# Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria
## 3 Results
### Fate of ppGpp-Defective S. Gallinarum Injected into Tumor-Bearing Mice  
For bacteria-mediated cancer therapy, _Salmonella_ spp. that trigger effective IL-1\(\beta\)/TNF-\(\alpha\)-mediated immune responses in the tumor leading to tumor regression are preferred over _E. coli_[9]. An attenuated strain of avian host-specific _S. enterica_ serovar Gallinarum was constructed by deleting relA and spoT, which encode enzymes that synthesize the bacterial signaling molecule ppGpp [26]. The ppGpp defect causes amino acid auxotrophy in _S. Typhimuriumium_ and in _E. coli_[35]. We observed that the ppGpp-defective \(S\). Gallinarum also required several amino acids to grow, including branched chain amino acids in addition to lysine and serine (Table S1). The AppGpp strain of \(S\). Gallinarum was attenuated by approximately 1000-fold in mice, which allowed injection of 10\({}^{8}\) CFU/mouse, resulting in regression of various tumors grafted in mice (manuscript in preparation). In this study, we examined the fate of AppGpp \(S\). Gallinarum after its injection into the tail vein of BALB/c mice bearing CT26 xenograft tumors. Similar to the _E. coli_, the bacterial counts in the tumor increased, whereas those in the RES decreased in a time-dependent manner (Figure 2). To obtain a clear picture of the fate of AppGpp \(S\). Gallinarum injected into tumor-bearing mice, we measured cell division using bacteria that were cross-linked with the reactive form of a fluorescent dye (Flamma(r) Fluors 552: NHS), which reacts readily with amine-modified oligonucleotides or amino groups of proteins on the bacterial surface [36; 37]. The bacteria incubated with the dye initially emitted strong red fluorescent signals when excited with a 550 nm laser light, which were visible under a fluorescence microscope (Figure 2A, 0 h). These bacteria were diluted in fresh LB medium (1/50) and grown with vigorous aeration (Figure 2B). Fluorescent signals from the bacterial cultures were detected at the indicated times and cell number was also estimated by determining optical density (A\({}_{600}\)). As the bacteria divided, the fraction of red fluorescent bacteria decreased, disappearing after approximately 2 h (four generations assuming g = 30 min) (Figure 2A,B). BALB/c marine bearing CT26 xenografts were injected with the fluorescent bacteria, and samples of the RES and tumor tissues were collected at the indicated times for the measurement of bacterial numbers and fluorescent signals using a fluorescence microscope (Figure 2C,D). Red fluorescent bacteria were observed in the RES even at 72 h after the injection, whereas they were rarely detected in tumor tissues after 12 h, indicating that the bacteria in the tumor tissue divided and diluted out the fluorescent dye (Figure 2E). In this experiment, the same tissue samples were stained for F4/80\({}^{+}\) macrophages, and the results showed that most of the bacteria in the RES were associated with macrophages, whereas those in the tumor were not (Figure S3). Lastly, the activity of the exponential phase promoter rmB P1 in \(\Delta\)ppGpp \(S\). Gallinarum was measured in vitro (Figure S2B) and in vivo by qPCR analysis (Figure 2F). The activity of rmB P1 in the tumor increased up to 72 h, whereas that in RES decreased over time. Taken together, these results suggest that the \(S\). Gallinarum accumulating in tumor tissues proliferated, whereas those in the RES were cleared by phagocytic macrophages. Figure 2: _Cont._Figure 2: Fate of \(\Delta\)ppGpp \(S\). Gallinarum injected into tumor-bearing mice. **(A)** Images of \(\Delta\)ppGpp \(S\). Gallinarum cross-linked with a red fluorescent dye (Flamma®) Fluors 552 NHS ester) grown in LB medium for the indicated amounts of time. Bacteria stained with a fluorescent antibody are shown in green and DAPI-stained nuclei are shown in blue. Scale bar = 5 \(\mu\)m for 1000\(\times\) magnification. **(B)** The red fluorescent bacteria were grown in LB with vigorous aeration.