Engineering Salmonella for Cancer Treatment

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Magnified 20,000X, this colorized scanning electron micrograph (SEM) depicts a grouping of methicillin resistant Staphylococcus aureus (MRSA) bacteria. See PHIL 617 for a black and white view of this image. Photo credit: CDC.

Engineered Microbes for Cancer Treatment By Isaac Johnson

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Introduction

Cancer is perhaps the most notorious disease on Earth. Over decades of research, four major therapies have been developed: surgery, radiotherapy, chemotherapy, and immunotherapy (Hompland et al. 2021). However, cancerous cells that rapidly proliferate often outgrow the surrounding vasculature, developing regions of hypoxia (Chen et al. 2023). Hypoxic conditions directly lead to treatment resistance of all therapies but surgery. Radiotherapy relies on the presence of molecular oxygen to generate reactive oxygen species to induce cell damage (Menegakis et al. 2021), while chemo- and immunotherapeutic drugs are administered through the circulatory system, and therefore have inherently lower drug uptake with limited vasculature (Ho et al. 2022). Furthermore, hypoxia reduces the cytotoxicity of these drugs and can trigger metastasis (Ho et al. 2022).

A solution may lie in bacterial-based treatments. Far from being a new discovery, bacterial treatments were observed by Dr. William Coley in the 19th century when injections of Streptococcus pyogenes led to tumor regression (McCarthy 2006). While the results reported by Coley were promising, many highlighted inconsistencies in his methods and doubted their legitimacy. Later, with the advent of radiotherapy in the 1890s and chemotherapy in the 1930s, bacterial treatments were forgotten. In hindsight, Coley’s experiments were an early, rudimentary form of immunotherapy. Tumors evade or suppress a patient’s immune system through various molecular pathways (Tie et al. 2022), but an infection can naturally stimulate the immune system and trigger the body’s defense mechanisms against a tumor.

Unsurprisingly, bacteria alone are insufficient cancer treatments, but they have several advantages that are not found in current methods. Unlike chemotherapy and immunotherapy, many bacteria do not require the circulatory system to propagate within a host. Additionally, many bacteria are facultative anaerobes and thrive in hypoxic conditions. Not only would certain species be able to penetrate a tumor, but they could grow within the tumor microenvironment as well (Zhou et al. 2018). Unfortunately, bacteria’s innate stimulation of the immune system is often inefficient in eliminating cancer, unpredictable from patient to patient, and can cause several negative effects which may outweigh the possible benefits. However, genetic engineering is enabling researchers to inhibit a bacterium’s virulence, increase its localization to tumors, and implement novel treatment strategies based on chemo- and immunotherapeutic approaches (Gurbatri et al. 2022).

This article focuses on recent engineering efforts on Salmonella typhimurium, a gram-negative, rod-shaped pathogen. S. typhimurium is one of the leading causes of foodborne illness, known to cause gastroenteritis in humans and animals (Fàbrega and Vila, 2013). However, several characteristics make it a promising candidate for cancer therapy. It has flagella allowing for movement and is facultatively anaerobic, allowing it to thrive in hypoxic conditions. Additionally, it is an intracellular pathogen capable of infiltrating and reproducing inside host cells (Guo et al. 2020). Lastly, and most importantly, it is a well-studied species with a fully sequenced genome (McClelland et al. 2001).

Safety and Specificity

Salmonella typhimurium is naturally retained in tumor microenvironments (TMEs) due to selective colonization: after initial dosing, the bacteria delivered to the tumor is approximately equal to the amount of bacteria colonizing surrounding tissues. However, outside of the immunosuppressive environment of the TME, the surrounding bacteria die while bacteria in the TME proliferate (Zhou et al. 2018). This results in an approximate 1,000-fold increase of tumor colonization compared to other tissues (Gurbatri et al. 2022). While this is clearly beneficial, S. typhimurium will also accumulate in healthy organs. For this reason, genetically-engineered strategies have been developed to increase specificity and safety.

Virulence Knockouts

One such strategy involves knocking out certain genes of S. typhimurium. Gram-negative bacteria like Salmonella have a lipopolysaccharide (LPS) outer membrane—polysaccharide chains extending from the cell—that protects the cell from exterior toxins and bile salts (Bertani and Ruiz, 2018). These polysaccharides are rooted in the membrane by lipid A, a toxic molecule responsible for much of S. typhimurium’s pathogenicity. Upon bacterial lysis by the host’s immune system, lipid A is released from the membrane and triggers the expression of inflammatory proteins like TNFɑ (Raetz and Whitfield, 2002). While this pathway is essential for stimulating the immune system and eliminating infections, it is dangerous in cases of severe infection. A surplus of lipid A will prompt an overproduction of inflammatory factors, triggering blood clotting, septic shock, and possibly death (Esmon et al. 2000).

Since the LPS membrane is critical for bacterial survival, a given knockout strain must disarm the toxic capabilities of lipid A while retaining its role in membrane structure. By measuring TNFɑ induction between strains, researchers found that the deletion of the msbB gene in S. typhimurium produced viable cells with a 10-fold decrease in pathogenicity, along with retaining all of its previous tumor-targeting abilities (Low et al. 1999). Loss of the msbB gene prevents the myristoylation of lipid A, a lipid modification reaction crucial for downstream pathways.

This attenuated strain of S. typhimurium, named VNP20009, was isolated and tested in a mouse melanoma model, causing tumor regression (Low et al. 1999). It was subsequently implemented in clinical trials. These trials revealed that while VNP20009 was safe for patients, tumors were weakly colonized and no significant regressive effects were observed (Toso et al. 2002). These results suggested that TNFɑ induction may have been critical to Salmonella’s antitumor properties, emphasizing the difficulty of achieving both safe and efficacious treatments (Gurbatri et al. 2022).

Section 3

Include some current research, with at least one figure showing data.

References

Authored for BIOL 238 Microbiology, taught by Joan Slonczewski,at Kenyon College,2024