Part:BBa_K4737011

prpsM-SopE-FLAG-GOx

To express SopE-FLAG-GOx constitutively

Usage and Biology

Ferroptosis is an iron-dependent programmed form of cell death that is strongly associated with abnormal iron metabolism and lipid peroxide accumulation. Lipid-based reactive oxygen species/phospholipid hydroperoxide (PLOOH) are thought to be the executor molecules of ferroptosis. We design to use the engineered attenuated Salmonella VNP20009 to express the proteins relating to this metabolism pathway. After the engineered bacteria VNP20009 targeting tumor cells, the proteins will be injected into tumor cells through the type III secretion system (T3SS) to induce ferroptosis in tumor cells, and then achieve the aim of killing tumor cells. As an important nutrient, glucose plays a vital role in tumor growth. The proliferation of tumor cells is primarily dependent on aerobic glycolysis, which leads tumor cells to be more sensitive to changes in glucose concentration than normal cells. Therefore, we plan to inject glucose oxidase (GOx) into tumor cells through the T3SS system when the engineered bacteria VNP20009 successfully infect tumor cells. Glucose oxidase (GOx) can catalyze the reaction of glucose and oxygen in cells to produce hydrogen peroxide (H₂O₂) and gluconic acid. The consumption of glucose can cut off the nutrient source of cancer cells, thereby inhibiting their proliferation and achieving the effect of starvation treatment. While achieving the starvation therapeutic effect, the hydrogen peroxide produced by this reaction can be used as one of the reactants to mediate the Fenton reaction. The essence of the Fenton reaction is that redox-active Fe²⁺ catalyze hydrogen peroxide (H₂O₂) to produce hydroxyl radicals (·OH) and other reactive oxygen species (ROS). The accumulation of ROS will directly promote the production of PLOOH. If the reduction of PLOOH into corresponding phospholipid alcohols (PLOH) by GPX4 and GSH didn’t work efficiently, these substances will continue to oxidize the polyunsaturated fatty acyl moieties in phospholipids (PUFA-PL) to produce more PLOOH. This chain reaction may eventually disrupt the integrity of the cell membrane, leading to the breakdown of the organelle and cell membranes. However, at the same time, the antioxidant mechanism in tumor cells may also inhibit the occurrence of ferroptosis and decline its effect. System Xc^-/GSH/GPX4 axis mediate ferroptosis defense in tumor cells, thereby maintaining the infinite proliferation of cells. So, we also designed shRNA modules to silence SLC7A11 to better achieve the effect of killing tumor cells.

Figure 1: The SopE-FLAG-GOx expression pathway.

Figure 2: GOx is injected into the tumor cells by T3SS for promoting Fenton reaction.

Recombinant S. typhimurium (VNP-SopE-GOx) was grown at appropriate conditions. The proteins from the culture supernatant and bacterial lysates were prepared for an immunoblotting assay. The results of Western blot showed that VNP20009 electroporated with plasmid pFPV25.1-SopE-FLAG-GOx expressing SopE-FLAG-GOx protein successfully (Figure 3).

Figure 3: Western blot analysis shows the expression of gene of interest(SopE-FLAG-GOx) in VNP-SopE-GOx. The theoretical molecular weight of SopE-FLAG-GOx is about 76 kDa, and the theoretical molecular weight of GAPDH is about 36 kDa.

We selected a common gastric cancer cell line, BGC-823, for bacterial infection experiments. We used VNP-SopE-GOx to infect BGC-823, while BGC-823 cells without bacterial infection were set up as a blank control and VNP20009 infected BGC-823 cells were used as a negative control. After the bacterial infection experiment, we collected the cell culture medium supernatant and the cell precipitate separately. We disrupted the cell precipitate, collected the protein. All samples were performed SDS-PAGE and Western blot experiments (Figure 4). According to Western blot results (Figure 4), after bacterial infection experiment, only the BGC-823 cells infected by VNP-SopE-GOx had been injected FLAG-GOx, which means that VNP-SopE-GOx successfully injected FLAG-GOx into tumor cells through T3SS.

Figure 4: Western blot analysis shows the injection of FLAG-GOx into BGC-823 cells by VNP-SopE-GOx via T3SS. The theoretical molecular weight of FLAG-GOx is about 64 kDa, and the theoretical molecular weight of GAPDH is about 36 kDa.

We inoculated BGC-823 cells in 24-well plates according to the number of 2.5×10⁵ cells per well. VNP20009 and VNP-SopE-GOx were used to infect BGC-823 cells for 5 h according to the protocol of bacterial infection, while using DMEM medium without antibiotics and with 5% FBS to culture the control group cells for 5 h. Finally, the medium was all switched to DMEM complete medium containing 50 μg/mL gentamicin to culture the BGC-823 cells for 24 h. After 24 h of incubation, we used the hydrogen peroxide detection kit (Beyotime) to detect the H₂O₂ level of the cell culture medium supernatant. According to the experiment results, it can be seen that the H₂O₂ level of the culture medium supernatant which belongs to tumor cell infected by VNP-SopE-GOx is significantly greater than that which belongs to control group and that which belongs to tumor cell infected by VNP20009. Moreover, because H₂O₂ can pass through the cell membrane in a free diffusion manner, our experimental results can demonstrate that FLAG-GOx is successfully expressed and injected into tumor cells by T3SS to successfully consume glucose and oxygen in the medium to produce H₂O₂.

Figure 5: H₂O₂ levels in the cell culture medium supernatant.

Ferrostatin-1 (Fer-1), a potent and selective ferroptosis inhibitor, suppresses Erastin-induced ferroptosis in tumor cells. Fer-1, a synthetic antioxidant, acts via a reductive mechanism to prevent damage to membrane lipids and thereby inhibits cell death. Ferrostatin-1 exhibits antifungal activity. GOx consumes glucose and oxygen in tumor cells to produce gluconic acid and H2O2. Under the catalysis of Fe²⁺, H₂O₂ will generate hydroxyl radicals, which will cause lipid peroxidation of tumor cell membranes, resulting in ferroptosis in tumor cells. We inoculated BGC-823 cells in 96-well plates according to the number of 5×10⁴ cells per well. In the group without Fer-1 treatment, VNP20009 and VNP-SopE-GOx were used to infect BGC-823 cells for 5 h according to the protocol of bacterial infection, while using DMEM medium without antibiotics and with 5% FBS and 20 μM Fer-1 to culture the control group cells for 5 h. Finally, the medium was all switched to DMEM complete medium containing 50 μg/mL gentamicin to culture the BGC-823 cells for 24 h. In the group with Fer-1 treatment, the cells were pretreated with DMEM without antibiotics and with 5% FBS and 20 μM Fer-1 for 30 minutes. VNP20009 and VNP-SopE-GOx were used to infect BGC-823 cells for 5 h according to the protocol of bacterial infection, while using DMEM medium without antibiotics and with 5% FBS and 20 μM Fer-1 to culture the control group cells for 5 h. Finally, the medium was all switched to DMEM complete medium containing 50 μg/mL gentamicin and 20 μM Fer-1 to culture the BGC-823 cells for 24 h. After 24 h of culture, we used the CCK-8 cell counting kit (Vazyme) to detect the viability of the cells. From the experiment results, it can be seen that the viability of tumor cells infected by VNP-SopE-GOx is much lower than that of tumor cells without being infected and that of tumor cells infected with VNP20009. Compared with the tumor cells which were treated with Fer-1 and infected by VNP-SopE-GOx and the tumor cells which were only infected by VNP-SopE-GOx, the cell viability was significantly increased, and there was no significant difference in cell viability between tumor cells infected by VNP20009 without Fer-1 treatment and the tumor cells which were treated with Fer-1 and infected by VNP20009. Therefore, we demonstrated that VNP-SopE-GOx infection can significantly cause ferroptosis in tumor cells. In summary, we demonstrated that the viability of tumor cells infected by VNP-SopE-GOx is significantly reduced, and ferroptosis is the main cause of tumor cell death.

Figure 6: Cell viability of cells treated with bacterial infection and co-treated with bacterial infection and Fer-1.

H₂O₂ generation and Fenton reaction mediated by glucose oxidation

1.Consumption of glucose mediated by glucose oxidase

1.1 Introduction

Glucose plays a crucial role as an important nutrient in tumor growth. The proliferation of tumor cells mainly relies on aerobic glycolysis, making them more sensitive to changes in glucose concentration. The catalytic glycolysis process of glucose oxidase（GOx）can regulate the tumor microenvironment (TME).

On the one hand, GOx consumes oxygen, further increasing the hypoxia level, which is beneficial for better targeted and selective colonization of engineered bacteria. On the other hand, the generated H₂O₂ not only significantly enhances tumor oxidative stress but also can be converted into hydroxyl radicals to target cancer cells. The glucose and oxygen reaction, resulting in the formation of gluconic acid and hydrogen peroxide, is catalyzed by glucose oxidase (GOx).

Initially, GOx(FAD) binds with glucose and oxidizes it into gluconic acid. This oxidation process reduces GOx(FAD) to GOx(FADH₂). Subsequently, oxygen oxidizes GOx(FADH₂) back to GOx(FAD), generating hydrogen peroxide as a byproduct.
According to the data collected from wet lab, the glucose concentration in tumor cells is typically 5 mmol/L. The oxygen content is typically 0.8 mmol/L. In reality, the glucose diffuses randomly, so that the glucose concentration can be approximately maintained at 5 mmol/L over a longer period of time. in the image, we can see close to 1 mmol/L hydrogen peroxide being generated in a time of 0.01s. So, the tumor cells can produce sufficient amount of hydrogen peroxide.

We modeled the reaction with the following set of ordinary differential equations.

Chemical kinetic equation
Parameters

Diagram

1.2 Fenton reation and lipid peroxidation

In order to test whether the generated hydrogen peroxide succeeded in mediating efficient generation of polyunsaturated fatty acids(PUFAs), we constructed the following model. In the Fenton reaction, hydrogen peroxide produces hydroxyl radicals in the presence of ferrous ions. Hydroxyl radicals react with polyunsaturated fatty acids to produce polyunsaturated fatty acid free radicals, which destroy the structure of cell membrane and cause ferroptosis of cancer cells.

We built the following model based on the literature and the experimental results from wet lab. It can be found that $H_2O_2$ is significantly reduced, and close to 0.2 mmol/L of polyunsaturated fatty acid is generated in a short period of time. In the tumor cells, the reaction products will diffuse, $Fe^{2+}$, and $H_2O_2$ is continuously generated. So the reaction will continue to generate a sufficient amount of polyunsaturated fatty acid. In the following formula, $PUFAs$ are denoted by $LH$. And peroxidized $PUFAs$ are denoted by $L·$ in the following equation.

Chemical kinetic equation
Parameters
Diagram

According to the modeling results, intracellular glucose can produce polyunsaturated fatty acid free radicals effectively, resulting in lipid peroxidation in tumor cells.

Sequence and Features

Assembly Compatibility:

• 10
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  Illegal PstI site found at 34
  
• 12
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• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

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• 25
  INCOMPATIBLE WITH RFC[25]

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• 1000
  COMPATIBLE WITH RFC[1000]