# Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria
## 4 Discussion  
In this study, we determined the fate of attenuated noninvasive \(\Delta\)ppGpp \(S\). Gallinarum and the common laboratory strain of _E. coli_ MG1655 after intravenous injection into tumor-bearing mice (10\({}^{8}\) CFU). Approximately 10% of the injected bacteria were detected initially in the RES, whereas approximately 0.01% were in tumor tissues. The bacteria in the tumor tissue proliferated vigorously to up to 10\({}^{9}\) CFU/g tissue, whereas those in the RES died off. (Figures 1 and 2). The proliferation of bacteria in tumor tissues can be attributed to the unique immunosuppressive and biochemical environment of the tumor [46]. The rmB P1 promoter in proliferating bacteria in tumor tissues was active, whereas that in the dying bacteria in the RES was not. The rmB P1 promoter controls an operon that includes rrsB (16S rRNA), gltT (tRNA-glu), rrIB (23S rRNA), and rrfB (5S rRNA), which encode the three major rRNA building blocks of ribosomes [32]. In rapidly dividing bacteria, a large fraction of cellular energy and matter is devoted to the synthesis of ribosomes, accounting for 47% of the cell mass in _E. coli_ when grown fast with a generation time of <30 min [47]. Because the rate of ribosome synthesis is determined solely by the availability of rRNA, the decrease observed suggests that bacteria in the RES ceased to grow and thus did not  
Figure 4: Antitumor effect of \(\Delta\)ppGpp \(S\). Gallinarum carrying prrnBP1-psp-TP in BALB/c mice grafted with CT26 colon carcinoma and 4T1 murine breast cancer cells. BALB/c mice were subcutaneously implanted with 1 \(\times\) 10\({}^{6}\) CT26 colon carcinoma cells (\(n\) = 5 per group, (**A–C**)) or 1 \(\times\) 10\({}^{6}\) 4T1 murine breast carcinoma cells (\(n\) = 6 per group, (**D–F**)) on the high flank. Mice were treated with PBS (black line), \(\Delta\)ppGpp \(S\). Gallinarum (SG4023, green line), or \(S\). Gallinarum transformed with the therapeutic plasmid prrpmBP1-psp-TP (SMP4003, red line) by intravenous injection at a dose of 1 \(\times\) 10\({}^{8}\) CFU/mouse when tumor volumes reached 90–120 mm\({}^{3}\). Average tumor sizes in each group of mice grafted with CT26 (**A**) or 4T1 (**D**) were recorded every 2 days after treatment with the engineered bacteria until the volumes reached > 1500 mm\({}^{3}\) (two-way ANOVA with [PERSON]’s multiple comparisons test, \(p\) < 0.0001). Representative images of CT26 carcinoma (**B**) or 4T1 carcinoma (**E**) in the above mice. The survival of CT26 tumor-bearing mice (**C**) or 4T1 tumor-bearing mice (**F**) treated as described above was determined using Kaplan–[PERSON] curves (logrank Mantel–Cox test, \(p\) < 0.005 and \(p\) < 0.05 for (**C**,**F**), related to data shown in Figure S5. need ribosomes for de novo protein synthesis. It is likely that these bacteria were processed by phagocytic leukocytes, i.e., polymorphonuclear leukocytes (PMNs or neutrophils) and mononuclear phagocytes (monocytes, macrophages, and dendritic cells), providing a front line of defense against bacterial infection [48]. In this study, we showed that most of the bacteria in the RES were associated with phagocytic macrophages (Figure S3). These innate immune cells promote bacterial clearance through phagocytosis, generation of reactive oxygen and nitrogen species, extracellular trap formation, and production of proinflammatory cytokines [48]. The results presented in this study suggest that the confined expression of therapeutic payloads using a controllable system could be replaced by a constitutive expression system (Figures 3 and 4). Metabolically inert bacteria that died out in macrophages detected in the RES discontinued protein synthesis, and leakage of therapeutic cargo into the circulation would thus be impossible. Several inducible promoter systems have been developed for the controlled expression of therapeutic cargo, including an _E. coli_ promoter (pBAD) inducible with L-arabinose [20; 49; 50] and a tet promoter inducible with tetracycline [24; 49].