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Pathogenicity differences of Salmonella enterica serovars Typhimurium, Enteritidis, and Choleraesuis-specific virulence plasmids and clinical S. Choleraesuis strains with large plasmids to the human THP-1 cell death
Accepted Manuscript Pathogenicity differences of Salmonella enterica serovars Typhimurium, Enteritidis, and Choleraesuis-specific virulence plasmids and clinical S. Choleraesuis strains with large plasmids to the human THP-1 cell death Yao-Kuang Huang, Sheng-Ya Chen, Min Yi Wong, Cheng-Hsun Chiu, Chishih Chu PII:
S0882-4010(18)30288-2
DOI:
https://doi.org/10.1016/j.micpath.2018.12.035
Reference:
YMPAT 3328
To appear in:
Microbial Pathogenesis
Received Date: 19 February 2018 Revised Date:
17 November 2018
Accepted Date: 18 December 2018
Please cite this article as: Huang Y-K, Chen S-Y, Wong MY, Chiu C-H, Chu C, Pathogenicity differences of Salmonella enterica serovars Typhimurium, Enteritidis, and Choleraesuis-specific virulence plasmids and clinical S. Choleraesuis strains with large plasmids to the human THP-1 cell death, Microbial Pathogenesis (2019), doi: https://doi.org/10.1016/j.micpath.2018.12.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Pathogenicity differences of Salmonella enterica serovars Typhimurium, Enteritidis, and Choleraesuis-specific virulence plasmids and clinical S. Choleraesuis strains with large plasmids to the human THP-1 cell death
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Yao-Kuang Huanga, Sheng-Ya Chena,b, Min Yi Wonga, and Cheng-Hsun Chiuc,d, Chishih Chub*
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b
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d
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Division of Thoracic and Cardiovascular Surgery, Chiayi Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan
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Department of Microbiology, Immunology, and Biopharmaceuticals, National Chiayi University, Chiayi, Taiwan
Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan
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Division of Pediatric Infectious Diseases, Department of Pediatrics, Chang Gung Children’s Hospital and Chang Gung University, Taoyuan, Taiwan
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Running title: Different pathogenicity of Salmonella virulence plasmid
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*Corresponding author.
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Dr. Chishih Chu
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Department of Microbiology, Immunology, and Biopharmaceuticals
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National Chiayi University
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Address: No.300, University Rd., Chiayi City 60004, Taiwan
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Tel: 886-5-2717898
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Fax: 886-52717830
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E-mail: [email protected]
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Abstract Salmonella is a common foodborne and zoonotic pathogen. Only a few serovars carry a
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virulence plasmid (pSV), which enhances the pathogenicity of the host. Here, we investigated
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the pathogenicity roles of the pSVs among wild-type, plasmid-less, and complemented S.
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Typhimurium, S. Enteritidis S. Choleraesuis in invasion, phagocytosis, and intracellular
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bacterial survival in human THP-1 cells and cell death patterns by flow cytometry and
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difference in cell death patterns between pig and human S. Choleraesuis isolates with large
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pSCVs. Virulence plasmid (pSTV) led to slightly increasing cellular apoptosis for S.
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Typhimurium; virulence plasmid (pSEV) enhanced apoptosis and necrosis significantly for S.
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Enteritidis; and pSCV reduced apoptosis significantly for S. Choleraesuis. After
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complementation, pSTV increased the intracellular survival of pSCV-less Choleraesuis and
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the cytotoxicity against human THP-1 cells. Using the Cytochalasin D to differentiate the
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invasion of S. Choleraaesuis and phagocytosis of THP-1 cells determined that pSCV were
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responsible for invasion and phagocytosis at 0 h and inhibited intracellular replication in
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THP-1 cells, and pSTV were responsible for invasion and increased intracellular survival for
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S. Choleraesuis in THP-1 cells. The human isolates with large pSCV induced more cellular
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apoptosis and necrosis than the pig isolates. In conclusion, human S. Choleraesuis isolates
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carrying large pSCVs were more adapted to human THP-1 cells for more cell death than pig
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isolates with large pSCV. The role of pSVs in invasion, phagocytosis, intracellular survival
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and apoptosis differed among hosted serovars.
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Keywords: Salmonella, S. Choleraesuis, S. Enteritidis, S. Typhimurium, Virulence
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plasmid, Apoptosis, Necrosis, intracellular survival
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1. Introduction Salmonella is one of the most prevalent foodborne pathogens worldwide, and salmonellosis
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in humans is frequently associated with the consumption of food products of animal origin and their
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byproducts. Salmonella species constantly face different stress conditions in the environment
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and in the gastrointestinal tract, such as the extreme pH conditions that prevail in the stomach
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[1-3]. Among the nontyphoid serovars involved in salmonellosis, S. Choleraesuis and S. Dublin
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cause bacteremia rather than diarrhea [4]. In C3H/HeN mice, bacteremic non-typhoidal S.
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Schwarzengrund, S. Bredeney and S. Virchow are low in stimulating systemic disease, S.
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Schwarzengrund and S. Enteritidis are profound and elongated shedding, and S.
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Typhimurium cause chronic infection and lower intracellular growth [5], suggesting
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enhanced invasion and intracellular growth not enough to become bacteremic strains.
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Furthermore, SPI-2 allows the bacteria to reside and proliferate within macrophages,
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furthering their reach and survival within the lymphatic system and bloodstream [6-8].
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The innate immune system provides the first line of defense against invading
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microorganisms by inflammatory and antimicrobial responses [9,10]. Further, apoptosis plays
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an essential role in normal development, homeostasis, and pathogenic processes [11]. Signals
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such as phosphatidylserine are exposed on the outside of apoptotic bodies to attract
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macrophages for phagocytosis more efficiently [12]. In Salmonella, S. Dublin induces DNA
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fragmentation of early apoptosis in human THP-1 cell dependent on phoP expression, not
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spvB, and caspase-3, 8, and 9 [13]. By contrast, necrosis is morphologically characterized by
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the swelling of organelles, disruption of the plasma membrane, increase in cell volume, and
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loss of intracellular contents [14].
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The serovar-associated virulence plasmids (pSVs) in Salmonella may correlate with host
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specificity. These pSVs differ in gene composition, plasmid size and possibly origins [15,16], but
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all of them encode an 8-kb spvRABCD operon, which is positively regulated by the SpvR and the 3
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within host cells [17-19]. In zebrafish, the spv operon inhibits the functions of neutrophils and
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macrophages of early innate immune responses and accelerates the replication and
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dissemination of S. Typhimurium [20]. Furthermore, the spv locus inhibits transcription of
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the tbx21 gene and suppresses expression of the cytokines IFN-γ, IL-12, and TNF-α, but it
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enhances the expression of the gata3 gene and the cytokines IL-4, IL-10, and IL-13 [21].
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Furthermore, SpvB prevents actin polymerization through ADP-ribosylation, facilitates apoptosis
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[12] and inhibits early autophagy formation in zetafish [22], suppresses autophagosome
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formation and increases inflammation in murine macrophage-like cells J774A.1 and human
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epithelial HeLa cells during infection [23].
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Expression of rck gene on virulence plasmid is regulated by the temperature and quorum-
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sensing regulator SdiA on the chromosome [24], demonstrating the interaction between genome
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and virulence plasmid during infection. Previously, we reported that large pSCVs are evolved
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through recombination of the R plasmid and the 50-kb pSCV in human and pig isolates
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[25,26]. However, we did not determine the virulence among these clinical human and swine
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isolates with larger pSCV.
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2. Materials and Methods
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2.1 Bacterial strains
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The bacterial strains listed in Table 1 were employed to investigate the role of pSVs in three
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serovars and in clinical S. Choleraesuis swine strains CN30 and CN32, and human strains SCB52,
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SCB74, SCB97, SCB130, and MS22872 with large pSCV through pathogenicity assays. These
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strains included wild-type S. Typhimurium (OU5045), S. Enteritidis (OU7130), and S. Choleraesuis
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(OU7085) with 90-kb pSTV, 60-kb pSEV and 50-kb pSCV, respectively; their pSV-less strains
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OU5046, OU7067, and OU7266; and complemented strains OU5193, OU5194, OU7294, and 4
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strains were kindly provided by Dr. Jonathan T. Ou and examined by plasmid analysis and PCR
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amplification of invC for Salmonella and spvA for pSV (Fig. 1). All of the bacterial strains used for
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cell lysis assays grew routinely on xylose lysine deoxycholate (XLD) plates. A single black colony
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was grown overnight in Luria-Bertani (LB) broth at 37 °C. All strains for
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2.2 Cell and bacterial culture
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Murine RAW 264.7 cells grew in RPMI 1640 medium (Sigma, R6504) supplemented
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with 10% preheated fetal bovine serum (FBS, Sigma, SAB-1900SC-500 ml), D-glucose (J.T.
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Baker, 1916-01), sodium bicarbonate (Sigma, S5761), 10 mM HEPES (Sigma, H0887), 1
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mM sodium pyruvate (Sigma, S8636), and a 1% antibiotic solution of penicillin,
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streptomycin, and amphotericin (Sigma, A5955). Human monocyte THP-1 cells were
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incubated routinely in RPMI 1640 medium (GIBCO, ThermoFisher Scientific, Taiwan)
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supplements with 10% FBS, 10 mM HEPES buffer, 2.5 mM sodium pyruvate, 2 mM L-
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glutamine, and 100 µg/ml penicillin/streptomycin at 37 °C and 5% CO2.
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For the phagocytosis and intracellular survival tests, 50 µM of phorbol myristate acetate
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(Sigma, P8139) were added to THP-1 cells to stimulate the macrophage differentiation at
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37°C for 48 h. The bacterial strains and E. coli (ATCC 8739) were incubated at 37°C for 12 h,
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respectively. After centrifugation for 10 min at 3,000 x g (KUBOTA 2010, Kubota, Japan),
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the pellets were washed with phosphate-buffered saline (PBS) several times and were
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resuspended with PBS solution. The OD550 of the bacterial solutions was measured using a
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Bio-Rad SmartSpecTM 3000 (approximately 8.6 × 108 bacteria per mL at OD550 = 1). The
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phagocytosis, cell death, and intracellular survival tests were performed in three independent
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experiments with two repeats each.
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2.3 Intracellular survival and phagocytosis
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differentiated THP-1 cells (MOI = 10), respectively, in each well of a 24-well plate (Becton,
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Dickinson and Company) and incubated with RPMI 1640 medium without antibiotics at
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37°C with 5% CO2 for 30 min or 1 h. The extracellular bacteria were killed by treating with
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gentamicin at a final concentration of 50 µg/mL for 1 h (corresponding to time 0 h). After the
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cell lysis ith 0.5 mL of 1% sodium deoxycholate (Sigma-Aldrich), 100 µL aliquots of the
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solution were plated on individual LB plates. The viable bacteria were counted for each plate
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after incubation at 37°C for 24 h. Furthermore, the invasion of the differentiated THP-1 cells
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were evaluated by treating with 5 µg/ml of cytochalasin D for 1 h at 37°C to inhibit
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phagocytosis. Subsequent infection and intracellular bacteria number were performed as
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described above.
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2.4 Cytotoxicity of different serovars and clinical S. Choleraesuis strains to human
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THP-1 cells
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Human monocyte THP-1 cells were cultured routinely, as described earlier.
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Furthermore, the bacteria and cell mixtures at MOI = 10:1 as above were incubated at 37°C
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for different periods depending on the experiment and then washed twice with PBS. After
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centrifugation and wash with PBS twice, the cells were treated with 0.25% trypsin in 0.53
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mM EDTA solution for 5 min and then 800 µl of RPMI 1640 medium with 10% FBS were
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added to each well. The cells were pelleted at 800 x g for 10 min. After washing with PBS,
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the cells were stained with propidium iodide (PI) and annexin V using an AnnexinV-FITC
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Apoptosis Detection Kit (Cat. No. AVK250, Strong Biotech Corporation, Taiwan) for 10–15
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min. The fluorescence of PI and annexin V was measured by flow cytometry, and the data
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were analyzed using WinMDI software.
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2.5 Statistical analysis
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one-way ANOVA, and a post-hoc Tukey’s HSD test using SPSS 18.0 software. The results
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were considered statistically significant when p < 0.05, and all data were displayed as the
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mean ± SD.
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3. Results
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3.1 Interaction of wild-type Salmonella serovars with different monocyte cell types
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Intracellular bacterial analysis of three serovars demonstrated that the highest bacterial
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number was observed for broad-host S. Typhimurium and S. Enteritidis in mouse RAW 264.7
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cells and for S. Enteritidis in human THP-1 cells and the lowest number for narrow-host S.
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Choleraesuis in both cell lines (Table 2). However, intracellular number of S. Choleraesuis
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was not change in RAW 264.7 cells compared to a gradual increase from 1 h to 2 h in the
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human THP-1 cells. Furthermore, intracellular E. coli was not observed in the human THP-1
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cells, compared to lowest number in mouse RAW 264.7, suggesting intracellular bacterial
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number depending on cell lines. During intracellular bacterial survival analysis, we observed
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two cell populations at the invasion and intracellular survival stages, and changes in the ratios
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of the three aforementioned serovars were observed (Table 3). During the invasion stage, S.
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Choleraesuis exhibited the lowest ratio and S. Typhimurium exhibited the highest ratio. At the
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intracellular survival stage, the ratio increased gradually for S. Typhimuirum, and decreased
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for S. Enteritidis. However, S. Cholearesuis exhibited the highest ratio at 0 h, the lowest ratio
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at 1 h, and then an increase in ratio from 1 h to 4 h. This change in cell size may be related to
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cell death.
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3.2 Roles of the pSVs of three serovars in cell death
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All serovars induced more apoptosis than necrosis and differed in their effects on cell
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viability, apoptosis, and necrosis (Fig. 2). Three wild-type serovars caused different viable
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cell patterns. S. Typhimurium infection showed the highest number of viable cells at 0.5 h 7
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phenomenon of increasing the viable cell number. However, S. Enteritidis infection killed all
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cells. Apoptosis analysis showed that S. Enteritidis caused the highest rate of apoptosis, S.
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Typhimurium showed an increase of apoptosis from 0.5 h to 2 h and S. Choleraesuis showed
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an significant decrease of apoptosis.
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The patterns of cellular viability, apoptosis and necrosis differed between wild-type and
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pSTV-less S. Typhimurium. pSTV-less OU5046 increased cellular ability and decreased
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apoptosis positively associated with the incubation time, compared that both pSTV-
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complemented strains OU5193 and OU5194 increased cellular viability and reduced necrosis
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at time-dependent pattern. In the case of S. Enteritidis, the wild-type and complemented
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strains demonstrated identical effects: no viable cells and nearly 100% apoptosis. However,
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treatment with the pSEV-less strain yielded the highest rate of viable cells and necrosis, and
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the least apoptotic rate. The S. Choleraesuis wild-type OU7085 and the pSCV-complemented
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OU7294 demonstrated similar patterns of influence, increasing ratio of viable cells and
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decreasing the ratio of apoptosis as well as necrosis. By contrast, the pSCV-less OU7266
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decreased the rate of viable cells and increased apoptotic rate.
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The role of the pSVs differed among serovars with slightly increase of apoptosis and
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necrosis for pSTV of S. Tphimurium, a significantly increase in cell death for S. Enteritidis,
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and a decrease of cell death for S. Choleraesuis.
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3.3 Intracellular survival of wild-type, pSCV-less and pSCV-complementary S.
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Choleraesuis strains in human THP-1 cells and infection related cell death
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These three strains differed in cell death and intracellular bacterial patterns (Table 4).
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Wild-type strain displayed an increase of cell death and very few intracellular bacteria. By
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contrast, the pSCV-less OU7266 increased in rate of cell death and higher intracellular
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bacteria ranged from 2- to 7.6 x 103 bacteria per 103 cells. Nevertheless, Tn5-inserted pSCV 8
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from 0 h to 4 h, but with higher apoptosis rate at 8 h and significantly less intracellular
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bacteria than the pSCV-less strain and higher than the wild-type. These results confirmed
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above pathogenicity roles of the pSCV in apoptosis and displayed that pSCV may inhibited
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intracellular survival.
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3.4 The role of pSVs in the invasion, phagocytosis and intracellular survival
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Cytochalasin D treatment can inhibit actin polymerization and, subsequently, the
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phagocytosis of bacteria by human macrophage-like THP-1 cells. Therefore, such a treatment
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can differentiate Salmonella invasion from the phagocytosis of macrophages. At 0 h, wild-
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type OU7085 exhibited a limited intracellular bacterial number derived from phagocytosis,
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whereas pSCV-less OU7226 exhibited the highest intracellular number with almost equal
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ratios of invasion (48.4%) and phagocytosis (51.6%), suggesting pSCV may increase of
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phagocytosis and invasion. Furthermore, intracellular bacteria changed over time (Table 5).
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By contrast, the pSTV-complemented OU7194 demonstrated that invasion was the cause
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(100%) of intracellular bacteria, and an increase in intracellular survival occurred in a time-
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dependent manner. These results imply pSCV responsible for invasion and phagocytosis at o
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h and following by decreasing intracellular survival, and pSTV responsible for invasion and
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increase in intracellular survival in S. Choleraesuis.
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3.5 Cytotoxicity of clinical human and swine S. Choleraesuis isolates with large pSCV to
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human THP-1 cells
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Cytotoxicity against differentiated THP-1 cells differed among the human and pig
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isolates in comparison to the laboratory OU7085 derived from humans (Table 7). At 2-h post-
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infection, the human isolates displayed the lowest viable cells and the highest rate of
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apoptosis, whereas the pig isolates exhibited the highest rate of necrosis. At 4-h post-infection, 9
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viable cells compared with OU7085. At 8-h post-infection, the human isolates displayed the
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highest apoptotic rates and the lowest rate of viable cells and necrosis.
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4. Discussion
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Different host range may be regulated by the gene acquisition via horizontal gene
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transfer with plasmids, transposons, and phages, the loss of genes or their function
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(pseudogenes) and interactions with the host’s immune system and commensal organisms
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during infection [27]. S. Typhimurium, S. Enteritidis, and S. Choleraesuis differ in the host-
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specific symptoms and invasion types. Such difference may be resulted from gene
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inactivation more in narrow-host-range S. Choleraeuis than in broad-host-range S.
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Typhimurium [28], more intracellular bacterial number of broad-host S. Typhimurium and S.
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Enteritidis than S. Choleraesuis in mouse RAW 264.7 macrophage cells and human THP-1
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cells (Table 2), gene interaction between virulence plasmid and chromosome [24] and serovar-
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specific genes, such that S. Choleraesuis lacks the enzymes required for citrate metabolism,
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including tricarballylate dehydrogenase and oxaloacetate decarboxylase, to evade activation
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of the NLPR3 inflammasome and to promote pathogenicity [29]. In macrophages, Salmonella
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evolves mechanisms to avoid host immune responses. Salmonella can quench arginine away
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from the inducible NO synthase (iNOS) pathway via arginine transporters (ArgT) for growth
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and survival [30]. Thus, S. Typhimurium with ArgT expression can evade being killed by
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macrophages and cause a severe systemic infection.
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During intracellular growth, 20 pSTV genes in S. Typhimuriun were upregulated,
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including repA, ccdA, rsd, virE, spvRA, tlpA, samAB, traTD, and trbH [31]. However, a few
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of these genes are not present in pSEV and pSCV [10]. These missing genes may change of
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the distribution of human THP-1 cell populations (Table 3). Such change may be due to
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differences in apoptosis rather than necrosis (Figure 1) and conditions of intracellular bacteria. 10
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In S. Typhimurium, genes ydgT, recA, and corA are involved in the cytosolic proliferation
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and asmA reduced the cytosolic replication [32] and a 597-nt IesR-1 sRNA encoded by the
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pSTV in dormant S. Typhimurium persisting inside fibroblasts is upregulated [33]. Early study reported that the DNA fragmentation was related to phoP expression, not
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related to SPI-1 and SPI-2, spvB, and caspase gene expression [11]. In this study, the
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pathogenicity roles of the pSVs in their host serovars and pSTV in pSCV-less S. Choleraesuis
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differed. In the original hosted serovar, pSTV and pSEV differed the apoptotic ability with
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complete cell killing for S. Enteritidis and lesser apoptosis for S. Typhimurium, but pSCV
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inhibited the apoptotic ability (Figure 1) and intracellular survival of S. Choleraesuis (Table
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4). Furthermore, cytochalasin D demonstrated pSCV inhibited phagocytosis of the human
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THP-1 cells and S. Choleraesuis invasion mainly for intracellular bacteria. By contrast, pSTV
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enhanced both phenomena, although the increase was limited (Table 5). All above results
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imply that pSCV and pSTV may play different pathogenicity roles for S. Choleraesuis and
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co-evolution of the pSVs with their hosted serovars.
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Host adaptation and/or evolution may be associated with genome degradation, such as
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pseudogene formation and deletion. Investigation of multiple S. Enteritidis isolates with
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mutation in the mismatch repair gene mutS from an interleukin (IL)-12 β-1 receptor-deficient
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individual with a 15-year recurrent blood-borne infection demonstrated genome changes
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associated within-host evolution on the timescale [34]. Furthermore, the evolution of the 50-
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kb pSCV to become large pSCVs (75–140 kb) is through the recombination of the R plasmid
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[26] and the involvement of co-evolution of large pSCV associated with human isolates, not
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pig isolates, caused the higher apoptotic rates in human THP-1 cells (Table 6). Nevertheless,
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the pSTVs in human-specific S. Typhi and Paratyphi A did not enhance their pathogenicity
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in infant mice [35]. These data demonstrate that coevolution of pSVs with their host serovar
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plays an important role in host-specific infection. However, a deeper investigation is required
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to determine whether the exchanged pSVs remain serovar-specific.
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5. Conclusion The pSVs differed in size and genes as well as their pathogenicity role among S.
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Typhimuirum, S. Enteritidis, and S. Choleraesuis. The pSTV and pSEV enhanced the
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apoptosis with difference in ratio between these two, but pSCV inhibited the apoptosis.
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Further, pSCV promoted invasion and phagocytosis as well as decreased intracellular
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bacterial survival, and pSTV stimulated invasion and intracellular survival in S. Choleraesuis,
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demonstrating a difference in host adaptation and coevolution of chromosome characteristics
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and/or virulence plasmids for the virulence and adaptation to host species.
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Competing interests
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The authors declare that they have no competing interests.
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This work was supported by grants from the Ministry of Science and Technology (MOST 102-2320-
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B-415-004 for CC) Executive Yuan, and Chiayi Chang Gung Memorial Hospital (grant numbers:
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CMRPG6B0503, CMRPG6E0423, and CMRPG6G0101 for YKH), ROC (Taiwan).
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References
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1. J.W. Foster, H.K.Hall. Inducible pH homeostasis and the acid tolerance response of
294 295 296
Salmonella typhimurium. J Bacteriol. 173(1991)5129-5135. 2. Foster JW. The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins. J Bacteriol 175(1993)1981-1987.
12
ACCEPTED MANUSCRIPT 297
3. A. Álvarez-Ordóñez, M. Prieto, A. Bernardo, C. Hill, M. López. The Acid Tolerance
298
Response of Salmonella spp.: an adaptive strategy to survive in stressful environments
299
prevailing in foods and the host. Food Res Int. 45(2012)482-492. 4. M.J. Blaser, R.A. Feldman. Salmonella bacteremia: reports to the Centers for Disease Control, 1968-1979. J Infect Dis. 143(1981)743-746
301
RI PT
300
5. J. Suez J, Porwollik, A. Dagan, A. Marzel, Y.I. Schorr, P.T. Desai, V. Agmon, M.
303
McClelland, G. Rahav, O. Gal-Mor. Virulence gene profiling and pathogenicity
304
characterization of non-typhoidal Salmonella accounted for invasive disease in humans.
305
PLOS ONE 8 (2013) e58449.
SC
302
6. F. Garcia-del Portillo, M.B. Zwick, K.Y. Leung, B.B. Finlay. Salmonella induces the
307
formation of filamentous structures containing lysosomal membrane glycoproteins in
308
epithelial cells. Proc Natl Acad Sci U S A. 90 (1993)10544-10548.
M AN U
306
7. A. Gallois, J.R. Klein, L.A.H. Allen, B.D. Jones, W.M. Nauseef. Salmonella pathogenicity
310
island 2-encoded type III secretion system mediates exclusion of NADPH oxidase
311
assembly from the phagosomal membrane. J Immunol.166 (2001)5741-5748.
312
8.
TE D
309
D. Chakravortty, I. Hansen-Wester, M. Hensel. Salmonella pathogenicity island 2 mediates protection of intracellular Salmonella from reactive nitrogen intermediates. J
314
Exp Med. 195(2002)1155-1166.
316 317 318 319 320
9.
AC C
315
EP
313
P. Broz, M.B. Ohlson, D.M. Monack. Innate immune response to Salmonella
typhimurium, a model enteric pathogen. Gut Microbes. 3(2012)62-70. 10. D. Hurley, M.P. McCusker, S. Fanning, M. Martins. Salmonella-host interactions modulation of the host innate immune system. Front Immunol. 5(2014)481. 11. E. Valle, D.G. Guiney. Characterization of Salmonella-induced cell death in human macrophage-like THP-1 cells. Infect Immun. 73(2005)2835-2840.
13
ACCEPTED MANUSCRIPT 321
12. R.G. Deschesnes, J. Huot, K. Valerie, J. Landry. Involvement of p38 in apoptosis-
322
associated membrane blebbing and nuclear condensation. Mol Biol Cell 12(2001)1569-
323
1582.
325 326 327
13. I.K. Poon, C.D. Lucas, A.G. Rossi, K.S. Ravichandran. Apoptotic cell clearance: basic biology and therapeutic potential. Nat Rev Immunol. 14(2014)166-180.
RI PT
324
14. W. Wu, P. Liu, J. Li. Necroptosis: an emerging form of programmed cell death. Crit Rev Oncol Hematol. 82(2012):249-258.
15. C. Chu, S-F. Hong, C. Tsai, W-S. Lin, T-P. Liu, J.T. Ou. Comparative physical and
329
genetic maps of the virulence plasmids of Salmonella enterica serovars Typhimurium,
330
Enteritidis, Choleraesuis, and Dublin. Infect Immun. 67(1999)2611-2614.
332
M AN U
331
SC
328
16. C. Chu, C.H. Chiu. Evolution of the virulence plasmids of non-typhoid Salmonella and its association with antimicrobial resistance. Microbes Infect. 8(2006)1931-1936. 17. F.C. Fang, M. Krause, C. Roudier, J. Fierer, D. Guiney. Growth regulation of a
334
Salmonella plasmid gene essential for virulence. J Bacteriol. 173(1991)6783-6789.
335
18. M. Krause, F.C. Fang, D. Guiney D. Regulation of plasmid virulence gene expression in
336
Salmonella dublin involves an unusual operon structure. J Bacteriol. 174(1992)4482-
337
4489.
EP
TE D
333
19. F.C. Fang, S.J. Libby, N.A. Buchmeier, P.C. Loewen, J. Switala, J. Harwood, D.G.
339
Guiney. The alternative sigma factor katF (rpoS) regulates Salmonella virulence. Proc
340
Natl Acad Sci USA. 89(1992)11978-11982.
AC C
338
341
20. S.Y. Wu, L.D. Wang, J.L. Li, G.M. Xu, M.L. He, Y.Y. Li, R. Huang. Salmonella spv
342
locus suppresses host innate immune responses to bacterial infection. Fish Shellfish
343
Immunol. 58(2016)387-396.
14
ACCEPTED MANUSCRIPT 344
21. S.Y. Wu, L.D. Wang, G.M. Xu, S.D. Yang, Q.F. Deng, Y.Y. Li, R. Huang. spv locus
345
aggravates Salmonella infection of zebrafish adult by inducing Th1/Th2 shift to Th2
346
polarization. Fish Shellfish Immunol. 67(2017)684-691. 22. Y.Y. Li, T. Wang, S. Gao, G.M. Xu, H. Niu, R. Huang, S.Y. Wu. Salmonella plasmid
348
virulence gene spvB enhances bacterial virulence by inhibiting autophagy in a zebrafish
349
infection model. Fish Shellfish Immunol. 49(2016)252-259.
RI PT
347
23. Y. Chu, S. Gao, T. Wang, J. Yan, G. Xu, Y. Li, H. Niu, R. Huang, S. Wu. A novel
351
contribution of spvB to pathogenesis of Salmonella Typhimurium by inhibiting
352
autophagy in host cells. Oncotarget 7(2016)8295-8309.
SC
350
24. N. Abed, O. Grépinet, S. Canepa, G.A. Hurtado-Escobar, N. Guichard, A. Wiedemann, P.
354
Velge, I. Virlogeux-Payant. Direct regulation of the pefI-srgC operon encoding the Rck
355
invasin by the quorum-sensing regulator SdiA in Salmonella Typhimurium. Mol
356
Microbiol. 942(2014)254-271.
M AN U
353
25. C. Chu, C.H. Chiu, W.Y. Wu, C.H. Chu, T.P. Liu, J.T. Ou. Large drug resistance
358
virulence plasmids of clinical isolates of Salmonella enterica serovar Choleraesuis.
359
Antimicrob Agents Chemother. 45(2001)2299-2303.
TE D
357
26. J.I. Tzeng, C.H. Chu, S.W. Chen, C.M. Yeh, C.H. Chiu, C.S. Chiou, J.H. Lin, C. Chu.
361
Reduction of Salmonella enterica serovar Choleraesuis carrying large virulence
362
plasmids after the foot and mouth disease outbreak in swine in southern Taiwan, and
363
their independent evolution in human and pig. J Microbiol Immunol Infect.
364
45(2012)418-425.
AC C
EP
360
365
27. S.L. Foley, T.J. Johnson, S.C. Ricke, R. Nayak, J. Danzeisen. Salmonella pathogenicity
366
and host adaptation in chicken-associated serovars Microb Mol Bio Rev. 77(2013)582–
367
560
15
ACCEPTED MANUSCRIPT 368
28. C.H. Chiu, P. Tang, C. Chu, S. Hu, Q. Bao, L. Yu, Y.Y. Chou, H.S. Wang, Y.S. Lee. The
369
genome sequence of Salmonella enterica serovar Choleraesuis, a highly invasive and
370
resistant zoonotic pathogen. Nucleic Acids Res. 33(2005)1690-1698. 29. M.A. Wynosky-Dolfi, A.G. Snyder, N.H. Philip, P.J. Doonan, M.C. Poffenberger, D.
372
Avizonis, E.E. Zwack, A.M. Riblett, B. Hu, T. Strowig, R.A. Flavell, R.G. Jones, B.D.
373
Freedman, I.E. Brodsky. Oxidative metabolism enables Salmonella evasion of the
374
NLRP3 inflammasome. J Exp Med. 211(2014)653-668.
RI PT
371
30. P. Das, A. Lahiri, A. Lahiri, M. Sen, N. Iyer, N. Kapoor, K.N. Balaji, D. Chakravortty.
376
Cationic amino acid transporters and Salmonella Typhimurium ArgT collectively
377
regulate arginine availability towards intracellular Salmonella growth. PLoS One.
378
5(2010)e15466.
M AN U
SC
375
31. S. Eriksson, S. Lucchini, A. Thompson, M. Rhen, J.C. Hinton. Unravelling the biology
380
of macrophage infection by gene expression profiling of intracellular Salmonella
381
enterica. Mol Microbiol. 47(2003)103-118.
TE D
379
32. M. Wrande, H. Andrews-Polymenis, D.J. Twedt, O. Steele-Mortimer, S. Porwollik, M.
383
McClelland, L.A. Knodler. Genetic determinants of Salmonella enterica Serovar
384
Typhimurium poliferation in the ctosol of epithelial cells. Infect Immun. 84(2016)3517-
385
3526.
AC C
EP
382
386
33. J. Gonzalo-Asensio, A.D. Ortega, G. Rico-Perez, M.G. Pucciarelli, F. Garcıa-del Portillo.
387
A novel antisense RNA from the Salmonella virulence plasmid pSLT expressed by
388
non-growingbacteria inside eukaryotic cells. PLoS One 8(2013) e77939.
389
34. E.J. Klemm, E. Gkrania-Klotsas, J. Hadfield, J.L. Forbester, S.R. Harris, C. Hale, J.N.
390
Heath, T. Wileman, S. Clare, L. Kane, D. Goulding, T.D. Otto, S. Kay, R. Doffinger,
391
F.J. Cooke, A. Carmichael, A.M.L. Lever, J. Parkhill, C.A. MacLennan, D.
392
Kumararatne, G. Dougan, R.A. Kingsley. Emergence of host-adapted Salmonella 16
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Enteritidis through rapid evolution in an immunocompromised host. Nat Microbiol.
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1(2016):15023. 35. M.J. Santander, R. Curtiss III. Salmonella enterica Serovars Typhi and Paratyphi A are
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avirulent in newborn and infant mice even when expressing virulence plasmid genes of
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Salmonella Typhimurium. J Infect Dev Ctries. 4(2010)723.
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Legends
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Figure 1. Plasmid and PCR analysis of wildtype, virulence plasmid (pSV)less, and pSV
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complementatry strains of S.Typhimurium, S. Enteritidis, and S. Choleraesuis. A)
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Plasmid analysis. B) PCR identification of
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identification and 571-bp spvC fragment for pSV identification. M: 100 bp marker, N:
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Negative, 1: OU5045, 2: OU5046, 3: OU5193, 4: OU5194, 5: OU7130, 6: OU7067, 7:
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OU7415, 8: OU7085, 9: OU7266, 10: OU7194, 11: and OU7294.
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244-bp invA fragment for Salmonella
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Figure 2. Difference in death type and rate of human THP-1 cells infected by wildtype,
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virulence plasmid (pSV)less, and pSV complementatry strains of S.Typhimurium, S.
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Enteritidis, and S. Choleraesuis.
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Strains
The pSV Characteristics
OU5045
Wild type with a 90-kb pSTV
OU5046
pSTV-less strain derived from wild type
S. Typhimurium
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OU5193
Tn5-tagged pSTV complemented strains OU5194
OU7067
pSEV-less strain derived from wild type
OU7415
Tn5-tagged pSEV complemented strain
OU7085
Wild type with a 50-kb pSCV
OU7266 OU7194
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Wild type with a 60-kb pSEV
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S. Enteritidis
OU7130
pSCV-less strain derived from wild type
Tn5-tagged pSTV complemented strains OU7294
S. Choleraesuis
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CN30
CN32
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SCB52 SCB97
Clinical isolates with a large pSCV
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SCB130 SCB74
MS22872
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Table 2. Intracellular numbers of different serovars in mouse and human macrophage cells Mouse RAW 264.7 cells
Human THP-1 cells
Strains 2h
4h
1h
2h
4h
4 x101
9 x101
4 x101
7.2 x101
0
0
0
>3 x 103
OU5045 OU7085 2.3 x102
1.0 x 103
3.33 x 102 2.37 x 103 2.05 x 104
4.8 x 102 4.3 x 102
>3 x 103
7.67x101
3.47x103
3.17 x 103
1.52 x 104 7.30 x 104 4.10 x 105
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E. coli
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Intracellular survival
Serovars 0
1
2
4
OU5045
0.536
1.022
1.584
2.458
3.346
OU7085
0.441
1.664
0.960
1.205
1.469
OU7130
0.503
1.621
1.021
0.837
0.710
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ACCEPTED MANUSCRIPT Table 4. Intracellular number and cytotoxicity of S. Choleraesuis isolates in human THP-1 cells after infection with MOI=10 Item
0h
2h
4h
8h
Control
Cell death (%)
0
3
4
5
Cell death (%)
0
24
31
32
Intracellular No/103 cells)
0.03
0.17
1.9
2.8
Cell death (%)
0
27
34
45
Intracellular No/103 cells
2 x103
4 x103
7.6 x 103
3.6 x103
pSCV-complemented
Cell death (%)
0
25
37
58
OU7294
Intracellular No/103 cells
2.5 x102
5.6 x 101
Wild type OU7085
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pSCV-less OU7266
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Table 5. Invasion of different S. Choleraesuis strains and phagocytosis of human THP-1 cells Bacteria Cytochalasin D 0h 2h 4h 0
0
0
+
0
0
0
-
0.18/100
0.15/100
1.8/100
+
0.02/11.1
0/0
0.06/3.3
(-) – (+)
0.16/88.9
0.15/100
1.74/96.7
1.50/96.2
-
3.1 x 103/100
7.4 x 103/100
7.2 x 103/100
9.4 x 103/100
+
1.5 x 103/48.4
1.8 x103/24.3 2.7 x 103/37.5
2.7 x 103/28.7
1.6 x 103/51.6 5.6 x 103/75.7 4.5 x 103/62.5
6.6 x 103/71.3
-
1.7 x 101/100
6.9 x 102/100
4.7 x 102/100
+
2.3 x 101/100 6.9 x 101/40.6 1.4 x 102/20.2
2.1 x 102/44.7
1.2 x 101/59.4 5.5 x 102/79.8
2.6 x 102/55.3
(-) – (+)
-6/0
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(-) – (+)
0
2.6/100
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1.7 x 102/100
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pSTV-complemented OU7194
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pSCV-less OU7266
0
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Wild type OU7085
8h
6
0.1/3.8
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8h
Apoptosis
Necrosis
OU7085
89.0±0.31a
9.8±0.27b
1.3±0.20b
Pig isolates
87.2±0.98a
9.6±0.56b
3.2±0.67a
Human isolates
77.0±0.90b
22.0±0.83a
OU7085
90.5±0.46a
8.2±0.26c
Pig isolates
85.9±1.05b
10.1±0.51b
Human isolates
81.3±0.63c
17.6±0.56a
1.1±0.17b
OU7085
90.8±0.26a
7.5±0.45c
1.7±0.46a
Pig isolates
88.3±1.23ab
9.9±1.02b
1.9±0.32a
Human isolates
87.3±0.50b
12.4±0.48a
0.3±0.03b
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Viability
1.0±0.87b
1.3±0.24b 4.0±0.87a
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Source
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ACCEPTED MANUSCRIPT Highlight
The pSVs differed in size and genes as well as their pathogenicity role among S.
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Typhimuirum, S. Enteritidis, and S. Choleraesuis. The pSTV and pSEV enhanced the apoptosis with difference in ratio between these two, but pSCV inhibited the apoptosis. Further, pSCV promoted invasion and phagocytosis as well as decreased intracellular bacterial survival, and pSTV
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virulence and adaptation to host species.
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S0882-4010(18)30288-2
DOI:
https://doi.org/10.1016/j.micpath.2018.12.035
Reference:
YMPAT 3328
To appear in:
Microbial Pathogenesis
Received Date: 19 February 2018 Revised Date:
17 November 2018
Accepted Date: 18 December 2018
Please cite this article as: Huang Y-K, Chen S-Y, Wong MY, Chiu C-H, Chu C, Pathogenicity differences of Salmonella enterica serovars Typhimurium, Enteritidis, and Choleraesuis-specific virulence plasmids and clinical S. Choleraesuis strains with large plasmids to the human THP-1 cell death, Microbial Pathogenesis (2019), doi: https://doi.org/10.1016/j.micpath.2018.12.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Pathogenicity differences of Salmonella enterica serovars Typhimurium, Enteritidis, and Choleraesuis-specific virulence plasmids and clinical S. Choleraesuis strains with large plasmids to the human THP-1 cell death
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Yao-Kuang Huanga, Sheng-Ya Chena,b, Min Yi Wonga, and Cheng-Hsun Chiuc,d, Chishih Chub*
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a
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b
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c
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d
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Division of Thoracic and Cardiovascular Surgery, Chiayi Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan
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Department of Microbiology, Immunology, and Biopharmaceuticals, National Chiayi University, Chiayi, Taiwan
Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan
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Division of Pediatric Infectious Diseases, Department of Pediatrics, Chang Gung Children’s Hospital and Chang Gung University, Taoyuan, Taiwan
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Running title: Different pathogenicity of Salmonella virulence plasmid
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*Corresponding author.
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Dr. Chishih Chu
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Department of Microbiology, Immunology, and Biopharmaceuticals
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National Chiayi University
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Address: No.300, University Rd., Chiayi City 60004, Taiwan
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Tel: 886-5-2717898
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Fax: 886-52717830
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E-mail: [email protected]
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Abstract Salmonella is a common foodborne and zoonotic pathogen. Only a few serovars carry a
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virulence plasmid (pSV), which enhances the pathogenicity of the host. Here, we investigated
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the pathogenicity roles of the pSVs among wild-type, plasmid-less, and complemented S.
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Typhimurium, S. Enteritidis S. Choleraesuis in invasion, phagocytosis, and intracellular
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bacterial survival in human THP-1 cells and cell death patterns by flow cytometry and
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difference in cell death patterns between pig and human S. Choleraesuis isolates with large
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pSCVs. Virulence plasmid (pSTV) led to slightly increasing cellular apoptosis for S.
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Typhimurium; virulence plasmid (pSEV) enhanced apoptosis and necrosis significantly for S.
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Enteritidis; and pSCV reduced apoptosis significantly for S. Choleraesuis. After
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complementation, pSTV increased the intracellular survival of pSCV-less Choleraesuis and
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the cytotoxicity against human THP-1 cells. Using the Cytochalasin D to differentiate the
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invasion of S. Choleraaesuis and phagocytosis of THP-1 cells determined that pSCV were
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responsible for invasion and phagocytosis at 0 h and inhibited intracellular replication in
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THP-1 cells, and pSTV were responsible for invasion and increased intracellular survival for
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S. Choleraesuis in THP-1 cells. The human isolates with large pSCV induced more cellular
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apoptosis and necrosis than the pig isolates. In conclusion, human S. Choleraesuis isolates
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carrying large pSCVs were more adapted to human THP-1 cells for more cell death than pig
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isolates with large pSCV. The role of pSVs in invasion, phagocytosis, intracellular survival
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and apoptosis differed among hosted serovars.
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Keywords: Salmonella, S. Choleraesuis, S. Enteritidis, S. Typhimurium, Virulence
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plasmid, Apoptosis, Necrosis, intracellular survival
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1. Introduction Salmonella is one of the most prevalent foodborne pathogens worldwide, and salmonellosis
54
in humans is frequently associated with the consumption of food products of animal origin and their
55
byproducts. Salmonella species constantly face different stress conditions in the environment
56
and in the gastrointestinal tract, such as the extreme pH conditions that prevail in the stomach
57
[1-3]. Among the nontyphoid serovars involved in salmonellosis, S. Choleraesuis and S. Dublin
58
cause bacteremia rather than diarrhea [4]. In C3H/HeN mice, bacteremic non-typhoidal S.
59
Schwarzengrund, S. Bredeney and S. Virchow are low in stimulating systemic disease, S.
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Schwarzengrund and S. Enteritidis are profound and elongated shedding, and S.
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Typhimurium cause chronic infection and lower intracellular growth [5], suggesting
62
enhanced invasion and intracellular growth not enough to become bacteremic strains.
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Furthermore, SPI-2 allows the bacteria to reside and proliferate within macrophages,
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furthering their reach and survival within the lymphatic system and bloodstream [6-8].
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The innate immune system provides the first line of defense against invading
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microorganisms by inflammatory and antimicrobial responses [9,10]. Further, apoptosis plays
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an essential role in normal development, homeostasis, and pathogenic processes [11]. Signals
68
such as phosphatidylserine are exposed on the outside of apoptotic bodies to attract
69
macrophages for phagocytosis more efficiently [12]. In Salmonella, S. Dublin induces DNA
70
fragmentation of early apoptosis in human THP-1 cell dependent on phoP expression, not
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spvB, and caspase-3, 8, and 9 [13]. By contrast, necrosis is morphologically characterized by
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the swelling of organelles, disruption of the plasma membrane, increase in cell volume, and
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loss of intracellular contents [14].
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The serovar-associated virulence plasmids (pSVs) in Salmonella may correlate with host
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specificity. These pSVs differ in gene composition, plasmid size and possibly origins [15,16], but
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all of them encode an 8-kb spvRABCD operon, which is positively regulated by the SpvR and the 3
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within host cells [17-19]. In zebrafish, the spv operon inhibits the functions of neutrophils and
79
macrophages of early innate immune responses and accelerates the replication and
80
dissemination of S. Typhimurium [20]. Furthermore, the spv locus inhibits transcription of
81
the tbx21 gene and suppresses expression of the cytokines IFN-γ, IL-12, and TNF-α, but it
82
enhances the expression of the gata3 gene and the cytokines IL-4, IL-10, and IL-13 [21].
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Furthermore, SpvB prevents actin polymerization through ADP-ribosylation, facilitates apoptosis
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[12] and inhibits early autophagy formation in zetafish [22], suppresses autophagosome
85
formation and increases inflammation in murine macrophage-like cells J774A.1 and human
86
epithelial HeLa cells during infection [23].
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Expression of rck gene on virulence plasmid is regulated by the temperature and quorum-
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sensing regulator SdiA on the chromosome [24], demonstrating the interaction between genome
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and virulence plasmid during infection. Previously, we reported that large pSCVs are evolved
90
through recombination of the R plasmid and the 50-kb pSCV in human and pig isolates
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[25,26]. However, we did not determine the virulence among these clinical human and swine
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isolates with larger pSCV.
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2. Materials and Methods
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2.1 Bacterial strains
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The bacterial strains listed in Table 1 were employed to investigate the role of pSVs in three
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serovars and in clinical S. Choleraesuis swine strains CN30 and CN32, and human strains SCB52,
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SCB74, SCB97, SCB130, and MS22872 with large pSCV through pathogenicity assays. These
99
strains included wild-type S. Typhimurium (OU5045), S. Enteritidis (OU7130), and S. Choleraesuis
100
(OU7085) with 90-kb pSTV, 60-kb pSEV and 50-kb pSCV, respectively; their pSV-less strains
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OU5046, OU7067, and OU7266; and complemented strains OU5193, OU5194, OU7294, and 4
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103
strains were kindly provided by Dr. Jonathan T. Ou and examined by plasmid analysis and PCR
104
amplification of invC for Salmonella and spvA for pSV (Fig. 1). All of the bacterial strains used for
105
cell lysis assays grew routinely on xylose lysine deoxycholate (XLD) plates. A single black colony
106
was grown overnight in Luria-Bertani (LB) broth at 37 °C. All strains for
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2.2 Cell and bacterial culture
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Murine RAW 264.7 cells grew in RPMI 1640 medium (Sigma, R6504) supplemented
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with 10% preheated fetal bovine serum (FBS, Sigma, SAB-1900SC-500 ml), D-glucose (J.T.
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Baker, 1916-01), sodium bicarbonate (Sigma, S5761), 10 mM HEPES (Sigma, H0887), 1
111
mM sodium pyruvate (Sigma, S8636), and a 1% antibiotic solution of penicillin,
112
streptomycin, and amphotericin (Sigma, A5955). Human monocyte THP-1 cells were
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incubated routinely in RPMI 1640 medium (GIBCO, ThermoFisher Scientific, Taiwan)
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supplements with 10% FBS, 10 mM HEPES buffer, 2.5 mM sodium pyruvate, 2 mM L-
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glutamine, and 100 µg/ml penicillin/streptomycin at 37 °C and 5% CO2.
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For the phagocytosis and intracellular survival tests, 50 µM of phorbol myristate acetate
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(Sigma, P8139) were added to THP-1 cells to stimulate the macrophage differentiation at
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37°C for 48 h. The bacterial strains and E. coli (ATCC 8739) were incubated at 37°C for 12 h,
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respectively. After centrifugation for 10 min at 3,000 x g (KUBOTA 2010, Kubota, Japan),
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the pellets were washed with phosphate-buffered saline (PBS) several times and were
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resuspended with PBS solution. The OD550 of the bacterial solutions was measured using a
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Bio-Rad SmartSpecTM 3000 (approximately 8.6 × 108 bacteria per mL at OD550 = 1). The
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phagocytosis, cell death, and intracellular survival tests were performed in three independent
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experiments with two repeats each.
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2.3 Intracellular survival and phagocytosis
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differentiated THP-1 cells (MOI = 10), respectively, in each well of a 24-well plate (Becton,
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Dickinson and Company) and incubated with RPMI 1640 medium without antibiotics at
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37°C with 5% CO2 for 30 min or 1 h. The extracellular bacteria were killed by treating with
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gentamicin at a final concentration of 50 µg/mL for 1 h (corresponding to time 0 h). After the
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cell lysis ith 0.5 mL of 1% sodium deoxycholate (Sigma-Aldrich), 100 µL aliquots of the
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solution were plated on individual LB plates. The viable bacteria were counted for each plate
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after incubation at 37°C for 24 h. Furthermore, the invasion of the differentiated THP-1 cells
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were evaluated by treating with 5 µg/ml of cytochalasin D for 1 h at 37°C to inhibit
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phagocytosis. Subsequent infection and intracellular bacteria number were performed as
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described above.
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2.4 Cytotoxicity of different serovars and clinical S. Choleraesuis strains to human
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THP-1 cells
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Human monocyte THP-1 cells were cultured routinely, as described earlier.
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Furthermore, the bacteria and cell mixtures at MOI = 10:1 as above were incubated at 37°C
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for different periods depending on the experiment and then washed twice with PBS. After
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centrifugation and wash with PBS twice, the cells were treated with 0.25% trypsin in 0.53
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mM EDTA solution for 5 min and then 800 µl of RPMI 1640 medium with 10% FBS were
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added to each well. The cells were pelleted at 800 x g for 10 min. After washing with PBS,
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the cells were stained with propidium iodide (PI) and annexin V using an AnnexinV-FITC
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Apoptosis Detection Kit (Cat. No. AVK250, Strong Biotech Corporation, Taiwan) for 10–15
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min. The fluorescence of PI and annexin V was measured by flow cytometry, and the data
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were analyzed using WinMDI software.
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2.5 Statistical analysis
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one-way ANOVA, and a post-hoc Tukey’s HSD test using SPSS 18.0 software. The results
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were considered statistically significant when p < 0.05, and all data were displayed as the
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mean ± SD.
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3. Results
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3.1 Interaction of wild-type Salmonella serovars with different monocyte cell types
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Intracellular bacterial analysis of three serovars demonstrated that the highest bacterial
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number was observed for broad-host S. Typhimurium and S. Enteritidis in mouse RAW 264.7
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cells and for S. Enteritidis in human THP-1 cells and the lowest number for narrow-host S.
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Choleraesuis in both cell lines (Table 2). However, intracellular number of S. Choleraesuis
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was not change in RAW 264.7 cells compared to a gradual increase from 1 h to 2 h in the
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human THP-1 cells. Furthermore, intracellular E. coli was not observed in the human THP-1
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cells, compared to lowest number in mouse RAW 264.7, suggesting intracellular bacterial
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number depending on cell lines. During intracellular bacterial survival analysis, we observed
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two cell populations at the invasion and intracellular survival stages, and changes in the ratios
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of the three aforementioned serovars were observed (Table 3). During the invasion stage, S.
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Choleraesuis exhibited the lowest ratio and S. Typhimurium exhibited the highest ratio. At the
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intracellular survival stage, the ratio increased gradually for S. Typhimuirum, and decreased
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for S. Enteritidis. However, S. Cholearesuis exhibited the highest ratio at 0 h, the lowest ratio
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at 1 h, and then an increase in ratio from 1 h to 4 h. This change in cell size may be related to
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cell death.
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3.2 Roles of the pSVs of three serovars in cell death
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All serovars induced more apoptosis than necrosis and differed in their effects on cell
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viability, apoptosis, and necrosis (Fig. 2). Three wild-type serovars caused different viable
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cell patterns. S. Typhimurium infection showed the highest number of viable cells at 0.5 h 7
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phenomenon of increasing the viable cell number. However, S. Enteritidis infection killed all
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cells. Apoptosis analysis showed that S. Enteritidis caused the highest rate of apoptosis, S.
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Typhimurium showed an increase of apoptosis from 0.5 h to 2 h and S. Choleraesuis showed
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an significant decrease of apoptosis.
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The patterns of cellular viability, apoptosis and necrosis differed between wild-type and
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pSTV-less S. Typhimurium. pSTV-less OU5046 increased cellular ability and decreased
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apoptosis positively associated with the incubation time, compared that both pSTV-
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complemented strains OU5193 and OU5194 increased cellular viability and reduced necrosis
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at time-dependent pattern. In the case of S. Enteritidis, the wild-type and complemented
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strains demonstrated identical effects: no viable cells and nearly 100% apoptosis. However,
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treatment with the pSEV-less strain yielded the highest rate of viable cells and necrosis, and
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the least apoptotic rate. The S. Choleraesuis wild-type OU7085 and the pSCV-complemented
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OU7294 demonstrated similar patterns of influence, increasing ratio of viable cells and
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decreasing the ratio of apoptosis as well as necrosis. By contrast, the pSCV-less OU7266
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decreased the rate of viable cells and increased apoptotic rate.
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The role of the pSVs differed among serovars with slightly increase of apoptosis and
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necrosis for pSTV of S. Tphimurium, a significantly increase in cell death for S. Enteritidis,
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and a decrease of cell death for S. Choleraesuis.
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3.3 Intracellular survival of wild-type, pSCV-less and pSCV-complementary S.
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Choleraesuis strains in human THP-1 cells and infection related cell death
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These three strains differed in cell death and intracellular bacterial patterns (Table 4).
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Wild-type strain displayed an increase of cell death and very few intracellular bacteria. By
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contrast, the pSCV-less OU7266 increased in rate of cell death and higher intracellular
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bacteria ranged from 2- to 7.6 x 103 bacteria per 103 cells. Nevertheless, Tn5-inserted pSCV 8
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from 0 h to 4 h, but with higher apoptosis rate at 8 h and significantly less intracellular
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bacteria than the pSCV-less strain and higher than the wild-type. These results confirmed
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above pathogenicity roles of the pSCV in apoptosis and displayed that pSCV may inhibited
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intracellular survival.
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3.4 The role of pSVs in the invasion, phagocytosis and intracellular survival
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Cytochalasin D treatment can inhibit actin polymerization and, subsequently, the
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phagocytosis of bacteria by human macrophage-like THP-1 cells. Therefore, such a treatment
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can differentiate Salmonella invasion from the phagocytosis of macrophages. At 0 h, wild-
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type OU7085 exhibited a limited intracellular bacterial number derived from phagocytosis,
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whereas pSCV-less OU7226 exhibited the highest intracellular number with almost equal
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ratios of invasion (48.4%) and phagocytosis (51.6%), suggesting pSCV may increase of
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phagocytosis and invasion. Furthermore, intracellular bacteria changed over time (Table 5).
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By contrast, the pSTV-complemented OU7194 demonstrated that invasion was the cause
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(100%) of intracellular bacteria, and an increase in intracellular survival occurred in a time-
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dependent manner. These results imply pSCV responsible for invasion and phagocytosis at o
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h and following by decreasing intracellular survival, and pSTV responsible for invasion and
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increase in intracellular survival in S. Choleraesuis.
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3.5 Cytotoxicity of clinical human and swine S. Choleraesuis isolates with large pSCV to
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human THP-1 cells
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Cytotoxicity against differentiated THP-1 cells differed among the human and pig
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isolates in comparison to the laboratory OU7085 derived from humans (Table 7). At 2-h post-
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infection, the human isolates displayed the lowest viable cells and the highest rate of
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apoptosis, whereas the pig isolates exhibited the highest rate of necrosis. At 4-h post-infection, 9
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viable cells compared with OU7085. At 8-h post-infection, the human isolates displayed the
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highest apoptotic rates and the lowest rate of viable cells and necrosis.
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4. Discussion
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Different host range may be regulated by the gene acquisition via horizontal gene
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transfer with plasmids, transposons, and phages, the loss of genes or their function
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(pseudogenes) and interactions with the host’s immune system and commensal organisms
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during infection [27]. S. Typhimurium, S. Enteritidis, and S. Choleraesuis differ in the host-
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specific symptoms and invasion types. Such difference may be resulted from gene
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inactivation more in narrow-host-range S. Choleraeuis than in broad-host-range S.
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Typhimurium [28], more intracellular bacterial number of broad-host S. Typhimurium and S.
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Enteritidis than S. Choleraesuis in mouse RAW 264.7 macrophage cells and human THP-1
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cells (Table 2), gene interaction between virulence plasmid and chromosome [24] and serovar-
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specific genes, such that S. Choleraesuis lacks the enzymes required for citrate metabolism,
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including tricarballylate dehydrogenase and oxaloacetate decarboxylase, to evade activation
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of the NLPR3 inflammasome and to promote pathogenicity [29]. In macrophages, Salmonella
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evolves mechanisms to avoid host immune responses. Salmonella can quench arginine away
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from the inducible NO synthase (iNOS) pathway via arginine transporters (ArgT) for growth
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and survival [30]. Thus, S. Typhimurium with ArgT expression can evade being killed by
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macrophages and cause a severe systemic infection.
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During intracellular growth, 20 pSTV genes in S. Typhimuriun were upregulated,
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including repA, ccdA, rsd, virE, spvRA, tlpA, samAB, traTD, and trbH [31]. However, a few
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of these genes are not present in pSEV and pSCV [10]. These missing genes may change of
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the distribution of human THP-1 cell populations (Table 3). Such change may be due to
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differences in apoptosis rather than necrosis (Figure 1) and conditions of intracellular bacteria. 10
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In S. Typhimurium, genes ydgT, recA, and corA are involved in the cytosolic proliferation
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and asmA reduced the cytosolic replication [32] and a 597-nt IesR-1 sRNA encoded by the
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pSTV in dormant S. Typhimurium persisting inside fibroblasts is upregulated [33]. Early study reported that the DNA fragmentation was related to phoP expression, not
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related to SPI-1 and SPI-2, spvB, and caspase gene expression [11]. In this study, the
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pathogenicity roles of the pSVs in their host serovars and pSTV in pSCV-less S. Choleraesuis
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differed. In the original hosted serovar, pSTV and pSEV differed the apoptotic ability with
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complete cell killing for S. Enteritidis and lesser apoptosis for S. Typhimurium, but pSCV
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inhibited the apoptotic ability (Figure 1) and intracellular survival of S. Choleraesuis (Table
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4). Furthermore, cytochalasin D demonstrated pSCV inhibited phagocytosis of the human
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THP-1 cells and S. Choleraesuis invasion mainly for intracellular bacteria. By contrast, pSTV
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enhanced both phenomena, although the increase was limited (Table 5). All above results
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imply that pSCV and pSTV may play different pathogenicity roles for S. Choleraesuis and
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co-evolution of the pSVs with their hosted serovars.
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Host adaptation and/or evolution may be associated with genome degradation, such as
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pseudogene formation and deletion. Investigation of multiple S. Enteritidis isolates with
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mutation in the mismatch repair gene mutS from an interleukin (IL)-12 β-1 receptor-deficient
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individual with a 15-year recurrent blood-borne infection demonstrated genome changes
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associated within-host evolution on the timescale [34]. Furthermore, the evolution of the 50-
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kb pSCV to become large pSCVs (75–140 kb) is through the recombination of the R plasmid
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[26] and the involvement of co-evolution of large pSCV associated with human isolates, not
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pig isolates, caused the higher apoptotic rates in human THP-1 cells (Table 6). Nevertheless,
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the pSTVs in human-specific S. Typhi and Paratyphi A did not enhance their pathogenicity
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in infant mice [35]. These data demonstrate that coevolution of pSVs with their host serovar
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plays an important role in host-specific infection. However, a deeper investigation is required
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to determine whether the exchanged pSVs remain serovar-specific.
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5. Conclusion The pSVs differed in size and genes as well as their pathogenicity role among S.
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Typhimuirum, S. Enteritidis, and S. Choleraesuis. The pSTV and pSEV enhanced the
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apoptosis with difference in ratio between these two, but pSCV inhibited the apoptosis.
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Further, pSCV promoted invasion and phagocytosis as well as decreased intracellular
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bacterial survival, and pSTV stimulated invasion and intracellular survival in S. Choleraesuis,
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demonstrating a difference in host adaptation and coevolution of chromosome characteristics
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and/or virulence plasmids for the virulence and adaptation to host species.
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Competing interests
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The authors declare that they have no competing interests.
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This work was supported by grants from the Ministry of Science and Technology (MOST 102-2320-
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B-415-004 for CC) Executive Yuan, and Chiayi Chang Gung Memorial Hospital (grant numbers:
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CMRPG6B0503, CMRPG6E0423, and CMRPG6G0101 for YKH), ROC (Taiwan).
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References
293
1. J.W. Foster, H.K.Hall. Inducible pH homeostasis and the acid tolerance response of
294 295 296
Salmonella typhimurium. J Bacteriol. 173(1991)5129-5135. 2. Foster JW. The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins. J Bacteriol 175(1993)1981-1987.
12
ACCEPTED MANUSCRIPT 297
3. A. Álvarez-Ordóñez, M. Prieto, A. Bernardo, C. Hill, M. López. The Acid Tolerance
298
Response of Salmonella spp.: an adaptive strategy to survive in stressful environments
299
prevailing in foods and the host. Food Res Int. 45(2012)482-492. 4. M.J. Blaser, R.A. Feldman. Salmonella bacteremia: reports to the Centers for Disease Control, 1968-1979. J Infect Dis. 143(1981)743-746
301
RI PT
300
5. J. Suez J, Porwollik, A. Dagan, A. Marzel, Y.I. Schorr, P.T. Desai, V. Agmon, M.
303
McClelland, G. Rahav, O. Gal-Mor. Virulence gene profiling and pathogenicity
304
characterization of non-typhoidal Salmonella accounted for invasive disease in humans.
305
PLOS ONE 8 (2013) e58449.
SC
302
6. F. Garcia-del Portillo, M.B. Zwick, K.Y. Leung, B.B. Finlay. Salmonella induces the
307
formation of filamentous structures containing lysosomal membrane glycoproteins in
308
epithelial cells. Proc Natl Acad Sci U S A. 90 (1993)10544-10548.
M AN U
306
7. A. Gallois, J.R. Klein, L.A.H. Allen, B.D. Jones, W.M. Nauseef. Salmonella pathogenicity
310
island 2-encoded type III secretion system mediates exclusion of NADPH oxidase
311
assembly from the phagosomal membrane. J Immunol.166 (2001)5741-5748.
312
8.
TE D
309
D. Chakravortty, I. Hansen-Wester, M. Hensel. Salmonella pathogenicity island 2 mediates protection of intracellular Salmonella from reactive nitrogen intermediates. J
314
Exp Med. 195(2002)1155-1166.
316 317 318 319 320
9.
AC C
315
EP
313
P. Broz, M.B. Ohlson, D.M. Monack. Innate immune response to Salmonella
typhimurium, a model enteric pathogen. Gut Microbes. 3(2012)62-70. 10. D. Hurley, M.P. McCusker, S. Fanning, M. Martins. Salmonella-host interactions modulation of the host innate immune system. Front Immunol. 5(2014)481. 11. E. Valle, D.G. Guiney. Characterization of Salmonella-induced cell death in human macrophage-like THP-1 cells. Infect Immun. 73(2005)2835-2840.
13
ACCEPTED MANUSCRIPT 321
12. R.G. Deschesnes, J. Huot, K. Valerie, J. Landry. Involvement of p38 in apoptosis-
322
associated membrane blebbing and nuclear condensation. Mol Biol Cell 12(2001)1569-
323
1582.
325 326 327
13. I.K. Poon, C.D. Lucas, A.G. Rossi, K.S. Ravichandran. Apoptotic cell clearance: basic biology and therapeutic potential. Nat Rev Immunol. 14(2014)166-180.
RI PT
324
14. W. Wu, P. Liu, J. Li. Necroptosis: an emerging form of programmed cell death. Crit Rev Oncol Hematol. 82(2012):249-258.
15. C. Chu, S-F. Hong, C. Tsai, W-S. Lin, T-P. Liu, J.T. Ou. Comparative physical and
329
genetic maps of the virulence plasmids of Salmonella enterica serovars Typhimurium,
330
Enteritidis, Choleraesuis, and Dublin. Infect Immun. 67(1999)2611-2614.
332
M AN U
331
SC
328
16. C. Chu, C.H. Chiu. Evolution of the virulence plasmids of non-typhoid Salmonella and its association with antimicrobial resistance. Microbes Infect. 8(2006)1931-1936. 17. F.C. Fang, M. Krause, C. Roudier, J. Fierer, D. Guiney. Growth regulation of a
334
Salmonella plasmid gene essential for virulence. J Bacteriol. 173(1991)6783-6789.
335
18. M. Krause, F.C. Fang, D. Guiney D. Regulation of plasmid virulence gene expression in
336
Salmonella dublin involves an unusual operon structure. J Bacteriol. 174(1992)4482-
337
4489.
EP
TE D
333
19. F.C. Fang, S.J. Libby, N.A. Buchmeier, P.C. Loewen, J. Switala, J. Harwood, D.G.
339
Guiney. The alternative sigma factor katF (rpoS) regulates Salmonella virulence. Proc
340
Natl Acad Sci USA. 89(1992)11978-11982.
AC C
338
341
20. S.Y. Wu, L.D. Wang, J.L. Li, G.M. Xu, M.L. He, Y.Y. Li, R. Huang. Salmonella spv
342
locus suppresses host innate immune responses to bacterial infection. Fish Shellfish
343
Immunol. 58(2016)387-396.
14
ACCEPTED MANUSCRIPT 344
21. S.Y. Wu, L.D. Wang, G.M. Xu, S.D. Yang, Q.F. Deng, Y.Y. Li, R. Huang. spv locus
345
aggravates Salmonella infection of zebrafish adult by inducing Th1/Th2 shift to Th2
346
polarization. Fish Shellfish Immunol. 67(2017)684-691. 22. Y.Y. Li, T. Wang, S. Gao, G.M. Xu, H. Niu, R. Huang, S.Y. Wu. Salmonella plasmid
348
virulence gene spvB enhances bacterial virulence by inhibiting autophagy in a zebrafish
349
infection model. Fish Shellfish Immunol. 49(2016)252-259.
RI PT
347
23. Y. Chu, S. Gao, T. Wang, J. Yan, G. Xu, Y. Li, H. Niu, R. Huang, S. Wu. A novel
351
contribution of spvB to pathogenesis of Salmonella Typhimurium by inhibiting
352
autophagy in host cells. Oncotarget 7(2016)8295-8309.
SC
350
24. N. Abed, O. Grépinet, S. Canepa, G.A. Hurtado-Escobar, N. Guichard, A. Wiedemann, P.
354
Velge, I. Virlogeux-Payant. Direct regulation of the pefI-srgC operon encoding the Rck
355
invasin by the quorum-sensing regulator SdiA in Salmonella Typhimurium. Mol
356
Microbiol. 942(2014)254-271.
M AN U
353
25. C. Chu, C.H. Chiu, W.Y. Wu, C.H. Chu, T.P. Liu, J.T. Ou. Large drug resistance
358
virulence plasmids of clinical isolates of Salmonella enterica serovar Choleraesuis.
359
Antimicrob Agents Chemother. 45(2001)2299-2303.
TE D
357
26. J.I. Tzeng, C.H. Chu, S.W. Chen, C.M. Yeh, C.H. Chiu, C.S. Chiou, J.H. Lin, C. Chu.
361
Reduction of Salmonella enterica serovar Choleraesuis carrying large virulence
362
plasmids after the foot and mouth disease outbreak in swine in southern Taiwan, and
363
their independent evolution in human and pig. J Microbiol Immunol Infect.
364
45(2012)418-425.
AC C
EP
360
365
27. S.L. Foley, T.J. Johnson, S.C. Ricke, R. Nayak, J. Danzeisen. Salmonella pathogenicity
366
and host adaptation in chicken-associated serovars Microb Mol Bio Rev. 77(2013)582–
367
560
15
ACCEPTED MANUSCRIPT 368
28. C.H. Chiu, P. Tang, C. Chu, S. Hu, Q. Bao, L. Yu, Y.Y. Chou, H.S. Wang, Y.S. Lee. The
369
genome sequence of Salmonella enterica serovar Choleraesuis, a highly invasive and
370
resistant zoonotic pathogen. Nucleic Acids Res. 33(2005)1690-1698. 29. M.A. Wynosky-Dolfi, A.G. Snyder, N.H. Philip, P.J. Doonan, M.C. Poffenberger, D.
372
Avizonis, E.E. Zwack, A.M. Riblett, B. Hu, T. Strowig, R.A. Flavell, R.G. Jones, B.D.
373
Freedman, I.E. Brodsky. Oxidative metabolism enables Salmonella evasion of the
374
NLRP3 inflammasome. J Exp Med. 211(2014)653-668.
RI PT
371
30. P. Das, A. Lahiri, A. Lahiri, M. Sen, N. Iyer, N. Kapoor, K.N. Balaji, D. Chakravortty.
376
Cationic amino acid transporters and Salmonella Typhimurium ArgT collectively
377
regulate arginine availability towards intracellular Salmonella growth. PLoS One.
378
5(2010)e15466.
M AN U
SC
375
31. S. Eriksson, S. Lucchini, A. Thompson, M. Rhen, J.C. Hinton. Unravelling the biology
380
of macrophage infection by gene expression profiling of intracellular Salmonella
381
enterica. Mol Microbiol. 47(2003)103-118.
TE D
379
32. M. Wrande, H. Andrews-Polymenis, D.J. Twedt, O. Steele-Mortimer, S. Porwollik, M.
383
McClelland, L.A. Knodler. Genetic determinants of Salmonella enterica Serovar
384
Typhimurium poliferation in the ctosol of epithelial cells. Infect Immun. 84(2016)3517-
385
3526.
AC C
EP
382
386
33. J. Gonzalo-Asensio, A.D. Ortega, G. Rico-Perez, M.G. Pucciarelli, F. Garcıa-del Portillo.
387
A novel antisense RNA from the Salmonella virulence plasmid pSLT expressed by
388
non-growingbacteria inside eukaryotic cells. PLoS One 8(2013) e77939.
389
34. E.J. Klemm, E. Gkrania-Klotsas, J. Hadfield, J.L. Forbester, S.R. Harris, C. Hale, J.N.
390
Heath, T. Wileman, S. Clare, L. Kane, D. Goulding, T.D. Otto, S. Kay, R. Doffinger,
391
F.J. Cooke, A. Carmichael, A.M.L. Lever, J. Parkhill, C.A. MacLennan, D.
392
Kumararatne, G. Dougan, R.A. Kingsley. Emergence of host-adapted Salmonella 16
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Enteritidis through rapid evolution in an immunocompromised host. Nat Microbiol.
394
1(2016):15023. 35. M.J. Santander, R. Curtiss III. Salmonella enterica Serovars Typhi and Paratyphi A are
396
avirulent in newborn and infant mice even when expressing virulence plasmid genes of
397
Salmonella Typhimurium. J Infect Dev Ctries. 4(2010)723.
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Figure 1. Plasmid and PCR analysis of wildtype, virulence plasmid (pSV)less, and pSV
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complementatry strains of S.Typhimurium, S. Enteritidis, and S. Choleraesuis. A)
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Plasmid analysis. B) PCR identification of
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identification and 571-bp spvC fragment for pSV identification. M: 100 bp marker, N:
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Negative, 1: OU5045, 2: OU5046, 3: OU5193, 4: OU5194, 5: OU7130, 6: OU7067, 7:
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OU7415, 8: OU7085, 9: OU7266, 10: OU7194, 11: and OU7294.
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Figure 2. Difference in death type and rate of human THP-1 cells infected by wildtype,
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virulence plasmid (pSV)less, and pSV complementatry strains of S.Typhimurium, S.
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Enteritidis, and S. Choleraesuis.
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ACCEPTED MANUSCRIPT Table 1. Characteristics of S. Typhimurium, S. Enteritidis and S. Choleraesuis strains Serovars
Strains
The pSV Characteristics
OU5045
Wild type with a 90-kb pSTV
OU5046
pSTV-less strain derived from wild type
S. Typhimurium
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OU5193
Tn5-tagged pSTV complemented strains OU5194
OU7067
pSEV-less strain derived from wild type
OU7415
Tn5-tagged pSEV complemented strain
OU7085
Wild type with a 50-kb pSCV
OU7266 OU7194
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Wild type with a 60-kb pSEV
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OU7130
pSCV-less strain derived from wild type
Tn5-tagged pSTV complemented strains OU7294
S. Choleraesuis
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SCB52 SCB97
Clinical isolates with a large pSCV
AC C
SCB130 SCB74
MS22872
2
ACCEPTED MANUSCRIPT
Table 2. Intracellular numbers of different serovars in mouse and human macrophage cells Mouse RAW 264.7 cells
Human THP-1 cells
Strains 2h
4h
1h
2h
4h
4 x101
9 x101
4 x101
7.2 x101
0
0
0
>3 x 103
OU5045 OU7085 2.3 x102
1.0 x 103
3.33 x 102 2.37 x 103 2.05 x 104
4.8 x 102 4.3 x 102
>3 x 103
7.67x101
3.47x103
3.17 x 103
1.52 x 104 7.30 x 104 4.10 x 105
AC C
EP
TE D
M AN U
OU7130
RI PT
1h
SC
E. coli
0h
3
ACCEPTED MANUSCRIPT Table 3. Ratio of larger cell population over small cell population of human THP-1 cells Invasion
Intracellular survival
Serovars 0
1
2
4
OU5045
0.536
1.022
1.584
2.458
3.346
OU7085
0.441
1.664
0.960
1.205
1.469
OU7130
0.503
1.621
1.021
0.837
0.710
AC C
EP
TE D
M AN U
SC
RI PT
1h
4
ACCEPTED MANUSCRIPT Table 4. Intracellular number and cytotoxicity of S. Choleraesuis isolates in human THP-1 cells after infection with MOI=10 Item
0h
2h
4h
8h
Control
Cell death (%)
0
3
4
5
Cell death (%)
0
24
31
32
Intracellular No/103 cells)
0.03
0.17
1.9
2.8
Cell death (%)
0
27
34
45
Intracellular No/103 cells
2 x103
4 x103
7.6 x 103
3.6 x103
pSCV-complemented
Cell death (%)
0
25
37
58
OU7294
Intracellular No/103 cells
2.5 x102
5.6 x 101
Wild type OU7085
SC
pSCV-less OU7266
RI PT
Strain
AC C
EP
TE D
M AN U
2.8 x 101 3.4 x 102
5
ACCEPTED MANUSCRIPT
Table 5. Invasion of different S. Choleraesuis strains and phagocytosis of human THP-1 cells Bacteria Cytochalasin D 0h 2h 4h 0
0
0
+
0
0
0
-
0.18/100
0.15/100
1.8/100
+
0.02/11.1
0/0
0.06/3.3
(-) – (+)
0.16/88.9
0.15/100
1.74/96.7
1.50/96.2
-
3.1 x 103/100
7.4 x 103/100
7.2 x 103/100
9.4 x 103/100
+
1.5 x 103/48.4
1.8 x103/24.3 2.7 x 103/37.5
2.7 x 103/28.7
1.6 x 103/51.6 5.6 x 103/75.7 4.5 x 103/62.5
6.6 x 103/71.3
-
1.7 x 101/100
6.9 x 102/100
4.7 x 102/100
+
2.3 x 101/100 6.9 x 101/40.6 1.4 x 102/20.2
2.1 x 102/44.7
1.2 x 101/59.4 5.5 x 102/79.8
2.6 x 102/55.3
(-) – (+)
-6/0
AC C
(-) – (+)
0
2.6/100
SC
1.7 x 102/100
EP
pSTV-complemented OU7194
M AN U
TE D
pSCV-less OU7266
0
RI PT
None
Wild type OU7085
8h
6
0.1/3.8
ACCEPTED MANUSCRIPT Table 6. Cell deaths of human macrophage-like THP-1 cells infected with OU7085 and clinic swine isolates and human isolates with large pSCV in different periods
8h
Apoptosis
Necrosis
OU7085
89.0±0.31a
9.8±0.27b
1.3±0.20b
Pig isolates
87.2±0.98a
9.6±0.56b
3.2±0.67a
Human isolates
77.0±0.90b
22.0±0.83a
OU7085
90.5±0.46a
8.2±0.26c
Pig isolates
85.9±1.05b
10.1±0.51b
Human isolates
81.3±0.63c
17.6±0.56a
1.1±0.17b
OU7085
90.8±0.26a
7.5±0.45c
1.7±0.46a
Pig isolates
88.3±1.23ab
9.9±1.02b
1.9±0.32a
Human isolates
87.3±0.50b
12.4±0.48a
0.3±0.03b
RI PT
4h
Viability
1.0±0.87b
1.3±0.24b 4.0±0.87a
SC
2h
Source
M AN U
Time
AC C
EP
TE D
Pig isolates: CN30 and CN32; human isolates: SCB52, SCB74, SCB97, SCB130, and MS22872. All values are the mean ± SE. a-c indicate significant differences between sources.
7
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlight
The pSVs differed in size and genes as well as their pathogenicity role among S.
RI PT
Typhimuirum, S. Enteritidis, and S. Choleraesuis. The pSTV and pSEV enhanced the apoptosis with difference in ratio between these two, but pSCV inhibited the apoptosis. Further, pSCV promoted invasion and phagocytosis as well as decreased intracellular bacterial survival, and pSTV
SC
stimulated invasion and intracellular survival in S. Choleraesuis, demonstrating a difference in host
AC C
EP
TE D
virulence and adaptation to host species.
M AN U
adaptation and coevolution of chromosome characteristics and/or virulence plasmids for the