Lipid A mutant Salmonella with suppressed virulence and TNFα induction retain tumor-targeting in vivo

Abstract

37Salmonella strains have recently been developed as antitumor agents capable of both preferentially amplifying within tumors and expressing prodrug-converting enzymes such as the herpes simplex thymidine kinase 1 . These bacteria were attenuated by auxotrophic mutations that limited their pathogenesis in normal tissues but retained high-level replication within the tumors following systemic administration. The auxotrophic requirements of these Salmonella are apparently met within the tumor environment where they then replicate, reaching up to more than 1000 times the concentration found in normal tissues.A significant limitation for safe use of systemically administered bacteria in humans is the ability of the bacteria to induce tumor necrosis factor α (TNFα)-mediated septic shock 2,3 . However, modifications in bacterial components responsible for eliciting host immune responses such as TNFα induction could interfere with tumor targeting or antitumor activity.Several mutations in lipid biosynthesis are known in Escherichia coli and Salmonella sp. that lower TNFα induction and render the bacteria nontoxic. Some mutations, such as kdo -result in the production of lipid IV A , which substantially lowers TNFα induction and acts as an antagonist to the TNFα response from wild-type lipid A 4,5 . However, these and most other lipid mutations are temperature-sensitive and conditionally lethal to the bacteria 6 , limiting the potential for tumor-based amplification seen in auxotrophic Salmonella 1 .In E. coli, the msbB (mlt) gene 7,8 is involved in the terminal myristoylation of lipid A 9,10 . Genetic disruption of this gene in E. coli results in a stable nonconditional mutation that lowers TNFα induction up to 10-fold by whole bacteria or up to 10,000-fold by purified lipopolysaccharide (LPS) 9 . A similar toxicity profile is observed when the msbB gene is disrupted in Salmonella 11 . We generated a deletion in the coding sequence of msbB within a hyperinvasive strain of Salmonella we previously used for tumor-targeting as well as the parental wild type, and examined the effect on virulence and TNFα production both in vitro and in vivo. Results indicate that msbB -mutant Salmonella retain the properties of tumor accumulation and tumor suppression in the absence of eliciting high levels of TNFα. Results Isolation and genetic disruption of the Salmonella msbB gene.DNA sequence analysis of Salmonella msbB clones obtained by DNA/DNA hybridization indicated the presence of an msbB homolog with flanking gene organization (orfU, msbB, pykA, and zwf) identical to E. coli 8 . The DNA homology of the Salmonella msbB and the E. coli msbB was determined to be 75%, and the amino acid homology 98%, confirming that the cloned Salmonella gene is an msbB homolog.Putative knockouts obtained by transformation of the linearized deletion construct were confirmed by several criteria using Southern blot analysis (Fig. 1): Two bands corresponding to the tetracycline gene were observed in the knockout construct and in the knockout clones and w...

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

“…Tumor-targeted bacteria offer alternative solutions to this and other problems as these bacteria readily travel through the blood, with a portion of them avoiding the innate immune system and taking up residence within the tumor where they then persist despite a subsequent systemic immune response. The immunoprivileged nature of tumors is thought to contribute to the persistence and tumor-selective growth of the bacteria within the tumor (Bermudes et al, 2001b; Darveau, 1999; Low et al, 1999), including providing partial protection from antibody responses, and thus the effect of neutralizing antibodies against the chimeras within the tumors should also be diminished. Furthermore, targeted proteins delivered systemically have limitations for delivery deep inside of tumors, whereas the bacteria localize even within the necrotic regions and can produce therapeutic proteins directly in situ (Soghomonyan et al, 2005), which could overcome that limitation.…”

“…Among the live bacteria now implicated with potentially positive therapeutic effects against cancer is Salmonella , which is currently being studied by a number of investigators for its tumor-targeted properties (see Forbes, 2010 and Hoffman, 2015 for reviews). Remarkably, attenuated strains of Salmonella preferentially grow in tumor tissue greater than 1000-fold compared to normal healthy tissue, creating a tumor-localized infection (Low et al, 1999; Pawelek et al, 1997, 2003). In studies using mice, Salmonella preferentially grow within all major forms of solid tumors, including prostate, breast, lung, and colon tumors, presenting the opportunity to develop a broadly effective cancer treatment.…”

Salmonella pathogenicity island-2 and anticancer activity in mice

“…Three S. enterica servovar Typhimurium parental strains ( all derived from ATCC 14028 ) were employed in these studies: YS7212, 1 YS1456, 7 and YS1646 ( VNP20009 ). 3,7 All three carry attenuating auxotrophic mutations, and YS1456 and YS1646 carry further attenuating msbB À (altered lipid A ) mutations whereas YS7212 carries msbB + .…”

“…3,7 All three carry attenuating auxotrophic mutations, and YS1456 and YS1646 carry further attenuating msbB À (altered lipid A ) mutations whereas YS7212 carries msbB + . All three strains showed similar tumor targeting, amplification, and growth suppression when injected intravenously (i.v. )…”

“…6 Lipid A -modified (msbB À ) salmonellae auxotrophs ( purI À ) that were attenuated for toxicity in mice and swine were developed. 7 These mutants showed significantly reduced host TNF induction, yet retained the abilities for tumor-targeting, amplification, and growth suppression in mice, achieving tumor accumulations of 10 9 -10 10 colonyforming units ( cfu ) /g tumor with tumor-to -normal tissue ratios exceeding 1000:1. However, little is known about the mechanisms through which salmonellae exert their anticancer effects.…”