Attenuated Salmonella typhimurium

The attenuation of virulence has been achieved by auxotrophic mutations that affect the biosynthesis of purines or aromatic amino acids , by mutations in the biosynthesis of lipid A , or by deletions of the relA and spoT genes, which generate strains defective in guanosine 5′-diphosphate 3′-diphosphate (ppGpp) synthesis .

Salmonella typhimurium VNP20009 was genetically engineered by the partial deletion of the msbB and purI genes . The msbB gene is responsible for the addition of a terminal myristyl group to lipid A. The lipopolysaccharide (LPS) produced by these lipid A mutants has a much lower capacity for inducing tumor necrosis factor-α (TNF-α) . The purI gene can induce a growth requirement for external sources of purines , and a purI gene mutation results in purine deficiency and a limited replication rate .

Salmonella typhimurium A1 is auxotrophic for leucine and arginine, and it was developed to prevent continuous infections in normal tissues while maintaining virulence against tumors because it has no other attenuated mutations compared with S. typhimurium VNP20009 . To enhance its tumor virulence, S. typhimurium A1 was injected into nude mice that had been transplanted with an HT-29 human colon tumor. The bacteria isolated from the infected tumor were then cultured, and the re-isolated A1 was designated A1-R. The number of A1-R bacteria attached to HT-29 human colon cancer cells was approximately six times higher than that of the parent A1 .

ppGpp is a stringent signaling molecule that is considered to be a key molecule for the cessation of ribosome production when a bacterial culture enters the stationary phase . Recently, ppGpp was implicated in the stationary phase induction of Salmonella pathogenicity island type I (SPI-1) genes by the activator hilA, which is a master transcriptional regulator of SPI-1-encoded genes. During the course of infections, it has been suggested that Salmonella encounters various stressful environments, which are sensed and translated into the intracellular signal ppGpp, and this induces the expression of Salmonella virulence genes, including SPI-1 genes . A ppGpp-defective Salmonella mutant exhibited significant virulence attenuation, whereas the LD ₅₀ of an S. typhimurium mutant that was defective for ppGpp synthesis (STΔppGpp) was increased by five orders of magnitude, irrespective of the administration route . This strain has been investigated in mouse models .

Selective Targeting of Tumors

From the standpoint of the interaction between bacteria and tumor cells or tissues, tumors have several unique characteristics compared with normal tissue, including hypoxic or necrotic areas, immune-privileged microenvironments, and abundant nutrients.

As a facultative anaerobe, Salmonella is thought to use several mechanisms to accumulate in tumors, such as the entrapment of Salmonella in the tumor vasculature , flooding into tumors after TNF-α-mediated inflammation , chemotaxis toward compounds produced by tumors , preferential amplification in tumor-specific microenvironments , and protection from immune system clearance .

The attenuation of Salmonella by auxotrophy reduces its virulence in mice but also amplifies its targeting capacity by two or three orders of magnitude in tumors compared with the liver . Auxotrophic mutants require exogenous nutrients, so they are generally weakened in mice, which allows them to be controlled or eradicated by the host defense mechanisms. Tumors contain actively dividing cells and necrotic areas, and they appear to be an environment in which metabolites such as purines, pyrimidines, and amino acids are plentiful, thereby providing a reservoir of nutrients for bacteria.

Salmonella chemotaxis using surface chemoreceptors also supports the targeting mechanism. A study using knockout bacteria showed that the aspartate receptor initiates chemotaxis toward viable tumor tissue where the serine receptor initiated tissue penetration and the ribose/galactose receptor directed S. typhimurium toward necrotic areas .

By contrast, attenuated Salmonella with a partial deletion of the msbB gene had a lower potential for accumulating in tumor tissues due to a decline in TNF-α, which could also lead to an influx of blood into tumors . Therefore, an adequate balance between attenuation and immunogenicity must be achieved.

Effective Delivery of Genes or Gene-Related Materials

As a delivery vector, Salmonella has been applied to gene therapy via three delivery routes : (1) the direct transfer of genes (or DNA) into the target tissue , (2) the delivery of proteins that were originally expressed by bacteria , and (3) the transfer of plasmids encoding small hairpin RNAs (shRNAs) ( Figure 30.2 ) . The direct transfer of genes (also known as bactofection) is the bacteria-mediated transfer of plasmid DNA into mammalian cells. Bacteria disrupt and release a plasmid, encoding the therapeutic gene after escaping from the blood vessel and entering the target cell. The plasmid is transferred into the cell nucleus and a therapeutic protein, with cytotoxic activity or immune response, is expressed by the host cell . Bactofection of mammalian cells can be achieved via the active invasion of nonphagocytic mammalian cells or passive uptake by phagocytic immune cells . Compared with other delivery mechanisms, such as protein delivery, this approach has benefits and drawbacks. Bactofection may produce stronger, more stable expression because it uses the mammalian expression system. However, the expression of the transferred genes may be more difficult to control . A poor transfer efficiency could also limit the therapeutic response because the transferred genes may be expressed exclusively in infected cells rather than being distributed homogeneously in the tissues. Furthermore, plasmids generally cannot replicate in host mammalian cells .

Delivery mechanisms for genetically engineered Salmonella.

Salmonella can be used as a delivery vector to introduce genes or gene products into target cells or tissues via three mechanisms. (A) Salmonella can transfer genes into the target cell in the form of a plasmid that encodes the therapeutic gene. The plasmid is released from disrupted Salmonella after infecting the target cell, and it is transferred into the cellular nucleus. The therapeutic protein is expressed by the target cell’s expression system. (B) Salmonella can deliver therapeutic proteins instead of genes, where the therapeutic protein is expressed by the Salmonella expression system inside or outside the target cell. (C) Salmonella can also transfer plasmid-based small hairpin RNAs (shRNAs) into the target cell nucleus or express the shRNAs directly. The shRNAs are processed into small interfering RNAs (siRNAs), which induce the degradation of mRNAs transcribed from specific genes.

Another approach is the bacterial delivery of therapeutic agents, in the form of proteins instead of genes. Bacteria can be used as protein production factories, so they can continue to produce cargo proteins while proliferating in the target tissues, either in the extracellular spaces or inside the targeted cells . The persistent bacteria can produce the requisite polypeptide in situ , thereby ensuring the localized distribution of a therapeutic agent without affecting systemic nontarget organs . This approach may facilitate the specific regulation of protein expression using inducible promoters (discussed later). Furthermore, the bacteria do not integrate the delivered genetic material into the host genome, which greatly decreases the risk of delivery-induced mutagenesis and tumorigenesis .

Finally, bacteria can be engineered to deliver plasmid-based shRNAs into tumor cells, where they produce small interfering RNA (siRNAs) (also known as gene silencing) . siRNAs induce the degradation of mRNAs transcribed by a specific gene such as Bcl2 or the signal transducer and activator of transcription 3 (STAT3) gene . The proteins expressed by both genes inhibit apoptosis, and STAT3, in particular, promotes cancer cell growth. The combination of these two advanced approaches (i.e., bacterial delivery and RNA interference) is expected to be a promising tool for cancer therapy .

Prodrug Conversion Strategies

Prodrug-converting enzymes (or suicide enzymes) aim to improve the therapeutic efficiency (benefit vs. toxic side effects) of cancer chemotherapy . A gene, encoding an enzyme that is not naturally expressed in the host, is introduced into the target cells by Salmonella and the transformed cells express the enzyme .

Salmonella typhimurium has been engineered to express suicide enzymes, such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) , and administration of the appropriate prodrugs (5-fluorocytosine or ganciclovir, respectively) leads to selective localization of the active forms, leading to tumor growth suppression or regression .

Cytotoxic Approaches to Controlling Cancer Cells

This strategy involves the introduction of anti-angiogenic or cytotoxic genes and native bacterial toxic proteins, which kill cancer cells .

The direct transfer of anti-angiogenic genes such as endostatin and thrombospondin-1 can kill cancer cells by preventing new blood vessel formation and blocking the nutrient supply. Salmonella has been used as the carrier to deliver cytotoxic genes that encode the second mitochondria-derived activator of caspases (SMAC) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which can induce apoptosis in many types of cancer cells .

Furthermore, bacterial toxic proteins, such as cytolysin A (also known as HlyE) , and other cytotoxic proteins, such as FAS ligand , TNF-α, or TRAIL , can be delivered to cancer cells using engineered Salmonella , where they induce apoptosis. In particular, the extracellular domain of TRAIL is a potent apoptotic agent in tumor cells, whereas it has minimal toxicity against normal cells. Attenuated Salmonella has been used to express TRAIL under the control of a prokaryotic promoter, resulting in tumor growth reduction .

Activation of the Immune System Using Cytokines

Unlike normal cells, cancer cells are generally tolerant of their host’s immune system, and they prevent immune cells from recognizing them as cancer cells. Conventional therapies such as chemotherapy or radiation therapy are limited when increasing the dose to eliminate all tumor cells because they can cause severe side effects in normal tissues. Immune-based therapy (immunotherapy) using cytokines is considered a powerful therapeutic tool, but it has several problems, including a short in vivo duration and severe side effects after the systemic administration of cytokines . Therefore, immunotherapy using genetically engineered bacteria may be a promising tool for overcoming the limitations of existing therapeutic methods.

Immunotherapy using Salmonella includes the transfer of specific genes encoding cytokines and the delivery of cytokines expressed by bacteria, which can enhance the anticancer effects of various lymphocytes, such as B cells, T cells, and natural killer cells .

Several different types of cytokines and growth factors have been investigated via the direct transfer of genes. These molecules, including FLT3L , interleukin-12 (IL-12) , and granulocyte–macrophage colony-stimulating factor (GM-CSF) , can activate several types of lymphocytes.

Salmonella has also been studied with respect to the delivery of cytokines produced intrinsically by the bacteria. These include IL-2 , IL-18 , CCL21 , and LIGHT . These cytokines induce T cells, natural killer cell proliferation (IL-2 or IL-18), dendritic cell growth (LIGHT), and leukocyte and neutrophil infiltration (LIGHT or CCL21), and they control the migration of immune cells (CCL21) .

Tumor-Specific Antigens

The engineering of Salmonellae to produce a cancer vaccine is another type of immunotherapy used to break the preexisting tolerance of the immune system with regard to tumor-specific antigens. Several members of the Salmonella genus have been used in vaccination strategies in which they serve as DNA or antigen carriers .

DNA vaccine delivery by genetically engineered Salmonella can inhibit the growth of several types of tumors that express specific antigens, such as α-fetoprotein and vascular endothelial growth factor (VEGF) receptor 2 . Antibodies against these antigens can prevent tumor formation, tumor growth, and angiogenesis .

In addition to DNA delivery, vaccine therapy based on antigen delivery has also been investigated using Salmonella . Immune cells may be sensitized and the formation of tumors that express specific antigens can be prevented by the delivery of these antigens, such as prostate-specific antigen (PSA) , NY-ESO-1 , canine parvovirus (CPV) , and RAF-1 .