Salmonella enterica causes various diseases, particularly, nontyphoidal salmonellae are very important in reportable food-borne infections. Salmonella is an intracellular pathogen that can invade eukaryotic cells and manage to survive in the living host cell    . It is well known that this bacteria is able to invade via Type Three Secretion System (TTSS) encoded in Salmonella Pathogenicity Island 1 (SPI-1)  . When Salmonella invades to host cells, the SPI-1 effector protein of the TTSS is injected into epithelial cells, thereby causing rearrangement of actin cytoskeleton    , membrane ruffling and formation of micropinosomes  . As a case of cytoskeletal rearrangement, it is revealed that SPI-1 proteins SipABCD, SopE and SopE2 are involved  .
In the present, it has been found that the infection route of Salmonella Typhimurium against fibroblast cells was quite different from that of epithelial cells, in which the SPI-1 effectors SipB and SipC were unnecessary. And this strain is able to suppress cell growth by stopping cell division in fibroblast cells after invasion    . Based on these findings, it is considered that S. Typhimurium could alter the routes into fibroblasts, and persistent infection and asymptomatic carrier are caused.
Although it has been known the importance of salmonellosis to public health, the mechanism of the Salmonella carrier state hasn’t been well known still now. We examined the invasion of Salmonella into fibroblasts, because there was a risk of giving rise to the persistent infection. In addition, we show here invasion and proliferation of S. Typhimurium in the fibroblasts differs from that of S. Enteritidis.
2. Materials and Methods
2.1. Bacterial Strains and Swine Fibroblast Cell Line
Salmonella enterica serovar Enteritidis strain zSE1 isolated in Zambia and Typhimurium wild type strain st1wt were cultured properly in Trypticase Soy Broth (TSB) at 37˚C for 18 hr  . Pig embryonic fibroblasts (PEFs), which infected with simian vacuolating virus 40 large T fragment (PEFs-SV40) to achieve immortalization, used in this study  . Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque Inc., Kyoto, Japan) with 10% PBS and 1% antibiotic-antimycotic mixed stock solution (Nacalai Tesque Inc.) at 37˚C in 5% CO2. Cells were maintained under exponential growth condition and used as the host cells in further experiments.
2.2. Bacterial Infection Assays
PEFs were seeded in a 24-well-plate to reach a density of 1.0 × 105 cells/well at the time of infection. The medium was changed to DMEM with 10% FBS (without antibiotics) 2 hr before bacterial infection to eliminate any potential effects of the antibiotics. PEFs were infected with overnight cultured bacteria at a multiplicity of infection (MOI) of 5:1 (bacteria to eukaryotic cells). To count the adhesive bacteria at 0, 20, 60, and 100 min after the infection, wells were washed with PBS containing 0.1% (wt/vol) sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries Ltd., Osaka, Japan) and 1% (vol/vol) Triton X-100 (Wako Pure Chemical Industries Ltd.) as lysis buffer. The invasive bacteria were counted, and cells were washed with PBS repeatedly. Fresh culture medium containing 100 µg/ml gentamicin was added for 2 hr post-infection, and then the cultured cells were lysed with lysis buffer.
2.3. Immunofluorescence Microscopy
Extracellular (adherent) and intracellular bacteria were stained by immunofluorescent microscopy by the methods of Aiastui et al.  . Briefly, extracellular bacteria were stained in nonpermeabilized cells with polyclonal rabbit anti-Salmonella lipopolysaccharide (LPS) antibodies (S. Enteritidis O4 and S. Typhimurium O9, Denka Seiken Co., Ltd, Tokyo, Japan), followed by anti-rabbit Alexa Fluor 594 F(ab’)2 fragment antibody (Invitrogen, CA, USA). Upon permiabilization by treatment with 0.2% Triton X-100, intracellular bacteria were stained with anti-Salmonella LPS antibody above, followed by anti-rabbit Alxa-Fluor 488 F(ab’)2 fragment antibody (Invitrogen) as secondary antibodies.Cells were observed with a Fluorescent microscope (FSX 100, Olympus, Tokyo, Japan).
2.4. Intracellular Proliferation
Fresh DMEM medium containing 100 µg/ml gentamicin was added into 2 hr infected fibroblast. After cultivation for 24 hr, PEFs were dissolved in lysis buffer and intracellular bacterial numbers were counted.
2.5. Live Cell Count
Bacteria-infected PEFs (MOI = 5:1 as described above) were treated at 0, 20, 60, 100 min after the infection with 0.05% Triton X-100 and cells were collected. Cells were mixed with trypan blue (Invitrogen) and living cells were counted using a CSTI Counter (Cell Science & Technology Institute, Inc., Miyagi, Japan). In order to assess the effects of intracellular pathogens and long-term infection, the medium was changed to fresh medium containing 100 µg/ml gentamicin 2 hr after infection. After gentamicin treatment for 24 hr, live PEFs were counted according to mentioned.
2.6. MTT Assay
PEFs were seeded in a 96-well-plate and incubated for 24 hr to reach a density of 3.0 × 103 cells/well at the time of infection. The medium was changed to DMEM with 10% FBS (without antibiotics) 2 hr before bacterial infection to eliminate any antibiotic effects. Cells were infected with Salmonella (MOI = 5:1), and then 10 μl of MTT reagent from the MTT Cell Proliferation Assay kit (Funakoshi Co., Ltd., Tokyo, Japan) was added immediately after infection. After post-infection for 2 hr, the medium was changed to DMEM containing 100 µg/ml gentamicin, and MTT reagent was added to each samples at 0, 2, and 24 hr.
2.7. Apoptosis and Cell Cycle Assay
Apoptosis of infected cell was analyzed with MuseTM Cell Analyzer (Merck Millipore Inc., Darmstadt, Germany).PEFs were inoculated 0.1 × 105 cells per well. S. Enteritidis or S. Typhimurium was added into well in the ratio of 5:1 (MOI) and incubated for 2 hr. After infection, PEFs were washed with PBS and collected with 0.05% trypsin. And then PEFs were centrifuged (800 × g) and washed with PBS. Infected PEFs were col-
lected with 0.05% trypsin and added Muse Annexin & Dead Cell Reagent and standing for 30 min at room temperature in a dark place. Apoptotic cells were detected with the analyzer. Furthermore, apoptosis profile of PEFs infected with S. Enteritidis zSE1 after infection for 24 hr was monitored using the flow cytometry.
The cell cycle of PEFs was also analyzed by using the MuseTM Cell Cycle Assay Kit (Merck Millipore Inc.), according to the procedure described by the manufacturer. Infected PEFs were collected with 0.05% trypsin, centrifuged (800 × g), and washed with PBS. Collected cells were suspended in cold 70% ethanol and incubated for over 3 hr at −20˚C for fixation. Fixed cells were washed with PBS and stained with the cell cycle reagent for 30 min. Samples were measured by using the above analyzer.
2.8. Statistical Test
In this study, all of the experiments were carried out with at least triplicated samples. Mean and standard deviations were calculated from the multiple data. The statistical significance was evaluated unpaired t-test. After statistical analysis, p values of less than 0.05 were considered statistically significant.
3. Results and Discussion
3.1. Infection to Swine Fibroblasts
Although it has been revealed that fibroblasts are highly involved in the persistence of pathogenic Salmonella  , the mechanisms of long-term and persistent infection of Salmonella enterica are still unknown. In this study, we investigated Salmonella-fi- broblast interactions to clarify the survival strategy of Salmonella in the host fibroblasts.
We first confirmed the infection of Salmonella to PEFs by conducting infection assay and immunofluorescence. Each cell of S. Enteritidis and Typhimurium adhered approximately 1% for a start, and adherent cell number was increased time-dependently (Figure 1(a)). At 100 min, adherent number of S. Typhimurium was significantly higher than that of S. Enteritidis (P < 0.01). Cell invasion was observed after 60 min of the infection. S. Typhimurium invaded cells more aggressively than S. Enteritidis (Figure 1(b), P < 0.01 at 60 min after the infection). The states infected with S. Enteriditis or S. Typhimurium were photographed with a fluorescent microscope and typical photo-images at 24 hr after S. Typhimurium infection were showed (Figure 2). Furthermore, the intracellular proliferation of Salmonella cells was different between S. Enteritidis and S. Typhimurium, i.e., S. Enteritidis reproduced sharply after invasion to PEFs, on the other hand, S. Typhimurium was almost not (Figure 3, P < 0.01). Namely, S. Enteritidis zSE1 reached 1.28 × 104 cfu after infection for 24 hr, while S. Typhimurium st1wt couldn’t proliferate in host cells and only reached 3.3 × 102 cfu.
3.2. Influences to Viability and Lifespan of Host Fibroblast
In order to assess the influence of host cells infected with pathogenic Salmonella, we analyzed the viability and proliferation of infected PEFs. The number of living infected
Figure 1. Chronological changes of adhesion and invasion on Salmonella enterica. (a) Cell numbers of S. Enteritidis or Typhimurium which can adhere to PEFs. (b) Cell numbers of S. Enteritidis or Typhimurium which can invade to PEFs. *: P<0.01 unpaired t-test.
Figure 2. Fluorescent microscopy photographs of S. Typhimurium after infection for 24 hr. Bar: 50 μm. Red arrow: adhesion, green arrow: invasion.
Figure 3. Chronological changes of bacterial numbers on intracellular Salmonella enterica. *: P < 0.01, unpaired t-test.
PEFs was counted after trypan blue staining. As a result, it was found that living cells were increased after infection with both Salmonella strains compared to non-infected cells (Figure 4). The proliferation of living PEFs was measured by MTT assay after 0, 2 and 24 hr infection (Figure 5). The proliferation of PEFs infected for 24 hr was remarkably increased, particularly, a significant difference was revealed on S. Typhimurium. These results indicate that Salmonella is able to survive within host fibroblasts and enhance the proliferation of host cells. It is found that S. Typhimurium led to enhance the proliferation in epithelial cells  and suppress it in dendritic cells and fibroblasts   . Furthermore, S. Enteritidis into human fibroblasts increased during 1 day after infection and could survive until 14 to 28 days  . Based on these findings, it is considered that Salmonella grows rapidly in fibroblast and gradually controls
Figure 4. Proliferation of PEFs infected with zSE1 or st1wt. (a) The number of PEFs infected with S. Enteritidis or Typhimurium and non-infected-PEFs were counted using the trypan blue method. 0, 20, 60, 100 min after infection, cell number of PEFs infected with bacteria tended to be larger that of non-infected PEFs. (b) 24 hr after infection, PEFs infected with zSE1 or st1wt were about 1.52 fold or 1.47 fold respectively compared with non-infected PEFs. **: P < 0.05, unpaired t-test.
Figure 5. Time-lapse changes of cell proliferation on host PEFs. Cell viability was measured by MTT cell proliferation assay. Colorimetrical formazan produced from live cell metabolism were detected as absorbance at 570 nm. *: P < 0.01 unpaired t-test.
them after that, and Salmonella is possible long-term survival by repressing the proliferation in restricted host cells.
3.3. Effect to Viability of Fibroblast
We demonstrated the cell death pattern of host cells after Salmonella infection as shown in Figure 6. As shown in Figure 7, apoptotic cells counted was 1% - 1.5% (significantly higher than control) at the beginning of the infection. After 24 hr infection, the percentage of apoptotic cells significantly increased in non-infected cells; in contrast, the percentage of apoptotic cells was approximately 0.5% on Salmonella infected cells. Total dead cell number was also decreased by the Salmonella infection (data not shown).
To further explain such phenomenon, cell cycle of infected cells was analyzed by using a Cell Analyzer. The cell cycle of infected or non-infected PEFs was measured by flow cytometry 24 hr after addition of bacteria. The results revealed that G0/G1 phases of infected cells were reduced and the G2/M phases were prolonged compared with non-infected cells, respectively (Figure 8). It has been found that enteropathogenic Escherichia coli and enterohaemorrhagic E. coli infused effector protein Cif to eukaryotic host cells and led to arrest cell cycle, and the barrier of the epithelium cell became weak by stopping a cell cycle, and bacteria became easy to infect cells  . And the G0 phase was longer to repair DNA in cell cycle  , the apoptosis was caused when DNA damage could not repair   . In addition, it has been known that Salmonella led to induce apoptosis of host chicken fibroblasts  , and the ratio of apoptotic PEFs infected with Salmonella Enteritidis or Typhimurium might be increased. Based on these findings, it seems that Salmonella infection induces the alteration of the cell cycle on host PEFs, and interrupts apoptosis of the host cell to survive in host cells. Namely, Salmonella of the intracellular parasitism may lead to repress cell cycle after invasion.
Figure 6. Dead /live cells and apoptosis profile of PEFs infected with S. Enteritidis zSE1. (a) Infected cells. (b) Control: non-infected cells.
Figure 7. Proportion of apoptotic PEFs after infection with Salmonella enterica. Apoptosis caused by Salmonella enterica infection were detected by Annexin V using flow cytometry. *: P < 0.01 and **P < 0.05, unpaired t-test.
Figure 8. The ratio of cell cycle phase in PEFs after infection with Salmonella enterica. *: P < 0.01 and **: P < 0.05, unpaired t-test.
It has been known that bacteria are able to suit host cell environment by promoting the most successful conditions for infection  . In this study, we demonstrated that Salmonella was able to survive in fibroblasts by manipulating both lifespan and apoptosis of host cell. This phenomenon is intended to optimize the circumstances of Salmonella survival and promote the persistent infection of Salmonella in livestock. This series of strategy could be a notable fact that highlights new concepts of Salmonella infection of fibroblasts in domestic animals, and encourages people to reconsider the hidden issue in food safety.