# Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria
## 3 Results
### Expression of an Immunotoxin under the Control of the Ribosomal RNA Promoter (rmB P1)  
To determine whether the rrmB P1 promoter could drive the expression of cytotoxic anticancer proteins, the rrmB P1 promoter sequence was cloned in place of the _araBAD_ promoter in pBAD24 [38], which was fused to the open reading frame of the immunotoxin TP [25; 27]. This immunotoxin (TP) comprising TGF\(\alpha\) and a modified _Pseudomonas_ exotoxin A (PE38) derived from _Pseudomonas aeruginosa_ was developed for the treatment of EGFR-expressing malignant tumors such as brain tumors [39; 40; 41]. PE38 acts by inactivating protein synthesis in mammalian cells [42; 43]. PE38, which lacks an intrinsic cell-binding domain, binds to EGFR-expressing cancer cells via the TGF\(\kappa\) moiety in the recombinant toxin. The TP protein is cytotoxic to EGFR-expressing tumor cells in vitro and in xenograft mouse models [25; 44]. In this study, we used the ribosomal RNA promoter rrmB P1 to express TP constitutively. The psp secretion signal peptide composed of 32 amino acids [25] was fused in-frame to the N' end of TGF\(\alpha\)-PE38 in the plasmid named prrmB1-psp-TP. In addition, the plasmid contained the glmS gene to ensure the maintenance of the plasmid by a balanced-lethal host vector system in GlmS' mutant bacteria [27]. This mutant undergoes lysis when grown in the absence of N-acetyl-D-glucosamine (GlcNAcNac) unless complemented by a plasmid carrying the glmS gene. The \(\Delta\)ppGpp strain of \(S\). Gallinarum carrying the mutation in glmS was transformed with prrmBp1-psp-TP (SMP4003), grown in LB broth, and harvested at the indicated times to quantify the expression of TP. The bacterial cells and supernatant were separated and subjected to western blotting to detect TP expression (Figure 3A). Under the control of the rrmB P1 promoter, TP was expressed at high levels in the pellet in a constitutive manner, whereas it was detected in the supernatant at later time points, indicating that TP was secreted via the psp signal after a certain time. Next, we investigated the cytotoxic effect of the immunotoxin TP secreted from SMP4003 on cancer cell lines overexpressing EGFR, i.e., CT26 mouse colon carcinoma and 4T1 murine breast cancer cells (Figure S4) [25; 45]. The bacteria were grown in LB medium and harvested when the culture entered the stationary phase. The cultures were centrifuged, and the supernatants were collected and concentrated. The CT26 and 4T1 cancer cell lines were treated with PBS or concentrated bacterial supernatant (1 \(\upmu\)g protein). Approximately 70% of CT26 cells and 60% of 4T1 cells were killed after treatment with the supernatant of SMP4003 for 24 h. The supernatant from \(\Delta\)ppGpp \(S\). Gallinarum only (SG4023) was included as a control and showed a moderate effect. These data indicate that TP released from SMP4003 is cytotoxic to these cancer cells. The expression and secretion of TP from tumor targeted AppGpp \(S\). Gallinarum carrying the prrmBp1-psp-TP (SMP4003) plasmid were evaluated in BALB/c mice grafted with mouse colon cancer CT26 cells (Figure 3B). At the indicated days after tail vein injection of the bacteria (1 \(\times\) 10\({}^{8}\) CFU), the grafted tumors were isolated and homogenized, and the supernatant was separated by centrifugation and filtered through 0.2 \(\upmu\)m pores. The filtrate was analyzed for TP protein (43.3 kDa) expression by western blotting. TP was detected constantly throughout the course of the experiment from 1 to 5 dpi, suggesting that the protein was expressed constitutively from the rrmB P1 promoter and released from bacteria through the psp signal peptide. Next, we evaluated the antitumor effects of the immunotoxin on CT26 and mouse breast cancer 4T1 cells implanted into BALB/c mice (Figure 4). All mice received an intravenous injection of (i) PBS, (ii) \(\Delta\)ppGpp \(S\). Gallinarum (SG4023) alone, or (iii) SG4030 carrying prrmBp1-psp-TP (SMP4003).