Molecular basis of agrobacterium mediated gene transfer into mammalian cells 3

Chapter 3. Agrobacterium-mediated DNA delivery into mammalian cells 3.1. Introduction Horizontal gene transfer (HGT) might play an important role in the evolution of both eukaryotic and prokaryotic genome (Syvanen, 1994; Nelson et al., 1999; Syvanen and Kado, 2001). Although HGT in prokaryotic cells has been established, the role of HGT in eukaryotic genome evolution is not well elucidated because the existence of a possible pathway or mechanism for HGT involved eukaryotes is often questioned. A. tumefaciens is often used as a vector to generate transgenic plants. The ability of Agrobacterium to transfer part of its DNA (T-DNA) into plant cells and integrate such foreign genetic material into the plant genome, followed by the consequent gene expression to induce the tumors on plant, is the best understood case of horizontal gene transfer between bacteria and eukaryotic cells. The recent findings that Agrobacterium could also transform other eukaryotes including mammalians, yeast and fungi might shed light on the exact mechanism of HGT involving eukaryotes. Studies on the interaction of Agrobacterium and mammalian cells can provide information about the mechanism of gene transfer between bacterium and mammalian cells and help us to understand the role that HGT might play in eukaryote evolution. 3.2. Results 3.2.1. Attachment of Agrobacterium to mammalian cells An early step in the plant tumor induction by Agrobacterium is the attachment of bacterial cells to the host cells. Bacterial attachment to host cells is often an 72 essential step to initiate internalization because this process allows pathogens to locate the appropriate target tissues. This attachment appears to be required for DNA transfer from A. tumefaciens into the host plant cells. A. tumefaciens with mutations in the chromosomal genes chvA and chvB are defective in both the efficient attachment and the subsequent transformation of plant cells. To study the interaction between Agrobacterium and mammalian cells, a plasmid pQM49 was constructed (Fig. 3.1). This plasmid can express gfp constitutively in Agrobacterium. A. tumefaciens strains harboring pQM49 exhibited bright green fluorescence when illuminated with UV light and could be visualized easily under the confocal microscope. Then the ability of Agrobacterium to adhere to mammalian cells EcoPack2-293 was examined. Fig. 3.2 (panel B) showed that the wild type strain A348(pQM49) could specifically attach to the surface of mammalian cells and this result is consistent with the previous report (Kunik et al., 2001). To investigate whether the binding of Agrobacterium to mammalian cells requires the same bacterial factors as its attachment to plant cells, various Agrobacterium mutant strains harboring pQM49 were tested for their attachment ability to the EcoPack2-293 cells as described in the Materials and Methods. As shown in Fig. 3.2 (panel D), At12513, a chvB mutant, showed reduced ability to attach the mammalian cells after co-incubation. This indicates that the chvB gene is important for the attachment of Agrobacterium cells to both mammalian and plant cells. This is consistent with the previous study (Kunik et al., 2001). Interestingly, the results also showed that the virB mutant strain, MX243, was less efficient in binding to the mammalian cells (Fig. 3.2, panel C). Thus, this data suggest that unlike what was observed in the interaction between Agrobacterium and plant cells, in which 73 pQM49 Fig. 3.1. Plasmid used for attachment study. For complete construction details see Materials and Methods. Abbreviations: Plac,LacZ promoter sequence; atpE, the efficient translation region of atpE gene; gfpuv, green fluorescent protein; tetA, tetracycline resistance gene; OriV, R6K origin of replication. 74 A B C D Fig. 3.2. Confocal microscopy analysis of Agrobacterium attachment to EcoPack2-293 Packaging Cells. A. tumefaciens cells were incubated with EcoPack2-293 Packaging Cells as described in Material and Methods and observed under confocol microscopy at the excitation wavelength of 488 nm. A, 293 cells alone. B, A348 (pQM49) co-incubated with 293 cells. C, MX243 (pQM49) co-incubated with 293 cells. D, At12513 (pQM49) coincubated with 293 cells. 75 mutation in the virB gene does not affect the attachment to the plant cells, virB gene product does affect the bacterial attachment to mammalian cells. This might imply that the mechanism of Agrobacterium attachment to human cells is different from that to the plant cells. 3.2.2. Development of a system to detect the horizontal gene transfer between the bacteria and mammalian cells To observe inter-kingdom horizontal gene transfer directly, a sensitive system that could detect a few copies of gene transfer from the bacterial cells into human cells was developed. In this system, EcoPack2-293 Packaging Cell was used as the host for Agrobacterium gene transfer. This Packaging Cell Line is a human embryonic kidney (HEK 293)-derived cell line designed for the rapid and transient production of high-titer retrovirus. It can stably express the viral gag, pol and env genes, which are necessary for viral production. Then two gene transfer vectors, pQM52 (with T-borders) and pQM54 (without T-borders) (Fig. 3.3) were constructed. Both vectors contain an egfp gene under the control of a CMV immediate early promoter, a 5' and 3' long-terminal repeats (LTRs) which contain promoter, polyadenylation and a sequence termed Ψ +which is essential for virus packaging. Agrobacterium harboring these plasmids were used to infect EcoPack2-293 cells and the expression of egfp in these cells would indicate the successful transfer of bacterial DNA into the host cells. In this system, the expression of egfp could not be attributed to contaminated bacteria, because CMV immediate early promoter is only functional in the eukaryotic cells. In this system, once foreign genes as well as the transcript containing Ψ + are delivered into EcoPack2-293 cells, the viral elements harbored on 76 ϕ+ pQM52 ϕ+ pQM54 Fig. 3.3. Plasmids used for transfection study. For complete construction details see Materials and Methods. Abbreviations: LTR, long terminal repeat sequence; Ψ+, extended packaging signal; Neor, Neomycin resistance gene; PCMV, immediate early CMV promoter; egfp, enhanced green fluorescent protein; LB and RB, A. tumefaciens left and right T-DNA borders, respectively; nptIII, kanamycinresistance gene; tetA, tetracycline resistance gene; OriV, R6K origin of replication 77 the mammalian cell chromosome will facilitate the amplification and transcription of foreign gene in host cells, which will result in expression of the egfp gene. Therefore, only a few copies of the egfp gene need to be transferred in order to detect green fluorescence protein expression in host cells. The integration of the foreign gene into the recipient genome, which would result in stable transformant, is not required in this process. 3.2.2.1. A. tumefaciens can deliver its DNA into human cells First of all, whether A. tumefaciens could transfer T-DNA into mammalian cells such as EcoPack2-293 cells or not was determined. Co-incubation of human EcoPack2-293 cells (1 × 106) with Agrobacterium strain LBA4404(pQM52) (4 × 107) resulted in the expression of egfp in mammalian cells, indicating that the A.tumefaciens could deliver its plasmid DNA containing the egfp reporter gene into human cells (Fig. 3.4, panel B). The multiplicity of infection (MOI) in this experiment is about 40. On the contrary, when EcoPack2-293 cells (1 × 106) were incubated with DH5α(pQM52) (4 × 107), in which E. coli cells were used as the donor strain, no expression of egfp in those cells was observed in this experimental condition (Fig. 3.4, panel C). Similarly, no transformants were observed when 50 µg of the plasmid pQM52 was incubated directly with the recipient cells, suggesting that DNA transfer is not due to a direct uptake of free plasmid DNA released by the dead bacteria. Then the same Agrobacterium strain (LBA4404(pQM52) (4 × 107) was incubated with normal mammalian cell line such as HeLa (1 × 106), which does not encode viral gag, pol and env genes. As expected, no expression of egfp in these host cells was observed under the same experimental conditions (data not shown). In order to investigate whether the viral elements on vector (such as LTR sequence and Ψ +) 78 Fig. 3.4. Expression of egfp in human Cells ( EcoPack2-293). The mammalian cells were incubated with different A. tumefaciens or E. coli strains and the infected cells were viewed with blue light excitation (480 nm). A, 293 cells alone; B, LBA4404(pQM52); C, DH5α(pQM52); D, LBA4404(pQM54); E, AG6(pQM52); F, A348::Tn5; G, A136::Tn5; H; GMI::Tn5. 79 A B C D F E F G H 80 are required for this gene transfer, another vector pQM45 that contains only an egfp gene under the control of a CMV promoter (Fig. 3.3) was constructed. Compared to pQM52, pQM45 lacked the viral components. When EcoPack2-293 cells (1 ×106) was incubated with LBA4404(pQM45) strain (4 ×107), no expression of egfp in mammalian host cells was observed. These results showed that this system is much more sensitive in detecting DNA transfer due to the amplifying function of the viral elements. To further verify that EGFP was indeed synthesized by the host cells, RT-PCR was carried out to detect the egfp transcription in the infected human cells. The host cells (2 × 107) were treated as described in Material and Methods and total RNA of the host cells was extracted. RT-PCR was performed with a pair of egfp primers (EGFPF, 5’-CTAACGCAGTCAGTGCTTCTG-3’; EGFPR, 5’CAGTCATAGCCGAATAGCCTCTC-3’). One step RT-PCR Kit (QIAGEN) was used to conduct the standard RT-PCR. According to the instructions of the manufacturer, approximately 10-100 of target molecules should be sufficient for the detection. Forty cycles of the reaction was necessary to detect the egfp transcription in the host cells infected by Agrobacterium in this system. As shown in Fig. 3.5, a clear band of 700 bp RT-PCR product, which matched well with the predicted egfp transcript product, could be amplified from the total RNA extract of host cells coincubated with wild-type A. tumefaciens strain LBA4404(pQM52). On the contrary, no amplification product could be detected when RNA extract from cells incubated with E.coli strain DH5α(pQM52) was used. These data are consistent with our visual observations of the EGFP fluorescence. Taken together, these results suggest that A. tumefaciens could deliver genes carried on its plasmids into the mammalian cells, while E.coli could not mediate such DNA transfer in similar experimental conditions. 81 Table 3.4. Effect of Cytochalasin D on Agrobacterium invasion and gene transfer into HEK-293 cells Percentage of invasion* (%) Inhibitor Type of inhibitor DH5α(pQM54) LBA4404(pQM52) None Control [...]... Agrobacterium- mediated transformation of plant cells, three genetic components of Agrobacterium are required: T-DNA border sequences, virulence (vir) genes located on the Ti plasmid and chromosomal virulence (chv) genes Therefore the effect of these factors on Agrobacterium- mediated gene delivery into the mammalian cells was investigated 3. 2 .3. 1 Agrobacterium vir genes are not required for DNA transfer into mammalian. .. necessary for mammalian cell gene delivery These results indicate that some Agrobacterium chromosomal genes might play important role(s) in mammalian cell gene transfer Taken together, the data suggest that the DNA transfer mediated by Agrobacterium is not a process of passive uptake of foreign DNA by mammalian cells 3. 2 .3. 3.2 Effect of katA on DNA delivery into mammalian cells katA gene encodes a... ability of Rhizobium to transfer DNA into EcoPack2-2 93 cells was test R meliloti strain RCR2011(pQM52) was used to incubated with 2 93 cells under the same experimental conditions However, no green fluorescence was 83 Table 3. 1 Frequency of DNA transfer from Agrobacterium to EcoPack2-2 93 Packaging Cells Strain Genetic background Location of reporter gene Total number of egfp expressing cells Gene transfer. .. involved in mammalian cell gene transfer should be located on its chromosome 87 3. 2 .3. 3 Roles of Agrobacterium chromosomal gene in DNA transfer into mammalian cells A tumefaciens cells with mutations in certain chromosomal genes are defective in the transformation of plant cells Thus, several strains with mutation in the chromosomal virulence genes, such as At12512 (chvA-), At125 13 (chvB-), A 634 0 (chvG-),... Agrobacterium- mediated mammalian cell gene transfer 3. 2 .3. 3 .3 Combinatorial effect of mutations in chromosomal genes on DNA delivery into mammalian cells The chromosomal genes chvG, chvH and katA were indicated to be involved in the bacterial ability to deal with the stress condition of low pH, while the above results showed that they are also involved in the Agrobacterium- mediated mammalian cell gene. .. 1.22 0.42 0.11 2 .34 3. 34 0.75 0.22 1.48 2.24 0.62 0.18 3. 69 4. 23 0.65 0 .31 0.46 1.42 0 .38 0.19 3. 03 4.02 0 .36 0.24 0.88 1 .39 0 .33 0.12 A 634 0-AG6b A6880-AG6b 3. 52 3. 64 0.84 0. 13 2.68 2.84 0.61 0.12 3. 87 4.84 0 .35 0.14 2.71 2.91 0.27 0.09 8.8 9.1 2.1 0 .33 6.7 7.1 1.5 0 .3 12 15 1.1 0.42 8.4 9.2 8.4 0.28 Average number of bacteria per 2 93 cell Attachment 4 . the DNA transfer mediated by Agrobacterium is not a process of passive uptake of foreign DNA by mammalian cells. 3. 2 .3. 3.2. Effect of katA on DNA delivery into mammalian cells katA gene encodes. facilitate the Agrobacterium- mediated mammalian cell gene transfer. 3. 2 .3. 3 .3. Combinatorial effect of mutations in chromosomal genes on DNA delivery into mammalian cells The chromosomal genes chvG,. involved in mammalian cell gene transfer should be located on its chromosome. 87 3. 2 .3. 3. Roles of Agrobacterium chromosomal gene in DNA transfer into mammalian cells A. tumefaciens cells with