Salmonella enterica serovar Typhimurium sseK3 induce cell apoptosis and enhance glycolysis in macrophages

Background: Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important infectious disease pathogen. Previous studies have identified that S. Typhimurium secreted effector K3 (SseK3) is a novel translated and secreted protein, but it is unclear whether this protein exerts a significant role in the progress of apoptosis and glycolysis in macrophages. Results: The S. Typhimurium SL1344 wild-type (WT) group, ΔsseK3 mutant group and sseK3-complemented group were used to infect macrophages and the effects of sseK3 on apoptosis and glycolysis of macrophages were investigated. The adherence and invasion of ΔsseK3 mutant group for macrophages were similar to WT group and sseK3-complemented group, indicating that SseK3 did not play an important role in the adherence and invasion of S. Typhimurium for macrophages. However, the apoptosis percentage of the ΔsseK3 mutant group was much lower than WT group and sseK3-complemented group using flow cytometry. The caspase-3, caspase-8 and caspase-9 enzyme activity of the ΔsseK3 mutant group were decreased significantly compared with WT group and sseK3-complemented group, which suggested that sseK3 could improve the activities of caspase-3, caspase-8 and caspase-9 enzyme. We also found that the pyruvic acid level did not significantly change among ΔsseK3 mutant group, WT group and sseK3-complemented group, but the lactic acid level of ΔsseK3 mutant group was much lower than WT group and sseK3-complemented group. The ATP level of ΔsseK3 mutant group was remarkably higher than WT group and sseK3-complemented group. These indicated that the sseK3 enhanced the level of glycolysis of macrophages infected by S. Typhimurium. Conclusions: Our data showed that the sseK3 of S. Typhimurium can promote macrophages apoptosis and These results may sseK3. The results showed that the apoptosis percentage of Δ sseK3 mutant

3 influence glycolysis levels of macrophages. These results may give a better clue about the relationship between apoptosis and glycolysis in macrophages induced by S. Typhimurium sseK3. Keywords: S. Typhimurium, sseK3, macrophages apoptosis, glycolysis Background Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most significant zoonotic pathogens that pose a threat to humans [1]. After being contaminated by Salmonella, animal products can be transmitted to humans through the food chain, threatening human health, and potentially leading to deaths [1,2].
Besides, S. Typhimurium can survive and replicate in macrophages. The macrophages or dendritic cells were reported to be able to carry bacteria from the Peyer's patches to adjacent lymph nodes, spleen and liver in mouse models [3].
One of the most essential metabolic pathways in cells is glycolysis [4]. Under anaerobic condition, the pyruvate is eventually converted into lactic acid. However, under aerobic conditions, pyruvic acid enters the tricarboxylic acid cycle (TCA cycle) and eventually is oxidized to CO 2 and H 2 O [5]. Liu found that Resveratrol reduced the production of lactic acid to inhibit glycolysis and induce cell apoptosis [6]. Kok found that Sirtuin6 reduced the production of HK2 during glycolysis, thereby regulating apoptosis induced by hypoxia [7]. Hypoxia inducible factor-1α (HIF-1α) could inhibit the production of Adenosine triphosphate (ATP) and thus inhibit the glycolysis of mouse granulosa cells [8]. Moreover, studies have shown that S.
Typhimurium can induce cell apoptosis [9,10]. There is increasing evidence in recent years that the apoptosis is closely related to glycolysis activities [11][12][13].
Therefore, the glycolysis is closely related to cell apoptosis.

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The immune cells were able to detect the metabolic abnormalities caused by Salmonella with the help of inflammatory signals, and glycolysis was essential in S.
Typhimurium infection with macrophages [14,15]. Following invasion or phagocytic uptake into the host cell, one of the key virulence determinants is the Salmonella pathogenicity island 2 (SPI-2)-encoded type III secretion system 2 (T3SS2), which delivers 28 effector proteins into the host cell [16][17][18]. Previous studies have identified that Salmonella secreted effector K3 (SseK3) is a novel translated and secreted protein of S. Typhimurium, which is encoded by sseK3 gene [19]. SseK3 was a glycosyltransferase, which could transfer an N-acetyl-glucosamine moiety onto the guanidino group of a target arginine, regulating host cell function. The protein belongs to the glycosyltransferase type-A family of glycosyltransferase enzymes and binds the ligand in a metal ion-dependent manner via a DXD motif [20]. SseK3 was co-regulated with the T3SS2 inside host cells and was injected into infected host cells. [21]. However, the mechanism of activity of SseK3 during S.
Typhimurium infection was completely unclear, especially there is no research about sseK3 affecting macrophages apoptosis and glycolysis.
In this study, we determined that sseK3 from S. Typhimurium induced macrophages death and glycolysis. Our data showed that the sseK3 of S. Typhimurium could promote macrophage apoptosis and improve glycolysis levels. These results would supply a critical step to provide a better understanding of the relationship between glycolysis and apoptosis in S. Typhimurium-infected macrophages.

Analysis of adherence and invasion
Adherence and invasion to RAW264.7 cells of the host could ensure the same level 2.

Analysis of cell apoptosis
The apoptosis assay was performed as previously described [22,23]. Apoptosis assay showed that the apoptosis percent of ΔsseK3 mutant group was much lower than that of the WT and sseK3-complemented groups. In the ΔsseK3 mutant group, the FITC-Annexin V positive cells were 3.78%, while in the WT group, it was 14.64% ( Fig.2), suggesting that SseK3 encoded by sseK3 plays an important role in the process of inducing macrophages apoptosis by S. Typhimurium.

Analysis of caspase activity
In the process of apoptosis, the caspase played an essential role [24]. Caspase-3, 8, 9 activities were measured at different time points respectively (Fig.3). The caspase-3, caspase-8 and caspase-9 activities of mock group was much lower than infection groups (ΔsseK3 mutant, WT and sseK3-complemented groups), which indicated that the infection groups could stimulate the activities of caspase-3,8,9 in macrophages. However, it could be seen from Fig.3

Analysis of glycolysis
The analysis of glycolysis was performed as previously described [25][26][27]. To Many secreted proteins of Salmonella exert different virulence functions in the process of survival and replication in cells [28]. T3SS2 helps Salmonella transport virulence (effector) proteins in SCVs into host cells [29]. S. Typhimurium possesses the SseK family of proteins, including SseK1, SseK2 and SseK3, which are T3SS effectors [1,30]. SseK3 is an important protein during Salmonella infection of cells and plays a pivotal role in the natural host immune process [19,21]. Multiple secretory proteins of S. Typhimurium exerted enormous stress during the period of apoptosis of cells and related to cell apoptosis [28,29,31,32]. Nonetheless, the role of SseK3 in the process of cells apoptosis and glycolysis is unclear. This research is the first to show that SseK3 encoded by sseK3 of S. Typhimurium could improve cell apoptosis and boost glycolysis in macrophage.
Santo found that early sipB-dependent and delayed sipB-independent mechanisms could cause the apoptosis induced by S. Typhimurium [33]. Kasinskas investigated that the lacking ribose chemoreceptors of S. Typhimurium localized in tumor quiescence and induced apoptosis [34]. Besides, there had shown that the cAMP receptor protein of S. Typhimurium could induce the apopotosis of macrophages [35]. Therefore, in order to determine the relationship between S. Typhimurium sseK3 and macrophages apoptosis, we used the WT, ΔsseK3 mutant and sseK3complemented strains to infect the macrophages. In our study, we found that the adherence percentage and invasion percentage of ΔsseK3 mutant group were similar to WT group and sseK3-complemented group during the infecting period (P>0.05), which ensured that the WT, ΔsseK3 mutant and sseK3-complemented strains had the same level going through into the macrophages. Subsequently, the apoptosis in different infection groups were detected by the assay of flow cytometry. The results showed that the apoptosis percentage of ΔsseK3 mutant 8 group (3.78%) was much lower than WT group (14.64%) and sseK3-complemented group (11.81%). These indicated that deletion of the sseK3 gene significantly affected the cell apoptosis.
The caspases activities were further to be detected. The activation of caspases exerts essential role on the cells apoptosis induced by the loss of mitochondria membrane [36]. There had shown that the SopB of Salmonella could protect host cell from apoptosis induced by caspase-3 [37]. The activation of caspase-3 and caspase-9 showed that the apoptosis was activated in the macrophages infected by Escherichia coli bacteria [38]. Besides, the caspase-8 influenced the synthesis of pro-IL-1β and was essential for the apoptosis of cells induced by Salmonella [ 39]. As these previously researches, we investigated the caspase-3, caspase-8 and caspase-9 activity during the apoptosis of macrophages infected by S. Typhimurium. We found that caspase-3, caspase-8 and caspase-9 activity were remarkably increased in infection groups compared with mock group at 2 h, 4 h, 6 h and 8 h (***P<0.001), indicating that the apoptosis were activated in the macrophages apoptosis.
Furthermore, the activity of caspase-3, caspase-8 and caspase-9 in ΔsseK3 mutant group were much lower than in WT group and sseK3-complemented group, which suggested that SseK3 may be a pivotal component for S. Typhimurium activating apoptosis in macrophages.
There were increasing evidences that the apoptosis and glycolysis had closely relationship. Previously, Comin-Anduix found that fermented wheat germ extract (FWGE) inhibited the glycolysis and induced the apoptosis of tumor cells [40]. Jeong found that the modification of glycolysis could change the sensitivity of apoptosis through mitochondria pathway [41]. Recently, There has shown that the glycolysis could be inhibited and the cell death was further blocked [42]. Li found that methyl jasmonate could inhibit the glycolysis and induce apoptosis of tumor cells [43].
Sumi found that propofol could improve glycolysis of cells and lead to the apoptosis of cells [44]. The pyruvic acid, lactic acid and ATP were important components in the process of cell glycolysis. There have various metabolic pathway to produce pyruvic acid [45]. Pyruvic acid could enter the TCA cycle to be completely oxidized in aerobic condition, whereas it could become lactic acid under anaerobic oxygen condition [46,47]. HIF-1α could prohibit the production of ATP and thus regulate the The conclusion was consistent with Sumi's [44] and Ding's [35] results, but it was contradict with Li's [43] and Comin-Anduix's [40] results. We speculate that there may be other different pathways involved in the apoptosis and glycolysis of cells, which need more experiments to explain this problem. In our future work, we will further explore the relationship between glycolysis and apoptosis.

Conclusions
In summary, from what has been discussed above, we could conclude that the SseK3 of S. Typhimurium could induce cell apoptosis and boost glycolysis in macrophages.
This finding may help to illustrate the mechanism that S. Typhimurium induces macrophages apoptosis and provide a better understanding of the possible relationship between macrophage apoptosis and glycolysis induced by SseK3.

Adherence and invasion assay
Adhesion and invasion of RAW264.7 cells was assessed as previously described [48,49]. A 24-well cell culture plate was inoculated with 1×10 5 RAW264.7 cells per well and incubated for 16 h. WT, ΔsseK3 mutant and sseK3-complemented strains were coincubated with RAW264.7 cells at a multiplicity of infection (MOI) of 100:1, with three replicate wells per strain. To allow the bacteria to fully contact the RAW264.7 cells, the plates were centrifuged and incubated with 5% CO 2 for 2 h at 37 ℃. For the adherence assay, the supernatants were aspirated, and the cells were washed three times with PBS. Subsequently, the cells were digested with 0.25% trypsin and plated in a gradient dilution and counted. For the invasion assay, the supernatants were aspirated, the cells were washed three times with PBS, and gentamicincontaining medium (100 μg/mL) was added and incubated at 37 ℃ with 5% CO 2 .
After incubation, the supernatants were aspirated, and the cells were washed three times with PBS. Subsequently, the cells were lysed using 0.1% Triton X-100 and plated with a gradient dilution and counted.

Flow cytometry assay
A 6-well cell culture plate was inoculated with 1×10 6 RAW264.7 cells per well and

Caspase-3, caspase-8 and caspase-9 activity assay
The activitiy of caspase-3, caspase-8, and caspase-9 was measured by Caspase-3 Assay Kit, Caspase-8 Assay Kit, Caspase-9 Assay Kit (Beyotime, Shanghai, China), respectively. The cells of infected and mock groups were digested by trypsinization without EDTA and washed three times with ice-cold lysis buffer 3 times, followed by adding 100μL lysis buffer on ice. After incubated for 15 minutes, the concentration of protein was detected using the Bradford protein assay kit (Beyotime, Shanghai, China). Subsequently, after the cell lysates were incubated with Ac-DEVD-pNA for 4 h at 37℃, the samples were read at 405 nm.

Glycolysis level assay
The glycolysis levels were measured using pyruvic acid analysis kit, lactic acid analysis kit and ATP analysis kit respectively, which were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). WT, ΔsseK3 mutant and sseK3-13 complemented groups were processed based on manufacturer's instruction at 2 h, 4 h, 6 h and 8 h, respectively. The concentration of protein each group was detected using the Bradford protein assay kit (Beyotime, Shanghai, China). Finally, the absorbance values of pyruvic acid analysis kit, lactic acid analysis kit and ATP analysis kit were read at 505 nm, 530 nm and 636 nm in a microplate spectrophotometer, respectively.

Statistical analysis
The data are presented as the mean ± standard deviation (SD) of three independent experiments, as based on triplicates assays. Two-way analysis of variance (ANOVA) with a post-hoc test (Bonferroni's multiple-comparison test) was used to compare and assess significance of the differences among all groups. The value of *P<0.05, **P<0.01 or ***P<0.001 was considered significant.

Ethics approval and consent to participate
Not applicable.
corresponding author on reasonable request.

Consent for publication
Not applicable. The apoptosis percent of RAW264.1 cells infected by WT, ΔsseK3 mutant and its complement Figure 3 The activity of caspase-3, caspase-8 and caspase-9 in RAW264.7 cells of all groups. The activ 23 Figure 4 The glycolysis of RAW264.7 infected by WT, ΔsseK3 mutant and sseK3-complemented strains