Tryptophanase Expressed by Salmonella Halts Breast Cancer Cell Growth In Vitro and Inhibits Production of Immunosuppressive Kynurenine.

Tryptophan is an essential amino acid required for tumor cell growth and is also the precursor to kynurenine, an immunosuppressive molecule that plays a role in limiting anticancer immunity. Tryptophanase (TNase) is an enzyme expressed by different bacterial species that converts tryptophan into indole, pyruvate and ammonia, but is absent in the Salmonella strain VNP20009 that has been used as a therapeutic delivery vector. We cloned the Escherichia coli TNase operon tnaCAB into the VNP20009 (VNP20009-tnaCAB), and were able to detect linear production of indole over time, using Kovács reagent. In order to conduct further experiments using the whole bacteria, we added the antibiotic gentamicin to stop bacterial replication. Using a fixed number of bacteria, we found that there was no significant effect of gentamicin on stationary phase VNP20009-tnaCAB upon their ability to convert tryptophan to indole over time. We developed a procedure to extract indole from media while retaining tryptophan, and were able to measure tryptophan spectrophotometrically after exposure to gentamicin-inactivated whole bacterial cells. Using the tryptophan concentration equivalent to that present in DMEM cell culture media, a fixed number of bacteria were able to deplete 93.9% of the tryptophan in the culture media in 4 h. In VNP20009-tnaCAB depleted tissue culture media, MDA-MB-468 triple negative breast cancer cells were unable to divide, while those treated with media exposed only to VNP20009 continued cell division. Re-addition of tryptophan to conditioned culture media restored tumor cell growth. Treatment of tumor cells with molar equivalents of the TNase products indole, pyruvate and ammonia only caused a slight increase in tumor cell growth. Using an ELISA assay, we confirmed that TNase depletion of tryptophan also limits the production of immunosuppressive kynurenine in IFNγ-stimulated MDA-MB-468 cancer cells. Our results demonstrate that Salmonella VNP20009 expressing TNase has improved potential to stop tumor cell growth and reverse immunosuppression.

Tumour-targeting bacteria engineered to fight cancer.

Recent advances in targeted therapy and immunotherapy have once again raised the hope that a cure might be within reach for many cancer types. Yet, most late-stage cancers are either insensitive to the therapies to begin with or develop resistance later. Therapy with live tumour-targeting bacteria provides a unique option to meet these challenges. Compared with most other therapeutics, the effectiveness of tumour-targeting bacteria is not directly affected by the 'genetic makeup' of a tumour. Bacteria initiate their direct antitumour effects from deep within the tumour, followed by innate and adaptive antitumour immune responses. As microscopic 'robotic factories', bacterial vectors can be reprogrammed following simple genetic rules or sophisticated synthetic bioengineering principles to produce and deliver anticancer agents on the basis of clinical needs. Therapeutic approaches using live tumour-targeting bacteria can either be applied as a monotherapy or complement other anticancer therapies to achieve better clinical outcomes. In this Review, we summarize the potential benefits and challenges of this approach. We discuss how live bacteria selectively induce tumour regression and provide examples to illustrate different ways to engineer bacteria for improved safety and efficacy. Finally, we share our experience and insights on oncology clinical trials with tumour-targeting bacteria, including a discussion of the regulatory issues.

Co-Expression of a Chimeric Protease Inhibitor Secreted by a Tumor-Targeted Salmonella Protects Therapeutic Proteins from Proteolytic Degradation.

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.

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

Tumor-targeted Salmonella VNP20009 preferentially replicate within tumor tissue and partially suppress tumor growth in murine tumor models. These Salmonella have the ability to locally induce apoptosis when they are in direct contact with cancer cells but they lack significant bystander killing, which may correlate with their overall lack of antitumor activity in human clinical studies. In order to compensate for this deficiency without enhancing overall toxicity, we engineered the bacteria to express epidermal growth factor receptor (EGFR)-targeted cytotoxic proteins that are released into the extracellular milieu. In this study, we demonstrate the ability of the Salmonella strain VNP20009 to produce three different forms of the Pseudomonas exotoxin A (ToxA) chimeric with a tumor growth factor alpha (TGFα) which results in its producing culture supernatants that are cytotoxic and induce apoptosis in EGFR positive cancer cells as measured by the tetrazolium dye reduction, and Rhodamine 123 and JC-10 mitochondrial depolarization assays. In addition, exchange of the ToxA REDLK endoplasmic reticulum retention signal for KDEL and co-expression of the ColE3 lysis protein resulted in an overall increased cytotoxicity compared to the wild type toxin. This approach has the potential to significantly enhance the antitumor activity of VNP20009 while maintaining its previously established safety profile. Biotechnol. Bioeng. 2016;113: 2698-2711. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

Isolation and Analysis of Suppressor Mutations in Tumor-Targeted msbB Salmonella.

Tumor-targeted Salmonella offers a promising approach to the delivery of therapeutics for the treatment of cancer. The Salmonella strains used, however, must be stably attenuated in order to provide sufficient safety for administration. Approaches to the generation of attenuated Salmonella strains have included deletion of the msbB gene that is responsible for addition of the terminal myristol group to lipid A. In the absence of myristoylation, lipid A is no longer capable of inducing septic shock, resulting in a significant enhancement in safety. However, msbB Salmonella strains also exhibit an unusual set of additional physiological characteristics, including sensitivities to NaCl, EGTA, deoxycholate, polymyxin, and CO2. Suppressor mutations that compensate for these sensitivities include somA, Suwwan, pmrA (C), and zwf. We describe here methods for isolation of strains with compensatory mutations that suppress these types of sensitivities and techniques for determining their underlying genetic changes and analysis of their effects in murine tumor models.

Accumulation of single-stranded DNA in Escherichia coli carrying the colicin plasmid pColE3-CA38.

We sequenced the complete 7118 bp circular plasmid pColE3-CA38 (pColE3) from Escherichia coli, located the previously identified colicin components together with two new ORFs that have homology to mobilization and transfer proteins, and found that pColE3 is highly similar to a plasmid present in enterohemorrhagic E. coli O111. We also found that unusual aspects of the plasmid include the inability to be completely digested with restriction endonucleases and asymmetric Phred DNA sequencing quality scores, with significantly lower scores in the forward direction relative to the colicin and immunity proteins consistent with plus (+) strand DNA. Comparing the A260 with picogreen double-stranded DNA (dsDNA) fluorescence and oligreen single-stranded DNA (ssDNA) fluorescence as well as metachromatic staining by acridine orange, we found that the undigested pColE3 DNA stains preferentially as ssDNA and that it coexists with dsDNA. We also identified ssDNA in pColE5 and pColE9 but not in pColE1. Colicin plasmids producing ssDNA may represent a new subclass of rolling-circle replication plasmids and add to the known similarities between colicins and filamentous phage.

A culture-based method for determining the production of secreted protease inhibitors.

We have developed a culture-based method for determining the production of secreted protease inhibitors. The assay utilizes standard proteolysis detection plates to support microbial growth followed by infiltrating the plate with a protease and subsequently detecting the remaining protein by trichloroacetic acid (TCA) precipitation, or by bromocreosol green (BCG) or Ponseau S (PS) staining. The presence of a protease inhibitor can be observed in the form of a protected zone of protein around the protease inhibitor-producing strain. Using the protease inhibitors α-2-macroglobulin, aprotinin, leupeptin, and bestatin and the primary and secondary forms of Photorhabdus luminescens in combination with the protease trypsin, we were able to demonstrate that the assay is specific for the cognate inhibitor of the protease and for bacteria secreting protease inhibitors. In addition, when casein-containing plates were used, the size of the diffusion zone was inversely correlated with the molecular weight of the inhibitor allowing a relative estimation of the protease inhibitor molecular weight. This assay is useful for detecting the presence of microbial secreted protease inhibitors and may reveal their production by microorganisms that were not previously recognized to produce them.

Optimizing Liposomal Cisplatin Efficacy through Membrane Composition Manipulations.

The first liposomal formulation of cisplatin to be evaluated clinically was SPI-077. Although the formulation demonstrated enhanced cisplatin tumor accumulation in preclinical models it did not enhance clinical efficacy, possibly due to limited cisplatin release from the formulation localized within the tumor. We have examined a series of liposomal formulations to address the in vivo relationship between cisplatin release rate and formulation efficacy in the P388 murine leukemia model. The base formulation of phosphatidylcholine: phosphatidylglycerol: cholesterol was altered in the C18 and C16 phospholipid content to influence membrane fluidity and thereby impacting drug circulation lifetime and drug retention. Phase transition temperatures (T(m)) ranged from 42-55°C. The high T(m) formulations demonstrated enhanced drug retention properties accompanied by low antitumor activity while the lowest T(m) formulations released the drug too rapidly in the plasma, limiting drug delivery to the tumor which also resulted in low antitumor activity. A formulation composed of DSPC : DPPC : DSPG : Chol; (35 : 35 : 20 : 10) with an intermediate drug release rate and a cisplatin plasma half-life of 8.3 hours showed the greatest antitumor activity. This manuscript highlights the critical role that drug release rates play in the design of an optimized drug delivery vehicle.

Leukemia-selective uptake and cytotoxicity of CPX-351, a synergistic fixed-ratio cytarabine:daunorubicin formulation, in bone marrow xenografts.

The objective of this study was to examine the pharmacodynamic basis for the potent preclinical and clinical anti-leukemic activity of CPX-351, a nano-scale liposome formulation of cytarabine and daunorubicin co-encapsulated at a synergistic 5:1 molar ratio. A bone marrow-engrafting CCRF-CEM leukemia model in Rag2-M mice was utilized to correlate the therapeutic and myelosuppressive properties of CPX-351 with bone marrow delivery and drug uptake in leukemia cells relative to normal bone marrow cell populations. When administered to mice bearing CCRF-CEM human leukemia xenografts, CPX-351 ablated bone marrow (BM) leukemic cells to below detectable levels for multiple weeks, whereas the free-drug cocktail only transiently suppressed leukemia growth. In contrast to the activity against leukemia cells, CPX-351 and free-drug cocktail induced similar myelosuppression in non-tumor-bearing BM. In leukemia-laden BM, drug concentrations were markedly elevated for CPX-351 over free-drug cocktail and the first dose of CPX-351, but not free-drug cocktail, potentiated BM drug accumulation for subsequent doses. Confocal fluorescence microscopy revealed that CPX-351 liposomes are taken up by CCRF-CEM cells and subsequently release drugs intracellularly. The improved in vivo efficacy of CPX-351 appears related to increased and prolonged exposure of synergistic cytarabine:daunorubicin ratios in BM, and the selective killing of leukemia may arise from direct liposome-leukemia cell interactions. These features may also have broader applicability in the treatment of other haematological malignancies.

msbB deletion confers acute sensitivity to CO2 in Salmonella enterica serovar Typhimurium that can be suppressed by a loss-of-function mutation in zwf.

BACKGROUND: Pathogens tolerate stress conditions that include low pH, oxidative stress, high salt and high temperature in order to survive inside and outside their hosts. Lipopolysaccharide (LPS), which forms the outer-leaflet of the outer membrane in Gram-negative bacteria, acts as a permeability barrier. The lipid A moiety of LPS anchors it to the outer membrane bilayer. The MsbB enzyme myristoylates the lipid A precursor and loss of this enzyme, in Salmonella, is correlated with reduced virulence and severe growth defects that can both be compensated with extragenic suppressor mutations. RESULTS: We report here that msbB (or msbB somA) Salmonella are highly sensitive to physiological CO2 (5%), resulting in a 3-log reduction in plating efficiency. Under these conditions, msbB Salmonella form long filaments, bulge and lyse. These bacteria are also sensitive to acidic pH and high osmolarity. Although CO2 acidifies LB broth media, buffering LB to pH 7.5 did not restore growth of msbB mutants in CO2, indicating that the CO2-induced growth defects are not due to the effect of CO2 on the pH of the media. A transposon insertion in the glucose metabolism gene zwf compensates for the CO2 sensitivity of msbB Salmonella. The msbB zwf mutants grow on agar, or in broth, in the presence of 5% CO2. In addition, msbB zwf strains show improved growth in low pH or high osmolarity media compared to the single msbB mutant. CONCLUSION: These results demonstrate that msbB confers acute sensitivity to CO2, acidic pH, and high osmolarity. Disruption of zwf in msbB mutants restores growth in 5% CO2 and results in improved growth in acidic media or in media with high osmolarity. These results add to a growing list of phenotypes caused by msbB and mutations that suppress specific growth defects.

Drug ratio-dependent antitumor activity of irinotecan and cisplatin combinations in vitro and in vivo.

Irinotecan and cisplatin are two established anticancer drugs, which together constitute an effective combination for treating small-cell lung cancer. We investigated whether the efficacy of this combination could be improved by controlling drug ratios following in vivo administration. Irinotecan and cisplatin combinations were evaluated systematically for drug ratio-dependent synergy in vitro using a panel of 20 tumor cell lines. In vitro screening informatics on drug ratio-dependent cytotoxicity identified a consistently antagonistic region between irinotecan/cisplatin molar ratios of 1:2 to 4:1, which was bordered by two synergistic regions. Liposomal co-formulations of these two agents were developed that exhibited plasma drug half-lives of approximately 6 hours and maintained a fixed drug ratio for more than 24 hours. Drug ratio-dependent antitumor activity was shown in vivo for these liposome formulations, and irinotecan/cisplatin ratios between 5:1 and 10:1 were identified as therapeutically optimal. The relationship between irinotecan/cisplatin ratio and in vivo efficacy was consistent with in vitro drug ratio dependency results. Superior antitumor activity was observed for the liposome-encapsulated 7:1 molar ratio of irinotecan/cisplatin (designated CPX-571) compared with the free-drug cocktail in all models tested. Further efficacy studies in a range of human tumor xenografts, including an irinotecan-resistant model, showed that both liposomal agents contributed to the overall efficacy in a manner consistent with in vivo synergy. These results show the ability of drug delivery technology to enhance the therapeutic activity of irinotecan/cisplatin combination treatment by maintaining synergistic ratios in vivo. CPX-571, a fixed-ratio formulation of irinotecan and cisplatin, is a promising candidate for clinical development.

Tumor-targeted Salmonella typhimurium overexpressing cytosine deaminase: a novel, tumor-selective therapy.

The ideal anticancer regimen is one that is specific for cancer cells with limited toxicity to normal tissues. Genetically modified, nonpathogenic Salmonella offer a potential way to induce direct tumoricidal activity or to deliver tumoricidal agents to tumors. An attenuated strain of Salmonella typhimurium, called VNP20009, and its derivative TAPET-CD (which expresses Escherichia coli cytosine deaminase) are highly selective for tumor tissue and can deliver therapeutic proteins preferentially to tumors in preclinical models. Both VNP20009 and TAPET-CD have been investigated successfully in Phase 1 clinical trials in cancer patients.

In vivo maintenance of synergistic cytarabine:daunorubicin ratios greatly enhances therapeutic efficacy.

We demonstrate here that cytarabine and daunorubicin, a standard drug combination used in the treatment of leukaemia, exhibits drug ratio-dependent synergistic antitumor activity in vitro and in vivo. A cytarabine:daunorubicin molar ratio of 5:1 displayed the greatest degree of synergy and minimum antagonism in a panel of 15 tumor cell lines in vitro. Co-encapsulating cytarabine and daunorubicin inside liposomes maintained the synergistic drug ratio in plasma for 24h post-injection. Liposome-encapsulated cytarabine:daunorubicin combinations exhibited drug ratio-dependent in vivo efficacy with the 5:1 molar drug ratio (designated CPX-351) having the greatest therapeutic index, despite using sub-MTD daunorubicin doses. CPX-351 exhibited superior therapeutic activity compared to free-drug cocktails, with high proportions of long-term survivors, consistent with in vivo synergy. The therapeutic advantage of CPX-351 was associated with prolonged maintenance of synergistic drug ratios in bone marrow. These results indicate that in vitro informatics on cytarabine:daunorubicin cytotoxicity can be translated in vivo to optimize the efficacy of anticancer drug combinations by controlling the exposure of drug ratios with drug delivery vehicles.

Attenuated Salmonella targets prodrug activating enzyme carboxypeptidase G2 to mouse melanoma and human breast and colon carcinomas for effective suicide gene therapy.

PURPOSE: We engineered the oncolytic Salmonella typhimurium-derived bacterium VNP20009 as a vector to target delivery to tumors of the prodrug-activating enzyme carboxypeptidase G2 (CPG2) and to show enhanced antitumor efficacy on administration of different prodrugs. EXPERIMENTAL DESIGN: We characterized CPG2 expression in vectors by immunoblotting, immunofluorescence, and enzyme activity. We assessed prodrug activation by high-performance liquid chromatography. Target human tumor cell and bacterial vector cell cytotoxicity was measured by flow cytometry and colony-forming assays. Therapy was shown in two human tumor xenografts and one mouse allograft with postmortem analysis of bacterial and CPG2 concentration in the tumors. RESULTS: CPG2 is expressed within the bacterial periplasm. It activates prodrugs and induces cytotoxicity in human tumor cells but not in host bacteria. Following systemic administration, bacteria multiply within xenografts reaching 2 x 10(7)/g to 2 x 10(8)/g at 40 days postinoculation. The concentration of CPG2 in these tumors increases steadily to therapeutic levels of 1 to 6 units/g. The bacteria alone reduce the growth of the tumors. Subsequent administration of prodrugs further reduces significantly the growth of the xenografts. CONCLUSIONS: The bacteria multiply within tumors, resulting in a selective expression of CPG2. The CPG2-expressing bacteria alone reduce the growth of tumors. However, in the presence of prodrugs activated by CPG2, this oncolytic effect is greatly increased. We conclude that bacterial oncolytic therapy, combined with CPG2-mediated prodrug activation, has great potential in the treatment of a range of cancers.

Modulating the therapeutic activity of nanoparticle delivered paclitaxel by manipulating the hydrophobicity of prodrug conjugates.

A series of paclitaxel prodrugs designed for formulation in lipophilic nanoparticles are described. The hydrophobicity of paclitaxel was increased by conjugating a succession of increasingly hydrophobic lipid anchors to the drug using succinate or diglycolate cross-linkers. The prodrugs were formulated in well defined block copolymer-stabilized nanoparticles. These nanoparticles were shown to have an elimination half-life of approximately 24 h in vivo. The rate at which the prodrug was released from the nanoparticles could be controlled by adjusting the hydrophobicity of the lipid anchor, resulting in release half-lives ranging from 1 to 24 h. The diglycolate and succinate cross-linked prodrugs were 1-2 orders of magnitude less potent than paclitaxel in vitro. Nanoparticle formulations of the succinate prodrugs showed no evidence of efficacy in HT29 human colorectal tumor xenograph models. Efficacy of diglycolate prodrug nanoparticles increased as the anchor hydrophobicity increased. Long circulating diglycolate prodrug nanoparticles provided significantly enhanced therapeutic activity over commercially formulated paclitaxel at the maximum tolerated dose.

pmrA(Con) confers pmrHFIJKL-dependent EGTA and polymyxin resistance on msbB Salmonella by decorating lipid A with phosphoethanolamine.

Mutations in pmrA were recombined into Salmonella strain ATCC 14028 msbB to determine if pmrA-regulated modifications of lipopolysaccharide could suppress msbB growth defects. A mutation that functions to constitutively activate pmrA [pmrA(Con)] suppresses msbB growth defects on EGTA-containing media. Lipid A structural analysis showed that Salmonella msbB pmrA(Con) strains, compared to Salmonella msbB strains, have increased amounts of palmitate and phosphoethanolamine but no aminoarabinose addition, suggesting that aminoarabinose is not incorporated into msbB lipid A. Surprisingly, loss-of-function mutations in the aminoarabinose biosynthetic genes restored EGTA and polymyxin sensitivity to Salmonella msbB pmrA(Con) strains. These blocks in aminoarabinose biosynthesis also prevented lipid A phosphoethanolamine incorporation and reduced the levels of palmitate addition, indicating previously unknown roles for the aminoarabinose biosynthetic enzymes. Lipid A structural analysis of the EGTA- and polymyxin-resistant triple mutant msbB pmrA(Con) pagP::Tn10, which contains phosphoethanolamine but no palmitoylated lipid A, suggests that phosphoethanolamine addition is sufficient to confer EGTA and polymyxin resistance on Salmonella msbB strains. Additionally, palmitoylated lipid A was observed only in wild-type Salmonella grown in the presence of salt in rich media. Thus, we correlate EGTA resistance and polymyxin resistance with phosphoethanolamine-decorated lipid A and demonstrate that the aminoarabinose biosynthetic proteins play an essential role in lipid A phosphoethanolamine addition and affect lipid A palmitate addition in Salmonella msbB strains.

Positron emission tomography (PET) imaging of tumor-localized Salmonella expressing HSV1-TK.

In order to noninvasively detect Salmonella delivery vectors within tumors, we used a genetically modified Salmonella, VNP20009, that expresses the herpes simplex thymidine kinase (HSV1-tk) reporter gene. VNP20009-TK were able to selectively localize within murine tumor models and to effectively sequester a radiolabeled nucleoside analogue, 2'-fluoro-1-beta-D-arabino-furanosyl-5-iodo-uracil (FIAU). A quantitative relationship between the level of radioactivity accumulated and the number of bacteria in tumor and different tissues was demonstrated. The in vivo accumulation of [14C]FIAU measured in tissue sample homogenates and sections were related to Salmonella number and to immunohistochemical bacterial staining, respectively. Quantitative autoradiography (QAR) revealed the relative intensity of [14C]FIAU accumulation in a tumor cross-section, demonstrating that the peripheral region of the tumor was significantly less active than internal regions. [124I]FIAU positron emission tomography (PET) and subsequent tissue radioactivity and bacterial concentration measurements were compared. A log-log relationship was found, and the PET images could identify multiple tumor sites. The ability to noninvasively detect Salmonella vectors by PET imaging has the potential to be conducted in a clinical setting, and could aid in development of these vectors by demonstrating the efficiency and duration of targeting as well as indicating the locations of tumors.

Hot spot for a large deletion in the 18- to 19-centisome region confers a multiple phenotype in Salmonella enterica serovar Typhimurium strain ATCC 14028.

Loss of the Salmonella MsbB enzyme, which catalyzes the incorporation of myristate destined for lipopolysaccharide in the outer membrane, results in a strong phenotype of sensitivity to salt and chelators such as EGTA and greatly diminished endotoxic activity. MsbB- salmonellae mutate extragenically to EGTA-tolerant derivatives at a frequency of 10(-4) per division. One of these derivatives arose from inactivation of somA, which suppresses sensitivity to salt and EGTA. Here we show that a second mode of MsbB- suppression is a RecA-dependent deletion between two IS200 insertion elements present in Salmonella enterica serovar Typhimurium strain ATCC 14028 but not in two other wild-type strains, LT2 and SL1344, which lack one of the IS200 elements. This deletion occurs spontaneously in wild-type and MsbB- strain 14028 salmonellae and accounts for about one-third of all of the spontaneous suppressors of MsbB- in strain 14028. It spans the region corresponding to 17.7 to 19.9 centisomes, which includes somA, on the sequenced map of Salmonella LT2 (136 ORFs in that strain; ATCC 14028 and other strains showed variability in this region). In addition to conferring EGTA resistance correlated with somA, the deletion confers a MacConkey galactose resistance phenotype on MsbB- Salmonella, indicating that at least one additional gene (distinct from somA) within the deletion is responsible for this phenotype. In the wild type, the deletion mutant grows with normal exponential growth rate in Luria broth but is chlorate resistant and does not grow on citrate agar. The deletion strains have lost hydrogen sulfide production, nitrate reductase activity, and gas production from glucose fermentation.

Bacteria as tumour-targeting vectors.

Live bacteria were first actively used in the treatment of cancer nearly 150 years ago, work that ultimately led to the study of immunomodulation. Today, with the discovery of bacterial strains that specifically target tumours, and aided by genomic sequencing and genetic engineering, there is new interest in the use of bacteria as tumour vectors. Bifodobacterium, Clostridium, and Salmonella have all been shown to preferentially replicate within solid tumours when injected from a distal site, and all three types of bacteria have been used to transport and amplify genes encoding factors such as prodrug-converting enzymes, toxins, angiogenesis inhibitors, and cytokines. In this review we provide a historical discussion of this area, and describe the development of the bacteria, which are currently being prepared for use in clinical trials in patients with cancer.