# Microbes as Medicines: Harnessing the Power of Bacteria in Advancing Cancer Treatment
## 2 Mechanism of Tumor Suppression
### Genetically Engineered Bacteria for Anti-Cancer Therapy
#### 2.2.1 Virulence Attenuation  
Pathogenicity of bacteria is a major safety concern when employing them as vectors. Attenuation of pathogenicity and safety can be ensured by deleting certain bacterial virulence genes without affecting the anti-tumor activity. Liposaccharide (LPS) present in the outer membrane of Gram-negative bacteria is responsible for sepsis as it is a potent tumor necrosis factor-\(\alpha\) (TNF-\(\alpha\)) stimulator. Therefore, when using Gram-negative bacteria, various strategies are employed to address LPS-related complications. For example, safety profile of genus _Salmonella_ was raised by deleting the virulence gene mshB which leads to loss of myristoylation of lipid A, an essential component of LPS. Hence, the toxicity of _Salmonella_ was drastically reduced by minimizing the TNF-\(\alpha\) expression [48]. This marked the generation of an attenuated _S. typhimurium_ strain, VNP20009, by deleting msbB (lipid mutataion) and purl (purine auxotrophy) gene. The attenuated strain showed favorable tumor specificity and inhibitory action in various murine tumor models such as B16F10 melanoma and Lewis lung carcinoma [49]. However, in Phase I clinical trials, the strain lacked tumor specificity in metastatic melanoma patients. Tumor colonization was observed however, no anti-tumor effect was evident [50]. The failure was attributed to the presence of penta-acylated lipid A, an antagonist of toll-like receptor (TLR4) responsible for diminished immunostimulatory activity [51]. To address the limitation and introduce anti-tumor activity, mutant _Salmonella_ strains were generated by deleting pagP, pagL, and lpxR genes thus forming hexa-acetylated lipid A possessing high affinity for TLR4. The anti-tumor activity was introduced with loss of virulence [1]. Another strain was genetically designed to downregulate genes responsible for expression of endotoxin associated genes. _Salmonella_ relA- and spoT-mutant strains were altered causing malfunctioning in the synthesis of ppGpp, a signaling molecule involved in expression of toxic genes and thereby, improving the safety [2; 52]. Its excellent anti-tumor activity can be attributed to inflammasome release such as nucleotide binding domain, leucine rich containing, pyrin domain containing (NLRP3) and ice protease activating factor (IPAF, also referred to as NLRC4)  
Figure 2: Bactofection into tumor cells: Intereellular: Bacteria bearing the transgene penetrates into the tumor cells, undergo lysis and release the transgene inside the tumor cell; Extracellular: Bacteria bearing the transgene do not enter the cell but, expresses the transgene in the vicinity of the tumor cells.  
which induces the expression of proinflammatory cytokines, interleukins (IL-18, IL-18) and TNF-\(\alpha\) and consequently, causes tumor regression. This strain is defective in entering and replicating into the host cells, and hence acts as an extracellular bacterium [12].  
Tumor specific proliferation and virulence attenuation can also be attained by developing auxotrophic mutants with specific nutrient requirement found preferentially only in the TME. Salmonella A1-R strain is auxotrophic for leucine and arginine and preferentially colonizes in tumors enriched with these nutrients. The A1-R strain was found to be effective in various mouse models of cancers such as prostate [53], pancreatic [54], and ovarian cancer [55]. _S. typhimurium_ strain, YBI, was engineered from SL7207 by incorporating essential gene asd in the hypoxia induced promoter region. This enables the strain to selectively survive in hypoxic TME and hence, reduces the toxicity to the normal tissues. Moreover, the deletion of purI and purD genes gave rise to the need of exogenous adenine, which increased the bacteria's susceptibility to proliferate in purine rich tumor regions in CT26 colon carcinoma mice model [56].