Delivery of protein antigens and DNA by virulence-attenuated strains of Salmonella typhimurium and Listeria monocytogenes

Two different plasmid-vector systems were developed which allow the efficient production and presentation of protein antigens in antigen-presenting cells (APC) by means of virulence-attenuated bacteria. The first antigen-delivery system is based on the secretion machinery of the Escherichia coli hemolysin (HlyA-type I secretion system), which transports proteins, possessing the specific HlyA secretion signal (HlyA(s)) at the C-terminus, across both membranes of gram-negative bacteria. This system functions in all gram-negative bacteria that possess the TolC-analogous protein in the outer membrane. This outer membrane protein is necessary for the stable anchoring of the type I secretion apparatus in the cell envelope. Suitable HlyA(s)-fused antigens are secreted with high efficiency by E. coli and by virulence-attenuated strains of Salmonella, Shigella, Vibrio cholerae and Yersinia enterocolitica. The other vector system expresses the heterologous antigen under the control of an eukaryotic promoter in a similar fashion as in plasmids commonly used for vaccination with naked DNA. This plasmid DNA is introduced into APCs with the help of virulence-attenuated self-destructing Listeria monocytogenes mutants. After synthesis of the heterologous protein, epitopes of the antigen are presented by the APC together with MHC class I molecules. This system functions in macrophages and dendritic cells in vitro and can also be used in a modified form in animal models.     

A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase

Transfection efficiency of different cell types as well as promoter strength of cloned genes can be easily determined by direct assay of beta-galactosidase activity encoded from recombinant genes containing the E. coli beta-galactosidase gene. A substrate for beta-galactosidase, o-nitrophenyl-beta-D-galactopyranoside (ONPG), can be added to dishes containing the transfected cells, and the intensity of the colored enzyme product released from either the intact cell or cells lysed in the dishes can be determined. The results obtained by this assay are a reliable measure of transfection efficiency as well as promotor strength of the genes introduced into the cells. In addition, cells expressing the transfected gene can be identified and quantitated under a light microscope after incubation with X-gal. Thus, it is more convenient to use the E. coli beta-galactosidase gene than the chloramphenicol acetyltransferase gene as a reporter gene in the evaluation of DNA transfection.     

Shuttle vectors conferring hygromycin B resistance to E. coli and to mammalian cells. Differential expression of carboxyterminal fusion proteins.

Shuttle vectors expressing resistance to hygromycin B in both E. coli and in mammalian cells were constructed. A combination of the simian virus 40 early promoter upstream of the native bacterial promoter of the neo gene from transposon Tn5 was found to express hygromycin B resistance better in both types of host cells than a combination of the Tn5 promoter followed by the promoter of the Herpes simplex virus thymidine kinase gene. Hygromycin phosphotransferase fusion proteins with extensions at the carboxyterminus were also tested and found to be marginally less effective as selection markers in eukaryotic cells but virtually inactive in E. coli.     

Evaluation of homology between cloned Escherichia coli and yeast DNA photolyase genes and higher eukaryotic genomes

Repair of ultraviolet-induced pyrimidine dimers by photoreactivation is catalyzed by a single enzyme, DNA photolyase. However, the process of photoreactivation is difficult to detect reproducibly in cultured mammalian cells. We have used clones containing yeast and Escherichia coli DNA photolyase genes to determine whether their sequences are conserved and whether there is homology between either cloned sequence and chick or human genomic DNA and mRNA sequences. The cloned sequences failed to hybridize to each other even under nonstringent conditions, indicating little conservation of sequence between the yeast and E. coli genes. Furthermore, only weak hybridization under nonstringent conditions was found between the cloned photoreactivating genes and human or chick genomic DNA or mRNA. This indicates that there is negligible homology between the cloned probes and mammalian DNA, but we are unable to conclude whether this indicates sequence divergence for prokaryotic and eukaryotic photoreactivation genes or the absence of such genes from the mammalian genome.     

Plant tissue-specific promoters can drive gene expression in Escherichia coli

Plant transgenesis often requires the use of tissue-specific promoters to drive the transgene expression exclusively in targeted tissues. Although the eukaryotic promoters are expected to stay silent in Escherichia coli, when the promoter-transgene units within the plant transformation vectors are constructed and propagated, some eukaryotic promoters have been reported to be active in prokaryotes. The potential activity of plant promoter in E. coli cells should be considered in cases of expression of proteins that are toxic for host cells, environmental risk assessment or the stability in E. coli of plant vectors for specific Cre/loxP applications. In this study, DNA fragments harbouring four embryo- and/or pollen-specific Arabidopsis thaliana promoters were investigated for their ability to drive heterologous gene expression in E. coli cells. For this, they were fused to gfp:gus reporter genes in the pCAMBIA1304 vector. Although BPROM, bacterial sigma70 promoter recognition program identified several sequences with characteristics similar to bacterial promoters including -10 and -35 sequences in each of tested fragments, the experimental approach showed that only one promoter fragment was able to drive relatively strong- and one promoter fragment relatively weak-GUS expression in E. coli cells. Remaining two tested promoters did not drive any transgene expression in bacteria. Our results also showed that cloning of a shorter plant promoter sequence into vectors containing lacZ α-complementation system can increase the probability of gene expression driven by upstream located lac promoter. This should be considered when cloning of plant expression units, the expression of which is unwanted in E. coli.     

Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli.

Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA polymerase beta and E. coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria. We found that the expression of mammalian DNA polymerase beta in E. coli restored growth in a DNA polymerase I-defective bacterial mutant. Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.     

Environmentally controlled invasion of cancer cells by engineered bacteria

Bacteria can sense their environment, distinguish between cell types, and deliver proteins to eukaryotic cells. Here, we engineer the interaction between bacteria and cancer cells to depend on heterologous environmental signals. We have characterized invasin from Yersinia pseudotuburculosis as an output module that enables Escherichia coli to invade cancer-derived cells, including HeLa, HepG2, and U2OS lines. To environmentally restrict invasion, we placed this module under the control of heterologous sensors. With the Vibrio fischeri lux quorum sensing circuit, the hypoxia-responsive fdhF promoter, or the arabinose-inducible araBAD promoter, the bacteria invade cells at densities greater than 10(8)bacteria/ml, after growth in an anaerobic growth chamber or in the presence of 0.02% arabinose, respectively. In the process, we developed a technique to tune the linkage between a sensor and output gene using ribosome binding site libraries and genetic selection. This approach could be used to engineer bacteria to sense the microenvironment of a tumor and respond by invading cancerous cells and releasing a cytotoxic agent.     

Infection by bacterial pathogens expressing type III secretion decreases luciferase activity: ramifications for reporter gene studies

Pathogenic microbes influence gene regulation in eukaryotic hosts. Reporter gene studies can define the roles of promoter regulatory sequences. The effect of pathogenic bacteria on reporter genes has not been examined. The aim of this study was to identify which reporter genes are reliable in studies concerning host gene regulation by bacterial pathogens expressing type III secretory systems. Human intestinal epithelial cells, T84, Caco-2 and HT-29, were transfected with plasmids containing luciferase (luc), chloramphenicol acetyltransferase (CAT) or beta-galactosidase (beta-gal) as reporter genes driven by the inducible interleukin-8 (IL-8) or constitutively active simian virus 40 (SV40) promoter. Cells were infected with enteropathogenic E. coli or Salmonella typhimurium, and the reporter activity was assessed. Luc activity significantly decreased following infection, regardless of the promoter. The activity of recombinant luc was nearly ablated by incubation with either EPEC or Salmonella in a cell-free system. Activity was partially preserved by protease inhibitors, and immunoblot analysis showed a decreased amount and molecular weight of recombinant luc, suggesting protein degradation. Neither beta-gal nor CAT activity was altered by infection. Disruption of type III secretion prevented the loss of luc activity. We conclude that CAT or beta-gal, but not luc, can be used as reliable reporter genes to assess the impact of pathogenic microbes, especially those expressing type III secretion on host cell gene regulation.     

Bacteria-mediated transfer of eukaryotic expression plasmids into mammalian host cells

Invasive intracellular bacteria are able to transfer eukaryotic expression plasmids into mammalian host cells in vitro and in vivo. This can be used to induce immune responses toward protein antigens encoded by the plasmid or to complement genetic defects. Plasmid transfer takes place when the recombinant bacterium dies within the host cell, either due to metabolic attenuation or induction of autolysis. Alternatively, antibiotics can be used and spontaneous transfer has also been observed, indicating that this phenomenon might also occur under physiological conditions. Plasmid transfer has been reported for Shigella flexneri, Salmonella typhimurium and S. typhi, Listeria monocytogenes and recombinant Escherichia coli, but other invasive bacteria should also share this property. In vivo attempts were mainly directed toward vaccination using shigella and salmonella as carrier. So far a wide variety of antigens have been used succesfully in mice. Often this type of immunization was superior over direct application of antigen or using the same bacterium as a heterologous carrier expressing the antigen via a prokaryotic promoter. Characterization of the host cells revealed that macrophages and dendritic cells might be responsible for immune stimulation by either expressing the antigen or cross-presenting the antigen after uptake of apoptotic antigen expressing cells.     

Comparative study of the coupling between topoisomerase I activity and high-mobility group proteins in E. coli and mammalian cells

It is now well established that the HMG box DNA-binding motif can alter the topology of double-stranded DNA in several ways. Using the spermatid-specific tsHMG as a model protein of the HMG-1/-2 family, we have demonstrated that its expression in E. coli produces an increase in plasmid supercoiling density that is likely a consequence of its ability to constrain free supercoils in vivo. As demonstrated in vitro, stabilization of free DNA supercoils by tsHMG prevents topoisomerase I from gaining access to the template and could represent a mechanism for the apparent inhibition of topoisomerase I in bacteria. A similar modulation of eukaryotic topoisomerase I activity was not detected after expression of the tsHMG in mammalian cells. This differential response is discussed in terms of the marked difference in DNA packaging and accessibility of free supercoils in prokaryotic vs. eukaryotic cells.     

Functional coexpression of serine protein kinase SRPK1 and its substrate ASF/SF2 in Escherichia coli

Mammalian proteins expressed in Escherichia coli are used in a variety of applications. A major drawback in producing eukaryotic proteins in E.coli is that the bacteria lack most eukaryotic post-translational modification systems, including serine/threonine protein kinase(s). Here we show that a eukaryotic protein can be phosphorylated in E.coli by simultaneous expression of a mammalian protein kinase and its substrate. We show that in bacteria expressing SRPK1, ASF/SF2 becomes phosphorylated to a degree resembling native ASF/SF2 present in interphase HeLa cell nuclei. The E.coli phosphorylated ASF/SF2 is functional in splicing and, contrary to the unphosphorylated protein, soluble under native conditions.     

Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.     

Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells.

A recombinant baculovirus containing the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under the control of the Rous sarcoma virus long terminal repeat promoter and the E. coli beta-galactosidase gene under the control of the very late baculoviral polyhedrin promoter was used to determine if Autographa californica nuclear polyhedrosis virus, a baculovirus of Lepidoptera, can enter and express viral DNA in dipteran (Drosophila sp.) and mammalian (Mus sp.) cells that are considered refractory to baculovirus replication. Following infection, CAT gene expression was observed in both dipteran and mammalian cells, but expression in the mammalian cell line was less than 0.05% of that observed in either dipteran or lepidopteran cells. Although the level of CAT gene expression was similar in permissive lepidopteran and nonpermissive dipteran cells, expression of beta-galactosidase activity from the late polyhedrin promoter in dipteran or mammalian cells was less than 0.3% of the levels observed in lepidopteran cells. These results indicate that foreign gene expression in nonpermissive cells is promoter dependent and that late viral gene expression is restricted in these cells. The Rous sarcoma virus long terminal repeat allows substantial CAT gene expression in both a D. melanogaster cell line and Aedes aegypti midgut cells. Baculovirus DNA undergoes a limited number of replications in Drosophila cells. The results are relevant to baculovirus host range, the safety of baculoviruses as pesticides, and the development of baculovirus pesticides with expanded host ranges.     

Papaya ringspot virus coat protein gene for antigen presentation in Escherichia coli

The coat protein (CP) of Papaya ringspot virus (PRSV) was analyzed for presentation of the antigenic peptide of animal virus, Canine parvovirus (CPV), in Escherichia coli (E. coli). The 45 nucleotides fragment coding for the 15-aa peptide epitope of the CPV-VP2 protein was either inserted into the PRSV-cp gene at the 5', 3' ends, both 5' and 3' ends or substituted into the 3' end of the PRSV cp gene. Each of the chimeric PRSV cp genes was cloned into the pRSET B vector under the control of the T7 promoter and transformed into E. coli. The recombinant coat proteins expressed from different chimeric PRSV-cp genes were purified and intraperitoneally injected into mice. All of the recombinant coat proteins showed strong immunogenicity and stimulate mice immune response. The recombinant coat proteins containing the CPV epitope insertion at the C terminus and at both N and C termini elicited ten times higher specific antisera in immunized mice compared with the other two recombinant coat proteins which contain the CPV epitope insertion at the N terminus and substitution at the C terminus.     

Escherichia coli as a model system to study DNA repair genes of eukaryotic organisms

The bacteria Escherichia coli has been widely employed in studies of eukaryotic DNA repair genes. Several eukaryotic genes have been cloned by functional complementation of mutant lineages of E. coli. We examined the similarities and differences among bacterial and eukaryotic DNA repair systems. Based on these data, we examined tools used for gene cloning and functional studies of DNA repair in eukaryotes, using this bacterial system as a model.     

Sequence and S1 nuclease mapping of the 5'' region of the dihydrofolate reductase-thymidylate synthase gene of Leishmania major.

The 5' structure of mRNA transcribed from the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene of the protozoan parasite Leishmania major has been characterized. S1 nuclease mapping identifies a heterogenous 5' structure which is not affected by growth phase or developmental stage. The DNA sequence of the 5' region of the DHFR-TS gene does not reveal homology with other trypanosomatid genes, eukaryotic consensus genetic elements, or the mammalian DHFR promoter element. This latter finding is especially significant as we show that the 5' region of the E. coli DHFR gene exhibits homology to the mammalian DHFR promoter element, despite their greater evolutionary distance.