus Mini Kit (Qiagen) was used to extract the RNA, the amount,

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quality and quantity were measured using Nanodrop Spectrophotometer. PrimeScript™ RT reagent

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Kit with gDNA Ereaser (TaKaRa) was used to gain cDNA and stored at -20°C, all of laboratory

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procedures were performed on ice and under safety cabinet to avoid RNase contamination. qRT-PCR

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was conducted to detect the available cytokines. Real-time PCR analysis was performed in a

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fluorescence temperature cycler 7500 Real Time system (Applied Biosystems) using Fast start

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Universal SYBR Green Master (Rox). The qRT-PCR analysis was performed in triplicates, with

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three independent total RNA samples.

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2.10. Statistical analysis

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All data were expressed as mean ± standard error of the mean (SEM). Student’s t test was used to

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analyse the data, with pairwise comparison. A p value of less than 0.05 was considered statistically

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significant. All analyses were performed using the software GraphPad Prism version 5

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3. Results

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3.1. The biological characteristics of S. Enteritidis

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The growth curve of the three strains determined in LB liquid medium revealed that there was no

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significant difference among all strains (Fig.1). The deletion of sspH2 gene cannot effect the growth

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of S. Enteritidis in vitro.

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The survival and proliferation of S. Enteritidis in Caco2-BBE culture monolayer was assessed. The

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intracellular bacterial number was strongly increased at 8 h p.i, and mutant strain showed low

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number of internalized bacteria. The rate of proliferation was low in mutant group (24h) (Fig.2).

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The LD50 result for the three S. Enteritidis strains under study was 1x104 for C50336-WT, 7.9x104

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for C50336∆sspH2-psspH2 and 3.16x105 for the mutant strain C50336∆sspH2(Table 2).

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3.2. Bacteria colonization in tissues

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The result of the spleen tissue analysis showed there was no significant difference in CFU value

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among the three strain-infected groups at 2nd p.i, while at 3rd and 4th day p.i. the C50336∆sspH2 was

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significantly decreased compared with the C50336-WT and C50336∆sspH2-psspH2 infected groups

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(Fig.3). The result of the liver tissue showed that there was no significant difference among all groups

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at day 2 p.i, but the mutant group (C50336∆sspH2) displayed significantly decreased colonization

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compared to the other two groups at 3rd and 4th day p.i, and no significant difference was detected

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between the WT (C50336-WT) and the complementary group (C50336∆sspH2-psspH2) (Fig.3). The

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result of bacterial burden in cecum showed that significantly decreased colonization of the mutant was

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detected at 2nd day p.i., which is different from the results in liver and spleen (Fig.3). In addition, the

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complementary strain infected group showed significantly increased colonization compared to the

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mutant group at 2nd, 3rd and 4th day p.i (p < 0.001, p < 0.001 and p < 0.05), respectively (Fig.3).

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3.3. qRT-PCR detection of cytokines

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The result of qRT-PCR in vitro showed that no difference between groups infected by the wild type

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and complementary strain in the expression level of all cytokines. The mutant strain showed a

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significant decrease in the expression level of 1L-1β, IFN-γ, IL-6 and iNOS compared to that of the

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wild type and complementary strains. The groups infected by the wild type and complementary

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strains showed a significant increase in IL-8 expression level than the mutant infected group (Fig.4).

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However, no significant difference was detected in the expression level of IL-17A, IL-10 and IL-4

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among the three groups, respectively (unpublished).

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The qRT-PCR analysis in vivo (two-way ANOVA) showed that the mutant C50336∆sspH2 infected

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group displayed high expression level of IL-1β in spleen with significant difference at four days p.i.

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(Fig. 5). The mutant C50336∆sspH2 strain induced high expression level of IFN-γ in spleen at all

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days p.i., and in liver at three and four days p.i. (Fig. 5). IL-6 was only highly expressed in liver of

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the mutant infected group at two and three days p.i. (Fig. 5). The expression of iNOS was high in

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livers and spleens of mutant infected group compared to that of the WT and complementary strain

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infected groups (Fig. 5). With difference to the above results, the cytokine IL-8 was expressed higher

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in all the tissues from WT and complementary strain infected groups than in the mutant group (Fig.

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5).

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4. Discussion

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The main purpose of this study was to detect the effect of sspH2 gene in immune response in

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mammals infected with S. Enteritidis in vivo (C57BL6/mice) and in vitro (Caco-2 BBE cells). The

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function of the gene sspH2 in Salmonella is unclear. A review described the functions of many

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substrate proteins of the TTSSs of SPI1 and SPI2, and mentioned that the SspH2 had no specified

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function [19]. To determine the role of the gene sspH2 of S. Enteritidis, we constructed

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C50336∆sspH2 and C50336∆sspH2-psspH2 strains. The growth curve showed that deletion of

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sspH2 had no effect on the growth ability of S. Enteritidis. To study the virulence of SspH2 effector,

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the LD50 analysis demonstrated that deletion of the sspH2 reduced the virulence of S. Enteritidis

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(~30-fold). The previous study confirmed that the sspH1 and sspH2 were virulence factors involved

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in the Salmonella infection in calves, and both genes may serve additive or redundant functions [20].

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The expression of different cytokines reflects the host immune response against infection

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microoraginsm [21-23]. According to the previous study, 8 h p.i were selected as the target time

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point to detect the expression of cytokines affected by sspH2 gene in vitro and in vivo [24]. The

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qRT-PCR result analysis demonstrated that deletion of sspH2 could inhibit the S. Enteritidis C50336

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induced IL-8 synthesis. Similar results were detected in the human intestinal epithelial cells infected

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with S. Dublin, S. Typhimurium or S. Typhi, which displayed the increased expression of many

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cytokine genes, particularly IL-8 [25]. In mammalian cells, SspH2 could interact with Nod1 and

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induced Nod1-mediated IL-8 secretion when it is transiently expressed or delivered by S.

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Typhimurium [12]. In addition, treatment of human neutrophils with heat-killed S. Typhimurium

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also increased the secretion of IL-8 [26]. However, there was few study about the immune response

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induced by SspH2. In this study, the expression level of IL-1β and IFN-γ showed significant

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difference between C50336-WT and mutant strain both in vivo and in vitro, which supported that

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IFN-γ was central for successful host defense against Salmonella [27]. For example, the embryonic

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fibroblasts infected with S. Typhimurium displayed increased mRNA expression and secretion of

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IFN-γ, which may enhance the ability of these cells to resist against further Salmonella infection

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[25]. In addition, the secretion of IFN-γ by T cells and the subsequent activation of IFN-γ producing

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Th cells is considered to be vital to control S. Typhimurium infections [27]. The mutant strain also

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increased the level of iNOS expression significantly in vivo and in vitro. Many studies have

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confirmed the production of RNIs by rodent models infected with Salmonella. For examples, the

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plasma nitrite and nitrate levels (RNIs detection measure) have been shown to increase significantly

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in mice after systemic infection with S. Typhimurium [28, 29]. Induction of iNOS and production of

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NO in both murine peritoneal macrophages and murine macrophage-like cell lines has been detected

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in tissue culture experiments infected with Salmonella [30, 31]. In our study, the sspH2 mutant strain

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displayed the increased expression of iNOS in vitro and in vivo, which reflected the SspH2 may

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performed as an effector inhibiting the production of iNOS in S. Enteritidis infection process.

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The result in present study showed that the expression of IL-6 differed significantly in vivo and in

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vitro (was higher in mutant strain in compare with wild and complementary strains), this result was

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supported by previous studies, which showed that IL-6 is particularly important among cytokines

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produced in the intestinal mucosa during sepsis and endotoxemia because of its multiple significant

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biological effects [32-36]. Although commonly considered a proinflammatory cytokine [33], IL-6

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has also been confirmed to have important anti-inflammatory properties and may exert protective

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effects in various tissues [34-36].

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5. Conclusion

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We can conclude SspH2 effector play an important role in pathogenicity of S. Enteritidis and act as

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an anti-inflammatory effector in cell response, we can also describe SspH2 as an effector involved in

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persistence of S. Enteritidis in host cells through downregulation of the pro-inflammatory cytokines

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expression.

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Conflict of interest

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The authors declare no conflict of interest.

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Acknowledgements

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This study was supported by National Natural Science Foundation of China (31730094,

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31920103015); The National Key Research and Development Program of China (2017YFD0500100;

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2017YFD0500700); Jiangsu province agricultural science and technology independent innovation

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funds (CX(16)1028); The Priority Academic Program Development of Jiangsu Higher Education

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Institutions (PAPD).

264

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362

Figure legends:

363

Figure 1. Construction of sspH2 deletion mutant in S. Enteritidis strain C50336. a. PCR verification

364

of C50336∆sspH2 and C50336-WT. The wild type strain harbors the complete sspH2 gene, with a

365

PCR product length of 3074 bp, whereas the PCR product from C50336∆sspH2 has a length of 888

366

bp,(left). b. Growth

367

C50336∆sspH2-psspH2. Bacteria were grown in liquid LB medium at 37 °C for 16 h with agitation,

368

and the OD600 values of triplicate cultures in LB medium were determined in 1-h intervals.

369

Figure 2. Bacterial survival and replication in cells. S. Enteritidis invasion in the Human epithelial

370

Caco-2 BBE cells treated with C50336-WT, C50336∆sspH2 and C50336∆sspH2-psspH2. The

371

number of Salmonella internalized was determined and presented as 106 CFU/g. Data are expressed

372

as the mean ± SEM.*P≤0.05 and**P ≤ 0.01

373

Figure 3. Bacterial colonization in mice. Six-week-old C57BL/6 mice were orally gavage with

374

C50336-WT, C50336∆sspH2 or C50336∆sspH2-psspH2, 1×108 cfu/mouse. The uninfected mice

375

were used as control. 2nd, 3rdand 4th day p.i, the bacterial load was counted in mice spleen, liver and

376

cecum. *P≤0.05, **P ≤ 0.01, and ***P ≤ 0.001.

377

Figure 4. The relative expression of cytokines in Caco-2 BBE cells using qRT-PCR. Cells were

378

seeded on 96-well plates. The infection assay by Salmonella strains C50336-WT, C50336∆sspH2

379

and C50336∆sspH2-psspH2 was performed (at a concentration of 2 × 107 CFU, 125 µl per well were

380

added directly on washed Caco-2 BBE cells). . The plates were incubated at 42 °C for 1.5 h,

curves of wild-type S. Enteritidis C50336, C50336∆sspH2 and

381

Extracellular bacteria were removed. The bacteria were collected at 30 min, 1, 2, 4, 8, 12 and 24 h

382

post infection. The relative expression of cytokines was done.

383

Figure 5. The relative expression of cytokines in tissues of C57BL/6 mice infected with S.

384

Enteritidis. qRT-PCR was conducted to detect some cytokines available in tissues of mice infected

385

with S. Enteritidis C50336-WT, C50336∆sspH2 and C50336∆sspH2-psspH2, using Fast start

386

Universal SYBR Green Master (Rox). The real-time PCR experiments were performed in triplicates,

387

with three independent total RNA preparations.

388

389

Table 1. The plasmids and bacteria strains used in the study to construct mutant and complementary strains derived from C50336 strain Plasmid

pBR322

Relevant genotype and/or phenotype Red recombinase expression plasmid Template plasmid; FRT-sspH2-FRT FLP recombinase expression plasmid Cloning vectors

The strain C50336-WT

Background S. Enteritidis

Genetic information Wild type

C50336∆sspH2

S. Enteritidis ∆sspH2::cat

Mutant

Laboratory of Zoonoses and Immunology, Yangzhou University, China Reference Obtained from Chinese National Institute for the Control of Pharmaceutical and Biological Products.。 This study

Complementary

This study

pKD46 pKD3 pCP20

C50336∆sspH2-psspH2 390 391

S. Enteritidis

AmpR

Resistance

Reference

AmpR

Datsenko and wanner, 2000

CmR

Datsenko and wanner, 2000

CmR, AmpR

Datsenko and wanner, 2000

AmpR

392

Table 2 The primers used in the study to construct mutant and complementary strains 393 Primer

Sequence (5`-3`)

Size (bp)

pKD46-F

ACCGCAAGGACCGTAATC

641 394

pKD46-R

GCAGGGTGTGGAAGTAGGAC

pKD3-F

GCATCAGCACCTTGTCGC

pKD3-R

CTTCGCAGAATAAATAAATCC

pCP20-F

CATACTGGCTAAATACGGAAGG

pCP20-F

CATACTGGCTAAATACGGAAGG

G-SspH2-F

GCGAGTTCAGGGAGTGGA

G-SspH2-R

GCGCTCTGCATCGGTATT

R-SspH2-F

GTAAAGTCGTTCAAAGGGTT

R-SspH2-R

CACTGTTGCTGGATACCCTC

SspH2-Cat-F

TTTATTCGCCGGAAGAGCTGTGCAGGCAGGCAGAATC GAGTGTAGGCTGGAGCTGCTTC

SspH2-Cat-R

GAATATCTTTGTCGCACCGCACCTCATTCACCTGGTGC AATGGGAATTAGCCATGGTCC

395 737

554

3000

2600

1111

396

Table 3. LD50 of S. Enteritidis C50336, C50336∆sspH2 and C50336∆sspH2-psspH2 in BALB/c

397

mice

Strains

C50336-WT

C50336∆sspH2

C50336∆sspH2-psspH2

Inoculation doses (CFU) 1X103

Number of dead animals /total number of mice 0/5

Percentage of deaths/dose 0%

1X104

1/5

20%

1X105

1/5

20%

1X106

4/5

80%

1X107

5/5

80%

1X103

0/5

0%

1X104

0/5

0%

1X105

1/5

20%

1X106

4/5

80%

1X107

5/5

100%

1X10

3

0/5

0%

1X10

4

2/5

40%

1X10

5

2/5

40%

1X106

4/5

80%

7

5/5

100%

0/5

0%

1X10 PBS (Control) 398 399

----

LD50 (CFU)

1x104

3.16x105

7.9x104

Highlights:

SspH2 has no obvious effect on S. Enteritidis virulence SspH2 is involved in colonization of S. Enteritidis in host cells SspH2 performed as an anti-inflammatory effector