# Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria
## 1 Introduction  
Bacterial cancer therapy relies on the inherent traits of certain facultative anaerobic bacteria that are capable of intratumoral penetration and localization in hypoxic areas presumably because of chemotaxis toward molecules produced by tumors and the immune-privileged environment of tumors [1; 2; 3]. Among gram-negative anaerbes, _Salmonella_ spp. has prevailed as a therapeutic candidate because it is amenable to genetic manipulation and can trigger an immune response [4; 5; 6; 7; 8; 9]. The initial response to the bacterial colonization of tumors is the secretion of the proinflammatory cytokine TNF\(\alpha\) by innate immune cells, which causes a hemorrhage in the tumor and the formation of large necrotic regions [10; 11]. The hemorrhage and necrosis formation induce tumor growth retardation. This is followed by the strong adjuvant effect of tumor-specific T cells that are activated by the colonizing bacteria [12; 13; 14; 15; 16]. CD8+ cytotoxic T cells are the main type of cells that counteract tumor growth.  
Tumor-colonizing bacteria can be used as a delivery system for therapeutic molecules that promote tumor elimination. However, this type of application is problematic for pathogenic _Salmonella_ spp. that can invade animal cells and reside within membrane-bound compartments, namely, _Salmonella_-containing vacuoles (SCVs) [17; 18]. The transportation of anticancer proteins expressed by SCV-bound _Salmonella_ into the cancer cell cytosol is another potential complication; therefore, _Salmonella_ should be prevented from invading host cells. Another challenging aspect of this approach is the specific targeting of cytotoxic anticancer proteins to solid tumors but not to the reticuloendothelial system (RES), the liver and spleen, where most of the bacteria are initially trapped [19; 20; 21]. To overcome this problem, bacteria are often genetically engineered to express a specific cytotoxic anticancer gene only when they accumulate in tumor tissues after being eliminated from the RES to prevent damage to these organs by toxic therapeutic molecules. We previously used the P\({}_{\text{BAD}}\) promoter from the _E. coli_ arabinose operon, which can be activated by L-arabinose to induce the selective expression of cytotoxic anticancer proteins, although multiple injections of the inducer can cause problems for patients [20; 22; 23; 24; 25].  
In this study, we determined the fate of the _E. coli_ K12 strain MG1655 and an attenuated strain of _Salmonella enterica_ serovar Gallinarum (_S._ Gallinarum), in which ppGpp synthesis was disabled after intravenous injection into tumor-bearing mice. The results showed that the bacteria that colonized the tumor tissue proliferated vigorously, whereas those in the RES were metabolically inert and were cleared in a short time by the innate immune cells. The AppGpp strain of avian-specific _S._ Gallinarum, the antitumor characteristics of which will be described in a separate manuscript, is defective in host cell invasion or intracellular survival [26]. We engineered \(\Delta\)ppGpp _S._ Gallinarum to express an immunotoxin (TGF\(\alpha\)-PE38, TP) using the constitutive exponential phase promoter, the ribosomal RNA promoter rrnB P1, and found that it has remarkable antitumor effects on tumor-bearing mice without causing any adverse effects.