Thesis

Heterologous Expression and Secretion of Aggregatibacter Leukotoxin A by a Tumor-Targeted Salmonella

Despite the advancement of numerous treatment options, conventional cancer therapeutics generally fail to prevent tumor recurrence or successfully treat metastatic cells. A promising alternative involves the use of attenuated Salmonella strains, which preferentially replicate within tumor tissue and suppress tumor growth. Although the tumor-targeted strain VNP20009 localized to some tumors with high efficiency in human clinical trials, they failed to result in eradicating cancer cells or delay tumor growth. To enhance the tumoricidal efficacy of this strain, we engineered the bacteria to express leukotoxin A (LtxA), a pore-forming multigene toxin produced by Aggregatibacter actinomycetemcomitans with natural specificity for β2-integrins found on human leukocytes. Previous studies have shown that the overexpression of this surface antigen on cancer cells is associated with increased metastatic potential, suggesting that LtxA may represent a target for metastatic tumors, which are notoriously resistant to therapy. The LtxA-encoding genes were cloned into the inducible plasmid vector pAra99a and expressed in the tumor-targeted Salmonella strain VNP20009. LtxA expression at the mRNA transcript level was verified by RT-PCR. A codon-optimized LtxA sequence was generated and tested for its ability to enhance expression in the VNP20009 background. To improve the secretion of the LtxA protein into the culture supernatant, the wild-type LtxCA was co-expressed with the E. coli hemolysin-α (HlyA) secretion complex, HlyBD. C-terminal modifications were generated to facilitate interaction of the LtxA protein with the HlyBD complex. Protein expression of the wildtype and modified LtxA constructs generated in this study could not be consistently determined by immunoblot analyses, although Columbia agar hemolysis assays suggested that active LtxA protein was functionally expressed and secreted by the wild type LtxCABD construct and an LtxCA with a modified C-terminus together with the E. coli HlyBD expressed in trans. In preliminary experiments, the human monocytic cell line THP-1 was sensitive to wildtype LtxCABD and to the C-terminal fusion constructs when co-expressed with HlyBD by VNP20009.

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