Salmonella enterica serovar typhi plasmid pR ST98 enhances caspase-3 mediated macrophage apoptosis by suppressing nitric oxide production

Plasmid pR ST98 is a hybrid antibiotic resistance-virulence plasmid isolated from Salmonella enterica serovar typhi ( S. typhi ). Previously, we transferred pR ST98 into attenuated Salmonella enterica serovar typhimurium ( S. typhimurium ) strain RIA to create a transconjugant pR ST98 /RIA and indicated that pR ST98 could enhance apoptosis of infected murine macrophages J774A.1. However, S. typhimurium -mice interaction cannot reflect all characteristics of S. typhi -human interaction. The present study Human-derived macrophage-like cell line THP-1 was infected with wild-type (ST8), pR ST98 -deletion (ST8-ΔpR ST98 ) and complemented (ST8-c-pR ST98 ) S. typhi strains to gain more precise and further insight into the role of pR ST98 in interaction between S. typhi and human macrophage. Macrophage apoptosis, caspase-3 activity and nitric oxide (NO) production were assayed. The results demonstrated that macrophages infected with ST8 and ST8-c-pR ST98 displayed more extensive apoptosis than those infected with ST8-Δ pR ST98 . Further studies showed that pR ST98 could increase caspase-3 activity and suppress NO production. Pretreated macrophages with NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) enhanced S. Typhi -induced macrophage apoptosis. The research data indicate that the plasmid pR ST98 can enhance caspase-3 mediated macrophage apoptosis by suppressing NO production.


INTRODUCTION
Typhoid fever, also known as enteric fever, is caused by Salmonella typhi, a gram-negative bacterium classified as a serovar of the species Salmonella enterica. The symptoms of patients with typhoid fever usually present with a history of prolonged fever, headache, abdominal discomfort and general lethargy. Ingestion of polluted food or water is main transmission of this disease. With strict sanitary and hygienic measures, the incidence of *Corresponding author.  #These authors contributed equally to the work. typhoid fever has decreased in recent years. But it remains a serious public health problem in developing and even in developed countries (Crump and Mintz, 2010). According to the estimation of the World Health Organization (WHO), 22 million typhoid fever cases occur each year, 5% of which are fatal. What is worse, the treatment of infected patients has become complicated as S. typhi acquires a considerable variety of genes by plasmids moving between bacteria through conjugation, such as, antibiotic-resistance and virulence genes (Morita et al., 2010).
An outbreak of typhoid fever, affected 13 provinces and cities, took place in the mid to late 1980s in China. The patients of this outbreak had severe symptoms and exhibited complications with high mortality rates. Studies of our lab demonstrated the antibiotic-resistance S. typhi strains with a 98.6 mega-Dalton (159 kb) plasmid were the main causative agent for the epidemic. This plasmid mediated multi-drug resistance to chloramphenicol, streptomycin, trimethoprim, sulfonamide, gentamycin, neomycin, cephalosporin, ampicillin, carbenicillin and tetracycline and was named as pR ST98 (Huang and Mu, 1994). Further research indicated that pR ST98 is a hybrid resistance-virulence plasmid carrying both antibiotic resistance and virulence genes. Plasmid pR ST98 was associated with the virulence increasing in mice, such as lethality, infection of spleen, liver and mesenteric lymph nodes and serum resistance; pR ST98 could also cause its host bacteria resistance to the phagocytes and enhance the intracellular growth of Salmonella strains in phagocytes. In 2005, we identified Salmonella plasmid virulence (spv) homologous genes which were considered to be absent from S. typhi before identifying it on pR ST98 by Southern blot and DNA sequence analysis (Huang et al., 2005). Spv genes have been shown to be required for Salmonella intracellular growth and systemic infection (Libby et al., 2000).
Although, we identified spv homologous genes on pR ST98 , the mechanism of pR ST98 -increased bacterial virulence was still not fully elucidated. In previous studies, we transferred pR ST98 into an attenuated S. enterica serovar typhimurium (S. typhimurium) strain RIA to create a trans-conjugant pR ST98 /RIA and then cultured with murine macrophage-like cell line J774A.1. The result indicated that pR ST98 could enhance the virulence of its host bacteria by increasing macrophage apoptosis (Wu et al., 2010). Because S. typhi only causes infection in humans, S. typhimurium that has a high degree of genome homology with S. typhi and a broad host range has been used extensively as an experimental model for typhoid fever. However, recent reports have shown that S. typhimurium-mice interaction cannot reflect all characteristics of S. typhi-human interaction. For example, Salmonella pathogenicity island (SPI)-2 is essential for intracellular survival of S. typhimurium in mice (Waterman and Holde, 2003), but not required for survival of S. typhi in human macrophages (Forest et al., 2010). In an effort to gain more precise and further insight into the role of pR ST98 in the interaction between S. typhi and human macrophage, in the present study, we examined apoptosis, caspase-3 activity and NO production in human-derived macrophage-like cell line THP-1 infected with wild-type (ST8) and pR ST98 -deletion (ST8-ΔpR ST98 ) S. typhi strains.

Bacterial strains and culture
Wild-type S. typhi strain ST8 harboring with pRST98 was obtained from the blood of patients during a typhoid fever outbreak in Suzhou, Jiangsu Province, China, pRST98-deletion S. typhi strain ST8-ΔpRST98 and pRST98-complemented S. typhi strain ST8-c-pRST98 were constructed by our lab. Bacteria were grown to midlogarithmic phase at 37°C in Luria-Bertani (LB) broth, harvested by centrifugation, washed with phosphate buffered saline (PBS) and resuspended in RPMI 1640 medium prior to the addition to macrophages.

Cell culture and infection
THP-1 cells were maintained in RPMI 1640 with L-glutamine and 10% fetal bovine serum. Prior to infection, cells were treated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA; Sigma) to induce differentiation into adherent macrophage-like cells. The next day, medium and non-adherent cells was removed and replaced with fresh complete medium. Cells were rested 2 days following chemical differentiation. Mid-logarithmic phase growth cultures of S. typhi strains ST8, ST8-ΔpRST98 and ST8-c-pRST98 were added to THP-1 macrophages at a multiplicity of infection (MOI) of 10:1. Culture plates were centrifuged at 1500 rpm for 10 min and incubated at 37°C for 30 min (0 h time point). The medium was then removed, washed three times with PBS and replaced with medium containing 100 mg amikacin per ml to kill the remaining extracellular bacteria. After 2 h of further incubation at 37°C, the medium in culture plates was replaced with medium containing 10 mg amikacin per ml to prevent extracellular growth of bacteria released from infected THP-1 cells. All assays were conducted in triplicate and repeated at least three times.

Preparation of samples for transmission electron microscopy
THP-1 cells were cultured and infected as described previously. Time point at 2 and 6 h post-infection was examined. Briefly, the adherent cells were scraped with a cell scraper and pelleted by centrifugation. The cells were then fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, post-fixed in 1% osmium tetroxide, and dehydrated through a series of graded acetone washes. Samples were embedded in epoxy resin, sectioned and placed onto 200mesh copper grids. The grids were stained with uranyl acetate and lead citrate, and the samples were examined for the presence of apoptotic cells using a Hitachi TEM.

Flow cytometry analysis of apoptosis
Macrophage apoptosis was analyzed with flow cytometer after propidium iodide (PI) staining, apoptotic population appeared as a sub-G1 peak. THP-1 cells were harvested and washed with PBS. The cells were then fixed in 70% ethanol at 4°C for 24 h, treated with RNaseA at 37°C for 30 min and stained with PI in the dark for 30 min. The samples were analysed by a FACScan flow cytometer (FC500, Beckman Coulter, Brea, CA, USA).

Assay of caspase-3 activity
Caspase-3 activity was assayed using commercial colorimetric caspase assay kits (Beyotime, China). THP-1 cells were harvested and washed with PBS. The cells were then resuspended in lysis buffer and incubated on ice for 15 min. The cell debris was removed by centrifugation at 12000 rpm for 10 min at 4°C, and the supernatant was collected for the colorimetric assay of caspase-3 activity. Para-nitroanilide (pNA)-conjugated specific substrates for caspase-3 (DEVD-pNA) was used according to the manufacturer's instructions. Cleaved substrates were quantified by reading absorbance at 405 nm with a microplate spectrophotometer (uQuant, Bio-Tek Instruments Inc, Winooski, VT, USA).

Measurement of nitric oxide (NO)
Nitric oxide (NO) released from macrophages infected with S. typhi was measured by means of the Griess reaction, which detects the amount of nitrite accumulated (Green et al., 1982). The supernatant was collected and an equal volume of Griess reagent was added. The mixture was incubated for 10 min at room temperature and absorbance was recorded at 540 nm. Blank and standards were also run in parallel. In some experiments, THP-1 cells were pretreated with NO synthase inhibitor L-NAME for 30 min.

Statistical analysis
Data were presented as mean ± standard deviation (SD). Experimental results were analyzed for their significance by Student's t-test using a SPSS program. A value of P < 0.05 was considered to be statistically significant.

Morphological features of macrophage apoptosis
As shown in Figure 1, at 2 h post-infection, THP-1 cells infected with S. typhi ST8, ST8-ΔpR ST98 and ST8-c-pR ST98 exhibited morphological features of apoptosis, such as condenses and marginated nuclear chromatin. But there was no significant difference between these three groups in amount of apoptotic cells at this point. At 6 h post-infection, these changes were more obvious and it was observed that more cells infected with ST8 and ST8-c-pR ST98 exhibiting morphological features of apoptosis than cells infected with ST8-ΔpR ST98.

Percentage of macrophage apoptosis
Though observation of cell ultra-structure by TEM is the gold standard to determine apoptosis, it is a qualitative standard and cannot reflect amount of apoptotic cells very accurately. The relationship between pR ST98 and macrophage apoptosis was further confirmed by flow cytometer analysis after PI staining. As shown in Figure  2, the percentage of macrophage apoptosis induced by ST8, ST8-ΔpR ST98 and ST8-c-pR ST98 at 2 h post-infection was 23.05 ± 1.63, 22.10 ± 2.55 and 21.99 ± 1.89%, respectively, in contrast to 6.76 ± 1.09% in control. There was no significant difference between macrophage apoptosis induced by ST8, ST8-ΔpR ST98 and ST8-c-pR ST98 (P > 0.05). Amount of apoptotic cells displayed increasing tend over incubation time. The percentage of macrophage apoptosis induced by ST8, ST8-ΔpR ST98 and ST8-c-pR ST98 at 6 h post-infection was 70.31 ± 2.50%、53.02 ± 2.24% and 68.26 ± 2.32%, respectively, in contrast to 6.90 ± 1.12% in control. Significantly higher percentage of macrophage apoptosis induced by ST8 and ST8-c-pR ST98 than ST8-ΔpR ST98 was observed at this appoint (P < 0.01).

DISCUSSION
Interaction between pathogens and macrophages is essential for the pathogenesis of many infections. Macrophages have double roles in pathogenic infection (Gordon, 2007). On one hand, macrophages are key mediators in eliciting both innate and adaptive immune responses. As the scavengers of host, macrophages can engulf and digest off invading pathogens, present antigen to T lymphocytes and secret cytokines that regulate immune response. On the other hand, many pathogens reside and replicate in macrophages, even exploit macrophages as a crucial tool in the initiation of the disease. In order to colonize host successfully, pathogens evolve a variety of strategies to manipulate the fate of macrophages towards their benefits. Regarding Salmonella, bacteria mainly evolve two strategies: (i) Following invasion of macrophages, Salmonella resides in a membrane-bound compartment, known as the Salmonella-containing vacuole (SCV) that superficially resembles a phagosome. Unlike conventional phagosome, SCV is redirected away from the phagosomal maturation pathway and does not fuse with lysosome (Brumell and Grinstein, 2004), (ii) when replicating within macrophages, Salmonella also produces cytotoxins to stimulate macrophage apoptosis , thus eludes host innate immune responses and spreads easily. The ability of Salmonella to initiate death of infected macrophages contributes to the severity of disease caused by Salmonella strains.
Our previous study demonstrated that pR ST98 could increase apoptosis level of murine macrophage J774A.1. However, pR ST98 was originally isolated from S. typhi. S. typhimurium-mice interaction and S. typhi-human interaction may differ in a number of respects. In the present study, we use S. typhi infected human macrophage THP-1, it was observed that pR ST98 could increase the percentage of macrophage apoptosis induced by S. typhi, as demonstrated by the results of cell ultra-structure observation using transmission electron microscopy and flow cytometer analysis. This is consistent with our previous study. Additionally, we find an interesting phenomenon that the percentage of apoptosis between macrophages infected with ST8, ST8-ΔpR ST98 and ST8-c-pR ST98 was not significantly difference at early phase, and that the cytotoxicity of pR ST98 occurred later. Similar phenomenon of delayed cytotoxicity of virulent effectors was observed by Libby et al. (2000) in assaying the spv phenotype in macrophages and Paesold et al. (2002) in intestinal epithelial cells. Perhaps, the delayed cytotoxicity of pR ST98 caused S. typhi infection. It would allow sufficient time for S. typhi to adapt to the host intracellular environment, replicate extensively and express genes for deep infection.
Apoptosis is an active process of programmed cell death (PCD), which leads to cell destruction and death. Classical apoptosis is triggered by two separate pathways: one is extrinsic pathway initiated by cell surface death receptors, and the other is intrinsic pathway which involves the disruption of mitochondrial membrane integrity (Burz et al., 2009). Both pathways activate executioner caspases (members of cysteine protease family) to cleave cellular substrates to produce the features associated with apoptosis. Caspase-3 is a central effector of caspase, activated caspase-3 is a protease with many cellular targets. Certain pathogens, such as Salmonella, can deliver effector proteins through type III protein secretion systems (TTSSs) to alter important signal transduction pathways and affect the balance between pro-and anti-apoptotic factors in the host cell, with caspase-3 as the major executioner of characteristic apoptotic changes (Guiney, 2005). The results demonstrate that pR ST98 raised caspase-3 activity in infected cells, suggesting that macrophage apoptosis induced by pR ST98 is related to caspase-3 activation.
Generation of NO is a host defense mechanism during pathogenic infection. NO has potent host defense functions, including antimicrobial actions via its cytotoxic or cytostatic effects (Nathan and Shiloh, 2000) and antiapoptotic activity through inhibition of caspases activity via S-nitrosylation of the active cysteine in caspases (Slomiany and Slomiany, 2010). Alam et al. (2006Alam et al. ( , 2008 reported earlier that Salmonella infection caused higher bacterial burden and more extensive apoptosis in Peyer's patch tissue, liver tissue and peritoneal macrophages of inducible NO synthase (iNOS)-deficient mice than wild-type mice. It indicated that a decrease or eventual absence of NO production may result in a deleterious outcome for host during pathogenic infection. Our present study gave similar results that pR ST98 suppressed NO production and resulted in higher percentage of infected macrophage apoptosis. Blocking of NO production with NO synthase inhibitor L-NAME enhanced significantly ST8-ΔpR ST98 induced macrophage apoptosis, but its effect on ST8 and ST8-c-pR ST98 induced macrophage apoptosis was limited. However,  reported a contrary result that, NO induced macrophage apoptosis following infection with S. typhi. Appearance of this opposite results may not affect the validity of our results. NO is a multifunctional molecule, its biological effects depend on the concentration of intracellular NO in combination with the intracellular environment and its interaction with other biological molecules (Wang et al., 2010).
In conclusion, our present study demonstrated that the pR ST98 enhances caspase-3 mediated macrophage apoptosis by suppressing NO production. However, our knowledge of pR ST98 obtained from present study is still limited. The mechanisms of pR ST98 mediating NO production are unclear. Spv locus is a highly conserved region consisting of five genes, one transcriptional regulator spvR and four structural genes spvABCD. It was reported that spvC had phosphothreonine lyase activity that can remove phosphate groups from host cells mitogen-activated protein kinases (MAPKs) thereby inactivating them (Mazurkiewicz et al., 2008). MAPKs have been demonstrated to be key players in regulating production of NO (Gao et al., 2012). Though, spv homologous genes have been found on pR ST98 , pR ST98 is a large plasmid containing complex sequences of unknown functions. However, It is not clear whether pR ST98 mediating NO production is directly related to spv genes or a result of interplay of several genes. Further exploration will be focused on the details of pR ST98 regulatory pathways.