Molecular basis of agrobacterium mediated gene transfer into mammalian cells 3

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 expres

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

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 withUV light and couldbe visualized easily under the confocal

microscope Then theability 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 plantcells, 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

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

C D

A B

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) co-

incubated with 293 cells

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, adenylation and a sequencetermed Ψ +which is essential for virus packaging

poly-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

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; Neo r ,

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,

kanamycin-resistance gene; tetA, tetracycline kanamycin-resistance gene; OriV, R6K origin of

replication

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 +)

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

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, 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

co-incubated 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,

1 2 3 4 5 6 7 8 9 10

700 bpdimer

Then flow cytometry analysis was conducted to estimate the DNA transfer efficiency

of Agrobacterium–mediated gene delivery by counting host cells with high green fluorescence, as a result of egfp expression A total of 100,000 EcoPack2-293 cells

incubated with LBA4404(pQM52) were counted, while same number of the host cells incubated with DH5α(pQM52) were also counted as a negative control Each

analysis was repeated for three times independently As shown in Table 3.1, the average efficiency of LBA4404(pQM52) mediated gene transfer was 9 × 10-5 per recipient cell

3.2.2.2 A tumefaciens can also transfer DNA into mouse cells

To test whether a specific genetic background of the recipient mammalian cells was required for the gene transfer, PT67 cells were incubated with

LBA4404(pQM52) PT67 is a NIH 3T3-derived mouse cell line having been

integrated of viral gag, pol and env genes into its chromosome High green

fluorescence was observed in infected PT67 cells, suggesting that Agrobacterium

could also deliver its DNA into the mice cells (Fig 3.6) This result indicates that the

gene transfer mediated by Agrobacterium could occur not only in human cells but

also in mouse cells Thus, this observed DNA transfer might occur widely in

mammalian cells and it does not require specific genetic background of the host cells

3.2.2.3 R meliloti could not deliver its DNA into mammalian cells

As Rhizobium is closely related to Agrobacterium in many aspects during their interactions with plants, the ability of Rhizobium to transfer DNA into EcoPack2-293 cells was test R meliloti strain RCR2011(pQM52) was used to incubated with 293

cells under the same experimental conditions However, no green fluorescence was

Table 3.1 Frequency of DNA transfer from Agrobacterium to EcoPack2-293

(x10 -5 )

No bacterium NC* NC* 0 0

Naked DNA NC* plasmid with T-borders 0 0 DH5α(pQM52) E Coli plasmid with T-borders 0 0 RCR2011(pQM52) Rhizobium wild type plasmid with T-borders 0 0 LBA4404(pQM52) Agrobacterium wild type plasmid with T-borders 28 9 ± 2

LBA4404(pQM54) Agrobacterium wild type plasmid without T-borders 20 7 ± 2

GMI9023(pQM52) Agrobacterium without Ti and

cryptic plasmid

plasmid with borders 18 6 ± 1

T-A6880(pQM52) chvH - plasmid with

T-borders 7 2 ± 1 A6340(pQM52) chvG - plasmid with T-

borders 2 1 ± 1 AG6(pQM52) katA - plasmid with T-

borders 88 29 ± 6 A6340-

AG6(pQM52) chvG - , katA -

plasmid with borders 6 2±1 A6880-

T-AG6(pQM52) chvH - , katA - plasmid with T-borders 4 1±1

A348::Tn5 Agrobacterium wild type without T-borders Random insertion 25 8 ± 3

A136::Tn5 without Ti plasmid Agrobacterium without T-borders Random insertion 31 10 ± 3

GMI::Tn5

Agrobacterium

without Ti and cryptic plasmid

Chromosome without T-borders 19 6 ± 3

* NC: Negative Control

**The data were analyzed using Excel (Miscrosoft Software, USA)

detected(data not shown) This result suggests that the delivery of a foreign gene into

the mammalian cells might require some unique factor(s) from A tumefaciens

3.2.3 Roles of Agrobacterium genes and T-DNA border sequences in horizontal

gene transfer to mammalian cells

In 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 cells

Phenolics and monosaccharides, which serve as inducers of vir genes, are

required for T-DNA transfer into the plant cells To determine if they are also

required for A tumefaciens-mediated mammalian cell transformation, Agrobacterium

cells were cultured the in the absence of Acetosyringoue (AS) and incubated with

EcoPack2-293 cell Under such condition, the expression of vir genes is minimal The data showed that the gene transfer efficiency of these A tumefaciens cells was similar to that of the induced cells, indicating that vir gene induction is not necessary

for mammalian cell DNA delivery Then the gene transfer ability of different

virulence gene mutant strains was tested, including MX226 (virA - ), MX363 (virG-),

MX306 (virD2-) and MX358 (virE-) harboring pQM52 All these strains have

retained their ability to deliver DNA into mammalian cells and they showed similar gene transfer efficiency as the wild type strain, LBA4404(pQM52) (Table 3.1) Thus,

these results suggest that virulence genes are not required for A tumefaciens-mediated

gene delivery into mammalian cells under our assay system

3.2.3.2 T-DNA border sequences are not required for DNA transfer into

mammalian cells

T-DNA border sequences are necessary for the transfer of T-DNA from A

tumefaciens to plant cells but are not required for the conjugative transfer of the Ti

plasmid between A tumefaciens strains In order to find out whether T-DNA border

sequence is essential for mammalian cell gene transfer, a binary-vector pQM54, which lacks the T-DNA border sequences that presented in pQM52 (Fig 3.3), was

conducted Agrobacterium strain LBA4404 harboring pQM54 was incubated with

mammalian cells The efficiency of mammalian cell gene transfer was quite similar, 7×10-5 for LBA4404(pQM54) and 9×10-5 for LBA4404(pQM52) (Table 3.1) No significant reduction of mammalian cell gene transfer efficiency was observed,

indicating that T-border sequences are not required for the DNA transfer

Furthermore, the gene transfer efficiency of Agrobacterium strain

GMI9023(pQM52) was tested, as GMI9023 contains neither the Ti plasmid nor the crypticplasmid (Rosenberg and Huguet, 1984) The DNA delivery efficiency of GMI9023(pQM52) was 6×10-5, which is similar to that observed with

LBA4404(pQM52) (Table 3.1) Thus the data demonstrated undoubtedly that the

DNA delivery into mammalian cells mediated by A tumefaciens is independent of the

Ti plasmid, which indicate that the Agrobacterium factor(s) involved in mammalian

cell gene transfer should be located on its chromosome

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 - ), At12513 (chvB-), A6340

(chvG-), A6880 (chvH-), CGI1 (aopB-), B119 (acvB-) and AG6 (katA -), respectively

(Charles and Nester, 1993; Peng et al., 2001; Xu and Pan, 2000; Jia et al., 2002), were tested for their ability to deliver DNA into mammalian cells Agrobacterium strains with chvA and chvB mutation (At12512 and At12513) could not synthesize or

transport β-1,2-glucan, which appears to be required for the efficient attachment to the plant cells However, when At12512(pQM52) or At12513(pQM52) was incubated with EcoPack2-293 cells, no reduction in gene transfer efficiency was observed(data not shown) This data suggested that the early steps of interaction, such as

attachment, might not be critical for DNA delivery into mammalian cells in our

system,in which relatively small percentage of bacterial attachment may be enough for the occurrence of gene transfer due to the high MOI (~40) used

3.2.3.3.1 chvG and chvH are involved in Agrobacterium-mediated gene transfer

into mammalian cells

Interestingly, two chromosomal mutants, A6880(pQM52) (chvH -) and

A6340(pQM52) (chvG -), showed attenuated ability to gene transfer to the human cells (Table 3.1) The transient gene transfer efficiencies of these two mutants are 2 × 10-5and 1 × 10-5 per recipient cells, respectively, which are five to nine times lower in the

DNA transfer frequency than that of the wild type chvH encodes a homologue of an elongation factor P (efp) involved in protein synthesis (Peng et al., 2001) The wild

type chvH locus is essential for the full expression of not only vir genes but also some

chromosomal genes These genes mightcode for particular amino acid sequences, perhaps near thestart site of translation that are exceptionally dependent on elongation

factor P for translation ChvG encodes a global pH sensor protein that is important for the virulence of Agrobacterium tumefaciens It is involved in the regulation of many

acidic pH-inducible genes, including both the chromosomal genes and the Ti plasmid

encoded vir genes (see Chapter 4) In addition, both A6880 and A6340 are sensitive

to acidic extracellular pH Therefore, the low gene delivery efficiency of these strains might be due to the low expression of one or more important protein(s), which are 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 catalase, which is involved in the dismutation of hydrogen

peroxide to water and oxygen (Xu and Pan, 2000) It has been reported that the rapid production and accumulation of H2O2 might lead to the hypersensitive response (HR)

in the plant defense system Mutation in this gene will highly attenuate the bacterial ability to cause tumors on plants and to tolerate H2O2, but not the bacterial viability in the absence of exogenous H2O2 To determine if the same defense response is utilized

in mammalian cells, the katA mutant strain AG6 was used to infect 293 cells (Table

3.1 and Fig 3.4) Surprisingly, the transient gene transfer efficiency of AG6 was 2.9

× 10-4 per recipient cell, based on flow cytometry analysis, which was four times

higher than that of wild type As catalase plays an important role in protecting

bacterial cells from damages caused by H2O2 to cellular components, including

nucleic acids, proteins and cell membranes (Imlay and Linn, 1998; Storz and Imlay, 1999), thus it is possible that the incomplete bacterial membrane may 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, 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 transfer These prompted studies on the functional relationship of these genes in the gene delivery of mammalian cells Thus, the gene transfer efficiency of

the chvG and chvH mutant strains in the absence of any functional katA gene was

tested The double mutant strains were created by introducing the total DNA of AG6,

which is a katA - mutant (Xu et al, 2001), into A6880 and A6340 by electroporation (Charles et al, 1994) The resulting recombinants, A6880-AG6 and A6340-AG6, lacked functional katA genes in the A6880 and A6340 background, respectively, as

verified by Southern analysis (Fig 3.7) The gene transfer ability of these strains was tested The double mutant strains gave similar gene delivery efficiency as that of the

chvH or chvG single mutant strain (Table 3.1), instead of an increasing efficiencies in

mammalian cell gene transfer as that of katA mutant strain These suggest that the proteins regulated by chvG and chvH might play roles before the above proposed

bacterial membrane lysis Again, these data agree with the hypothesis that

Agrobacterium play active roles in the gene transfer process instead of gene being

transfer from bacteria lysed passively

1 2 3 4 5

Fig 3.7 Southern blot analysis of homologous recombinants The total

DNA of Agrobacterium strains were extracted and digested with PstI gfp uv

from mini-Tn5 was used as probe Lane 1, A6340; lane 2, A6880; lane 3, AG6; lane 4, A6880-AG6; lane 5, A6340-AG6

3.2.4 Agrobacterium can deliver its chromosomal DNA into human cells

In order to investigate whether genes located on the A tumefaciens chromosome can also be transferred into human cells, the egfp gene as well as the viral LTR gene were introduced into the A tumefaciens chromosome by transposon mutagenesis

The plasmid pQM61 was used as the transposon donor strain, which contains both the

egfp gene and the viral LTR gene located between the mini-Tn5 border sequences

(Lorenzo et al, 1990) Three Agrobacterium strains, wild type strain A348, A136

which lacks Ti-plasmid and GMI9023 which contains neither the Ti plasmid nor the crypticplasmid, were used as the recipients The transposon mutants were selected

on AB plates containing 50 µg/ml of gentamicin and more than 100 targeted mutants

of GMI9023, A136 and A348 were obtained The frequency of transposition was 10-7 per recipient cell, which agrees with the previous report (Berg and Berg, 1996) The structure of the transposon insert is shown in Fig 3.8

To confirm that the egfp as well as viral elements (LTR and Ψ +) were

integrated into the Agrobacterium genome via transposon mutagenesis, one colony for

each strain was picked up for Southern analysis The total DNA of these strains were

extracted and digested with HindIII and probe with pQM61 The bands detected in

mutant strains were apparently different from the original transposon donor plasmid

pQM61 (Fig 3.9), demonstrating that the egfp gene as well as transcript containing

Ψ + has been integrated into the recipient genome by random transposon mutagenesis rather than existing on a free plasmid

These mutant strains (A348::Tn5, A136::Tn5 and GMI::Tn5) were incubated with the human cells As shown in Fig 3.4 (F)-(H), these strains could also express

egfp 3’ LTR Gmr

Fig 3.8 Transposon insertion in mutant bacterial strains For random

transposon mutagenesis 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; GmR, gentamycin-resistance gene; I and O, inverted repeats

of IS50;

O 5’ LTR Ψ + Νeo r Pcmv

I

Fig 3.9 Southern blot analysis of genomic DNA of transposon

mutants Genomic DNA or plasmid was digested with HindIII and

separated on a 0.8% agarose gel Hybridization was performed

using pQM61 as probe The position and size in kilobase are

indicated on the left Lane 1, pQM61; lane 2, A136::Tn5; lane 3, A348::Tn5; lane 4, GMI::Tn5