Molecular basis of agrobacterium mediated gene transfer into mammalian cells 1

Chapter 1. Literature Review Agrobacterium tumefaciens is a Gram-negative, soil-borne plant pathogen that can cause crown gall disease, a tumorous disease at infection sites, on a wide range of plant species (Van et al, 1974; Waston et al, 1975). Initial research in Agrobacterium-plant interaction was intended to understand the molecular mechanism of Agrobacterium-mediated tumor formation and to shed light on animal tumors. Although no relationship was found between animal and plant tumors, the research effort has introduced a possible revolution in plant genetic engineering and transgenic technology. An overview on the mechanism of plant tumor formation is shown in Fig. 1.1. Briefly, on the wound site of the plant cell, part of the Agrobacterium DNA (TDNA) is processed from a large tumor-inducing (Ti) plasmid to form a T-complex with some vir gene products. The T-complex is then transferred into the plant cell where it will be integrated into the plant genome. In nature, the subsequent expression of these genes carried on the T-DNA will result in the formation of neoplastic growth, known as crown gall tumors that serves to provide the major sources of carbon and nitrogen for Agrobacterium (Kado, 1991; Sheng and Citovsky, 1996; Zupan and Zambryski, 1997; Stafford, 2000; Zhu et al., 2000). Besides its natural hosts, which are dicotyledonous plants such as fruit trees and grape vines, Agrobacterium has also been used to transform monocotyledonous plants like rice (Komari et al, 1998; Hiei et al, 1997). Furthermore, the accumulated knowledge of Agrobacterium has been applied to fungus, yeast and mammalian cells as well (Bundock et al, 1995; Relic et al, 1998). Undoubtedly, the development of Agrobacterium as a plant genetic vector has been one of the most important technical developments in the past 25 years. . Fig. 1.1. Schematic diagram of the Agrobacterium transformation process. Critical steps that occur to or within the bacterium (chemical signaling, vir gene induction and T-DNA processing) and within the plant cell (bacterial attachment, T-DNA transfer, nuclear targeting, and T-DNA integration) are highlighted, along with genes and/or proteins known to mediate these events (Cited from Gelvin, 2000). 1.1. Overview of T-DNA transfer from A. tumefaciens into plant Agrobacterium-plant interaction is the only well studied example of natural interkingdom horizontal gene transfer system. The process of T-DNA transfer consists of several critical steps: bacterium chemotaxis and attachment, vir gene induction, T-DNA processing, T-DNA transfer and nuclear targeting, T-DNA integration into the plant genome and transferred gene expression. Briefly, the TDNA transfer process is initialed when Agrobacterium perceives and responds to certain phenolic compounds, sugar, acidic pH and low phosphate level, which are present at plant wound sites. The signal perception is mediated by the VirA/VirG two-component transduction system. Autophosphorylation of VirA protein and the subsequent transphosphorylation of VirG protein result in the activation of vir gene transcription. Then the vir gene products are directly involved in the T-DNA processing from the Ti plasmid and the subsequent transfer of T-DNA from the bacterium into the plant cell nucleus (for reviews see Tzfira et al., 2000; Kado, 2000; Gelvin, 2000). The T-DNA transfer process from Agrobacterium into a plant cell involves many factors from both the bacterium and the host. There are three genetic components of Agrobacterium that are essential for plant cell transformation. The first component is T-DNA, the transferred segment, which is transported from the bacterium into the plant cell (Wang et al., 1984; 1987). The T-DNA is located on the 200 kb Ti plasmid of Agrobacterium and is delimited by flanking two 25-bp imperfect direct repeats known as the T-DNA borders. Border sequences of the T-DNA are the only cis elements necessary for effective transformation of the plant cell (Miranda et al., 1992). The second component is the virulence (vir) genes, which are also located on the Ti plasmid. This 35-kb region of DNA, which is not transferred to the plant cells, codes for proteins that are required for the sensing of plant wound metabolites as well as the processing, transfer, nuclear targeting and integration of T-DNA. There are eight major loci (virA, virB, virC, virD, virE, virG, virJ and virH) in this region. All of the vir operons are induced as a regulon via the virA/virG two-component system by plant phenolic compounds, such as acetosyringone (AS) and specific monosaccharides. The third component is a set of chromosomal virulence (chv) genes, which have been identified as necessary for tumorigenesis. Some of the chv genes are involved in bacterial chemotaxis and attachment to wounded plant cells (Uttaro et al., 1990; Thomashow et al., 1987; O'Connell and Handelsman, 1989; Kamoun et al., 1989; Sheng and Citovsky, 1996), while others might be involved in the regulation of vir gene expression. The latter two genetic components play important roles in the processing and transfer of the T-DNA from A. tumefaciens to the plant nucleus. In the following subsections, the characteristics and functions of Vir proteins as well as Chv proteins that are involved in the T-DNA transfer will be described in detail. 1.1.1. Roles of Ti-plasmid encoded virulence genes 1.1.1.1. VirA/VirG, member of highly conserved class of two-component regulatory system Sensing of signal molecules released by wounded plant cells is the first step of signal transduction, which leads to vir gene expression in Agrobacterium. The vir operons constitute a regulon which is strongly and coordinately induced in cells growing under acidic pH conditions by two classes of plant signal molecules: phenolic compounds, such as acetosyringone, and sugars such as glucose and glucuronic acid. The expression of virulence genes is under the control of a two- component regulatory system in A. tumefaciens, which is comprised of VirA and VirG (Winans, 1992; Olson, 1993). Based on protein sequence similarities, VirA and VirG have been assigned to a large group of His-Asp two-component regulatory systems, involving a sensor and a response regulator. VirA, an inner membrane histidine kinase, senses certain phenolic compounds released from the wounded plant cells and gets autophosphorylated at His474 (Lee et al., 1995; 1996). The phosphorylated VirA will, in turn, transfer the phosphate moiety to the response regulator VirG at Asp-52. Physical and genetic evidences indicated that VirA protein exists as a homodimer in the native conformation and the homodimer is the functional state in the plant-bacterium signal transduction (Pan et al., 1993). The VirA protein could be divided into four domains, which are the periplasmic, linker, kinase and receiver domains. The periplasmic domain has been found to sense a variety of monosaccharides required for vir gene induction. This domain can also interact with a periplasmic sugar-binding protein, ChvE (Cangelosi et al., 1990; 1991). This interaction alone does not induce vir gene expression, but it sensitizes the VirA molecule to the phenolic inducers. The VirA protein has variable efficiency in different strains of A. tumefacines, which suggests that different chromosomal backgrounds, especially ChvE, are not equivalent for the VirA function. The linker domain is necessary for perceiving phenolic compounds and acidity whereas the kinase domain contains the conserved phosphorylatable His-474, which is required for signal transduction in all sensor molecules. Changing this His-474 to Gln results in a protein that can no longer be phosphorylated and a mutant carrying this modification is avirulent and unable to induce vir gene expression in the presence of plant signal molecules (Huang et al, 1990; Jin et al., 1990a; 1990b; 1990c). The receiver domain is somewhat similar to the region of VirG, which is phosphorylated by VirA. The function of this domain is unclear. However, it is proposed to play an inhibitory role in signal transduction, because once deleted, monosaccharides alone could induce vir gene expression in the absence of phenolic compounds. The VirG protein is a cytoplasmic protein. An 12-bp conserved consensus, called vir-box, is present in the upstream region of most of the vir genes. VirG can bind specifically to this vir box and act as a transcriptional activator of vir genes. The C-terminus region of VirG is responsible for the DNA binding activity, while the Nterminal is the phosphorylation domain and shows high homology to the VirA receiver (sensor) domain. Mutants with non-phosphorylatable VirA or VirG protein fail to induce vir gene expression (Jin et al., 1990a; 1990b; 1990c). Both the number of copies and the types of virG gene can influence some biological properties of A. tumefaciens. For example, multiple copies of VirG in A. tumefaciens can greatly enhance vir gene expression and the transient transformation frequency of some plants tissues (Liu et al., 1992). Besides, multiple copies of VirG allow a high level of vir gene induction by acetosyringone (AS) even at alkaline pH (Liu et al., 1993). In addition, recent studies have revealed that quantitative differences exist in the interactions between VirG and vir boxes of different Ti plasmids, suggesting that efficient vir gene induction in octopine and nopaline strains requires virA, virG, and vir boxes from the respective Ti plasmids. 1.1.1.2. VirC, VirD and VirE, elements necessary for T-DNA processing 1.1.1.2.1. Roles of VirC, VirD and VirE in T-complex formation Proteins responsible for the production of T-complex are encoded by virD and virE operons (Grimsley et al., 1989; Toro et al., 1989; Citovsky et al., 1988; 1989; Gietl et al., 1987; Sen et al., 1989). The T-complex consists of T-DNA, which is a single-strand DNA segment processed from Ti plasmid, a molecule of VirD2 that is an endonuclease covalently bound to the 5’ end of the T-DNA, and a large number of VirE2 molecules, which is a single-strand DNA binding protein. The T-DNA is delimited by two 25-bp direct repeats, also known as the T border, at its ends. Any DNA between the T borders will be transferred into the plant cell as a single-strand DNA and integrated into the plant genome. In vivo, VirD2, along with VirD1, is sufficient for T-DNA processing in both E.coli and A. tumefaciens. virD2 encodes an endonuclease, which cleaves the bottom strand of the T-DNA at the T-borders and remains covalently bound to the 5’ end of the nicked DNA (Pansegrau, 1993; Jasper, 1994; Zupan et al., 2000; Gelvin, 2000). The endonuclease activity domain lies in the N-terminal 228 aa of VirD2. This domain, along with two short regions near the Cterminus, is the only known highly conserved domain in VirD2 protein. The possible role of VirD1 might be its interaction with the T-borders, where ssDNA is originated. This interaction can induce local double helix DNA destabilization and provide a single-stranded loop substrate for VirD2. In vitro studies have shown that VirD2 alone is enough for mediating the precise cleavage of T border sequence carried by ssDNA templates even in absence of VirD1 protein. In contrast, VirD1 is essential for the cleavage of supercolied strand substrate by VirD2. Another factor, VirC1, has been found to increase the efficiency of T-strand production when VirD1 and VirD2 proteins were limited (De Vos and Zambryski, 1989). The VirC1 protein can specifically recognize and bind to an enhancer or overdrive sequence next to the right T-border, which is necessary for optimal T-DNA formation. After T strand processing, VirE2 subsequently coats ss-T-DNA along its entire length (Citovsky et al., 1988; 1989; Gietl et al., 1987; Sen et al., 1989; Zupan et al., 2000), forming the so-called T-complex. In this manner, VirE2 can protect the TDNA from potential nucleolytic attacks. However, recent evidences have suggested that VirE2 protein might function primarily in the plant cell but not necessarily in the bacterium because plants expressing virE2 can be successfully transfected by A. tumefaciens lacking virE2 (Citovsky et al., 1992). Although VirE2 is associated with the T-strand in plant cells, it is still unclear whether this binding also occurs within the bacterial cell or VirE2 and T-strand molecules meet each other only inside the host plant cell. There are two proposed models for the VirE2 transport. On one hand, VirE2 is one of the most abundant Vir proteins in Agrobacterium and it can bind ssDNA strongly in a cooperative way. In addition, VirE2 and T-strand are transported from the bacterium into the plant cells through the same channel. These suggest that VirE2 should bind to the T-strand in the early steps of the infection process. Indeed, the T-strand and VirE2 can be coimmunoprecipitated from the extracts of vir-induced Agrobacterium. On the other hand, more and more evidence support that VirD2/T-DNA complexes and VirE2 might be exported into plant cells independently from the bacterium. Complementation and co-infection studies suggest that T-strand and VirE2 are exported from the bacterial cells independently and VirE2 is not required for the export of T-strand (Citovsky et al., 1992), while VirE2 export can be inhibited without affecting T-strand export. A recent biophysical report further suggested that VirE2 itself could form channels on the artificial membranes (Dumas, 2001). Based on the above result, Dumas et al. (2001) proposed that VirE2 is transported through the VirB/VirD4 channel or an alternative route and subsequently inserts into the plant plasma membrane, allowing the transport of the ss-T-DNA-VirD2 complex. As a specific molecular chaperone for VirE2, VirE1 is essential for the export of VirE2 to plant cells, but not that of the T strands (McBride and Knauf, 1988; Winans et al., 1987; Deng et al., 1999). VirE1 is a small, acidic protein with an amphipathic -helix at its C-terminus. Yeast two-hybrid studies and extracellular complementation suggest that VirE1 mediates T-complex formation in several possible ways: (i) Although VirE1 does not influence VirE2 transcription from the native PvirE promoter, virE1 indeed regulates the efficient translation of virE2; (ii) VirE1 stabilizes VirE2 via an interaction with the N-terminus of VirE2. VirE1-VirE2 complex is composed of one molecule of VirE2 and two molecules of VirE1; and (iii) the formation of VirE1VirE2 complex, which inhibits self-interacting of VirE2 to form aggregates, might help to maintain the VirE2 molecule in an export-competent state. 1.1.1.2.2. Roles of VirD2 and VirE2 in nuclear localization T-complex nuclear localization is the critical step of tumorigenesis. Since TDNA itself does not contain any specific sequence, any DNA fragment located between T-DNA borders can be transported into the plant cells and subsequently integrated into the plant genome. This implies that VirD2 and VirE2, which are thought to associate directly with T-DNA molecule, are able to specifically mediate T-complex nuclear localization instead of the nucleic acid molecule itself. Both proteins contain conserved bipartite nuclear localization sequence (NLS), which can direct the T-complex into the plant nucleus through the nuclear pores (Tinland et al., 1992; Citovsky et al., 1992; 1994). VirD2 mutants with mutations at the nuclear localization sequence have been shown to have a reduced capability to cause tumorigenesis, while the VirE2 mutants were completely avirulent. For the import of short ssDNA, VirD2 alone was sufficient, but the import of long ssDNA required VirE2 additionally (Ziemienowicz et al, 2000; 2001). These imply that the NLS of two proteins might play different roles in nuclear localization. The targeting of the T-DNA to nucleus is thought to occur in a polar fashion (Zupan and Zambryski, 1997). VirD2, which is attached to the 5' end of the T-strand, may provide this piloting function. VirD2 molecule contains two NLS sequences, one at each end of the molecule (Herrera-Estrella et al., 1990; Howard et al., 1992). The N-terminal sequence possesses the monopartite type that resembles the NLS found in the SV40 large T-antigen, whereas the C-terminal sequence belongs to the bipartite NLS group which is characterized by two adjacent basic amino acids, a variablelength spacer region and a basic cluster in which any three out of the five contiguous amino acids must be basic (Dingwall and Laskey, 1991, Howard et al., 1992). The N-terminal half of VirD2 required for nicking at the border sequences may be involved in T-DNA integration in the plant nucleus, but it is not required for TDNA transfer because mutations in this domain could not affect T-DNA transfer significantly (Koukolikova-Nicola et al., 1993; Shurvinton et al., 1992). It has been reported that the N-terminal NLS of VirD2 might be occluded by the covalently bound T-DNA because the tyrosine 29, with which VirD2 is bound to T-DNA, is only 10 approach, a type2C serine/threonine protein phosphatase (PP2C) was shown to negatively affect the nuclear localization (Gelvin, 2000). Co-electroporation of GUS (β-glucuronidase)-VirD2 NLS gene together with PP2C resulted in the cytoplasmic localization of GUS in the majority of cells. Another plant-encoded protein identified to interact with VirD2 is Arabdopsis cyclophilins (Deng et al, 1998). Since some cyclophilins have peptidyl-prolyl isomerase activity, it has been hypothesized that this protein might serve as a chaperone for VirD2 during the T-strand trafficking in the plant cell. An Arabidopsis protein, VIP1, was identified to interact with VirE2 and implicated to be required for the nuclear import of VirE2 and early stages of T-DNA gene expression (Tzfira et al., 2001). The VIP1 protein contains a β-ZIP motif made up a long basic domain followed by a leucine zipper, which is composed of seven leucine repeats evenly separated from each other by six amino acid residues. Using a recently developed genetic assay for nuclear import and export (Rhee et al., 2000), VIP1 was shown to facilitate the transport of VirE2 into the nuclei of yeast and mammalian cells and participate in the early stages of T-DNA expression (Tzfira et al., 2001). Another protein, VIP2, was also demonstrated to interact with VirE2 and VIP1 but had no effect on intracellular localization of VirE2 when co-expressed in yeast or mammalian cells. To define the precise roles of this protein in nuclear localization in plant cells, new nuclear import system using purified plant nuclei and fractionated cellular extracts has to be developed (Gelvin, 2000). 1.1.3.5. Plant factors participating in T-DNA integration 30 The roles of plant proteins in the T-DNA integration process are only beginning to be defined recently. When inoculated with Agrobacterium, one of the rat mutants (rat5) was found to be deficient in T-DNA integration, even though the T-DNA encoded reporter gene was expressed in the plant cell. Genetic analysis showed that rat5 contains two tandem copies of T-DNA integrated into the 3' untranslated region of a histone H2A gene. Complementation of the rat5 mutant with histone H2A gene resulted in restored tumorigenesis phenotype. The histone H2A genes comprise a small multigene family in Arabidopsis. It might potentially specify the conformation at the T-DNA integration site. The exact mechanism of the involvement of histone H2A in T-DNA integration needs further investigation. 1.1.4. Environmental factors involved in the tumorigenesis of A. tumefaciens Growth conditions, such as pH, temperature and ionic composition of the external medium, affect the virulence functions of many pathogenic bacteria including A. tumefaciens. Virulence gene expression in plant and animal pathogenic bacteria shifts in concert with incubated conditions, reflecting their adaptation to the host environment. In many cases, regulation occurs at the level of gene expression by modulating the activity of specific two-component regulatory systems. Previous studies indicated that a high level of vir gene induction could be obtained at a pH below 6.0 and a temperature below 28°C. It has been reported that environmental acidity plays an important role in inducing the virulence gene expression in Agrobacterium (Olson, 1993; Foster, 1999). Acidic pH in the minimal medium resembles the plant environment that Agrobacterium usually encounters during the infection. There are at least two proposed independent regulatory pathways required for vir gene induction by acidic 31 pH (Winans et al., 1988; Winans, 1990; Chen and Winans, 1991; Mantis and Winans, 1992). They are: (i) the pH-inducible promoter of virG , and (ii) the maintenance of an active conformation of VirA in acidic media that affects the VirA periplasmic domain. Transcription of virG is initiated at two promoters, called P1 and P2. The upstream promoter P1 is induced by phenolic compounds in a VirA-VirG–dependent manner and by phosphate starvation. P2 is primarily induced by low pH and is secondarily responsive to certain stress stimuli (Mantis and Winans, 1992). The activation of vir system also depends on external temperature. Tumor formation induced by Agrobacterium on plant wound site was strongly reduced at temperatures above 29°C when compared to that at 22°C. Inefficient expression of some vir genes and the denaturation of a protein complex could be the cause for this phenomenon. The expression of some virulence genes is specifically inhibited at temperature above 32°C, even when the virA and virG are expressed under a constitutive promoter instead of their native ones. This suggests that the signal transduction mediated by VirA and the subsequent transfer of phosphate to VirG might be sensitive to ambient temperature above 32°C. Jin et al. (1993) proposed that the conformational change of VirA protein at high temperature was responsible for the thermal sensitivity of vir gene expression. virB secretory machinery of A. tumefaciens was also affected by high temperature (Fullner and Nester, 1996). Pili could be readily observed on the surface of Agrobacterium cells grown at 19 °C but not at 28°C. The reasonable explanation for this phenomenon is that the degradation of a limited set of virulence proteins prevents the assembly of the type IV transporter at elevated temperatures. Interestingly, a low temperature also enhances the virB-independent secretion of 32 VirE2 and VirD2. At least fivefold more of VirE2 and VirD2 proteins were shown to be present in the supernatant fraction of cells grown at 19°C when compared to that at 28°C. 1.2. Overview of T-DNA transfer from A. tumefaciens into other eukaryotic cells The host range of A. tumefaciens is not restricted to plant species. It has been proven that A. tumefaciens can also transfer its T-DNA into yeasts such as Saccharomyces cerevsiae, fungi such as Kluyveromyces lactis, as well as some mammalian cells. 1.2.1. T-DNA transfer from A. tumefaciens into yeast T-DNA transfer from A. tumefaciens into S. cerevisiae is very similar to T-DNA transfer into plant cells (Bundock et al., 1995; Piers et al., 1996). The Ti plasmidencoded vir genes that are required for T-DNA transfer into plant cells are also required for T-DNA transfer into S. cerevisiae and vir gene induction is also necessary. The frequency of A. tumefaciens-mediated transformation of S. cerevisiae is approximately 10-3–10-6 transformants per recovered recipient. However, the mechanisms of transformation are not entirely conserved. Mutations in the chromosomal virulence genes of A. tumefaciens involved in attachment and subsequent transformation of plant cells have no effect on the efficiency of T-DNA transfer into S. cerevisiae. This suggests that the yeast transformation system does not emulate plant cell transformation in the attachment step. If the T-DNA shares homology with the genome of S. cerevisiae, it is able to efficiently integrate into the host genomic DNA via homologous recombination. T-DNA lacking homology with S. cerevisiae’s genome could integrate via illegitimate recombination, although at a very low frequency. This is not the case in plants, where gene targeting is difficult to 33 achieve and T-DNA that shares extensive homology with the plant genome integrates primarily via illegitimate recombination. 1.2.2. T-DNA transfer from A. tumefaciens into fungi de Groot et al (1998) demonstrated that the host range of Agrobacterium includes the filamentous fungi. The mechanism of this transfer is not fully understood, but it has been proven that this transfer is depended on the induction of the bacterial virulence genes, which leads to the processing of the T-complex and the establishment of VirB pillus that can mediate the transfer of T-strand into fungi cells (de Groot et al., 1998). In nature, A. tumefaciens and certain species of filamentous fungi share the same habitat. If T-DNA transfer from A. tumefaciens to filamentous fungi indeed occurs in nature when these organisms encounter each other, then the inter-kingdom horizontal DNA transfer between kingdoms may be more extensive than expected. 1.2.3. T-DNA transfer from A. tumefaciens into mammalian cells Kunik et al (2001) first found that the host range of Agrobacterium could be expanded to mammalian cells. They have confirmed that Agrobacterium could transport its T-DNA into human cells and integrate it into their genomes. The efficiency of Agrobacterium-dependent stable transfection is about 1.6 ± × 10-5 cells. In stably transformed HeLa cells, the integration event occurred at the right border of T-DNA, suggesting T-DNA transfer supports the notion that Agrobacterium transforms human cells by a mechanism similar to that which it uses to transform the plant cells. Mutant strains with mutations in the vir or chv genes (virA, virB, virG, 34 virD, virE, chvA and chvB) lost their transforming ability, producing no geneticinresistant cells under their experimental conditions. However, this Agrobacterium-mediated mammalian cell transformation does not always agree with that of plant transformation because mammalian cell transformation could occur at 37°C and uninduced Agrobacterium could still transform HeLa cells. In these conditions, the expression of virA, which is involved in perceiving the vir-inducing plant signals and other components of the T-DNA transfer machinery, is inhibited (Winans et al., 1994). Thus, additional experiments have to be performed to elucidate the exact mechanism by which Agrobacterium transforms mammalian cells. 1.3. Overview of horizontal gene transfer in nature Several mechanisms could be responsible for the evolution and variation of life, such as point mutation and inactivation or differential regulation of existing genes. However, accumulation of point mutations alone is not enough to explain the ability of organisms to exploit new environments. Another mechanism, horizontal (also known as lateral) gene transfer (HGT), might also play an important role in the evolution of both eukaryotic and prokaryotic genome (Syvanen and Kado, 2001; Syvanen, 1994; Nelson et al., 1999). HGT is the gene exchange among different species, even those assigned to different kingdoms. The most common criterion for (or against) horizontal gene transfer derives from a molecular genetic analysis of the DNA sequence database because horizontal transfer can result in genes that display an unusually high degree of similarity to those in a distantly related lineage. In addition to information obtained from the sequences of the genes themselves, the regions adjacent to genes identified as being horizontally transferred often contain vestiges of 35 the sequences affecting their integration, such as remnants of translocatable elements, transfer origins of plasmids or known attachment sites of phage integrases, which further attest to their foreign origin in the genome. Genes acquired from HGT has three requirements: Firstly, there needs to be a means for the donor DNA to be delivered into the recipient cell. Secondly, the acquired sequences must be incorporated into the recipient's genome (or become associated with an autonomous replicating element). And thirdly, the incorporated genes must be expressed in a manner that benefits or at least not deleterious to the recipient microorganism. However, “direct proofs ” of HGT may be difficult to obtain due to rare observation of the actual transfer events. HGT in prokaryotic cells has been regarded as a major, if not the sole, evolutionary factor of microbial genome: novel enzymatic pathways, novel membrane transporter capacities and novel energetics. More recently, the role of HGT in eukaryotic genome evolution has also been exploited. International Human Genome Sequencing reported that 113 human genes appear to be incidents of direct HGT between bacteria and vertebrates, without any apparent occurrence of evolutionary intermediates that is non-vertebrate eukaryotes (Ponting, 2001). 1.3.1. Horizontal gene transfer between bacteria Horizontal gene transfer between bacteria has been confirmed and thoughtfully studied since 1950’s, as in the spread of antibiotic resistance among bacteria and the symbiotic establishment of plastids and mitochondria. The phenomenon that certain bacteria developed resistance to the same antibiotics indicated that these traits were being transferred laterally among species instead of that being generated de novo by each lineage (Davies, 1996). Comparison of multiple, complete prokaryotic genomes 36 indicated that bacteria have obtained a significant proportion of their genes through the acquisition of sequences from other distantly related organisms. The acquisition of foreign genes and gene clusters can explain some of the striking differences in the properties of bacterial species (or strains within a species) that, by other criteria, are almost identical. Large (>20%) differences in genetic make-up between pathogenic and benign strains of the same species (Escherichia coli O157:H7 and K12, for instance) can best be understood with models that invoke evolutionarily rapid gene acquisition (by HGT) and loss. There are three mechanisms through which HGT can occur in microbial genome: transformation, conjugation and transduction. Transformation is a process by which prokaryotes can obtain foreign DNA from their surrounding. Some organisms such as B. subtilis are naturally competent in uptaking naked DNA in the environment from certain physiological stage. Conjugation, known as bacterial sex, involves physical contact between donor and recipient cells and can mediate the transfer of genetic material between them. The conjugation mechanism is often related to the means of transfer of DNA from bacteria to other spices. DNA transfer from E.coli to S. cerevisiae occurs by a mechanism analogous to conjugation and the transfer of T-DNA from Agrobacterium into a plant cell also resembles the bacterial conjugation. Transduction is known as the gene transfer between species via viruses known as bacteriophage in bacteria. Broad host range viruses have the capability to laterally move their DNA across vast phylogenetic distance. By examining the amelioration of atypical sequence characteristics, Lawrence and Ochman (1997, 1998) calculated that the rate of sequence acquisition into the E. 37 coli genome is 16 kb per million years. The widespread evidences for HGT among many bacterial genomes demonstrate that this process has had a strong impact in the overall microbial evolution. 1.3.2. Horizontal gene transfer between bacteria and eukaryotic cells Although the occurrence of horizontal gene transfer among prokaryotes is well established, the gene transfer between bacteria and eukaryotic cells is not so well elucidated. The assertion that HGT does not occur or is not important among eukaryotes is still being made regularly. As HGT involving eukaryotic cells has mainly been inferred from comparative data only, the existence of a possible pathway or mechanism is often questioned. The findings of transposable elements in eukatyotic cell such as the P element in Drosophila and Mariner metazoans has provided actual examples of HGT involving eukaryotes. Moreover the discovery of the two human Mariner transposons demonstrated that these elements might have contributed to the evolution of our genome through HGT (Auge-gouillou et al., 1995). In addition, 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 case of horizontal gene transfer between bacteria and eukaryotic cells. Under such prevalent conditions, as in pathogenesis, the direction of horizontal gene transfer between bacteria and eukaryotic cells may be dominant in one direction (from eukaryotic cell to bacteria), but the opposite direction (retrotransfer) may also occur under different circumstances. 38 1.3.2.1. Acquisition of eukaryotic genes by bacteria There have been many reported cases of proteobacteria acquiring eukaryotic genes based on database and phylogenetic analysis (Copley and Dhillon, 2002). The GAP dehydrogenases of thypanosoma and E. coli share 88% of homology (Hidalgo et al., 1996). Such high resemblance infers that HGT must have been a quite recent event, perhaps within the last few hundred million years. In another case, the 70% identity between the glucose phosphate isomerase of animals and proteobacteria has revealed that it was the eukaryotic sequence that was introduced into the proteobacteria. The kingdom of Proteobacteria contains a large number of bacteria with diverse morphological, metabolic and reproductive characteristics. Although most have lost their ability to perform photosynthesis, this group of bacteria has a common photosynthetic ancestor. The morphologies of the member of kingdom Proteobacteria are very diverse. Their only common characteristic is that they are all Gram negative. Based on 16S RNA analysis, this kingdom is further divided into the αproteobacteria, β- proteobacteria, γ- proteobacteria, δ- proteobacteria and εproteobacteria. The α-proteobacteria is a very large and diverse group of organisms that live within various media including water, soil and other living organisms. Some of these microbes are harmless, while others create a symbiotic relationship with other organisms, and some are pathogenic. Many bacteria in this group can infect and invade other eukaryotic cells including both plant and mammalian and Agrobacterium tumefaciens is one of such bacteria that can so. 39 There is a chance that these α-proteobacteria meet the eukaryotes and acquired their genes by direct transformation. Although the transfer of T-DNA from Agrobacterium to the plant cell is unidirectional, someone has proposed that the same DNA deliver system might also pick up ancillary DNA from host and transfer it back into the donor Agrobacterium (Chou et al., 1998 ) . If true, eukaryotic genes might have been acquired by a retrotransfer mechanism. There are many such candidate genes that were retrotransferred into Agrobacterium, such as virF and ros. The fact that Ros contains a C2H2 zinc finger that is known to exist in eukaryotes, together with the fact that it can recognize the typical eukaryotic transcription element (TATA box), suggest that the zinc finger of Ros might have been acquired from a higher organism by means of HGT. In addition, the gene homology analysis has confirmed a distant evolutionary relationship between the bacterial ros gene and its eukaryotic counterparts. It is interesting to note that the zinc finger in Ros is akin to the zinc finger domain in animal cells rather than in plant cell, which is the native host of Agrobacterium. This suggests that the ancestor of ros might come from an animal source. It can be indirectly inferred that Agrobacterium might also retain the ability to infect animal cells. The bi-directional DNA transfer would also account for the presence of eukaryotic genes located on the Ti plasmid and chromosome of Agrobacterium. 1.3.2.2. Acquisition of horizontal transferred genes by eukaryotes Unlike the uptake of genetic materials by prokaryotes, which are mostly single cell organisms, the introduction of foreign DNA into a multicellular eukaryotic cell's cytoplasm does not ensure successful gene transfer unless the transferred sequences 40 are stably maintained in the recipient germ cell line. Thus, eukaryotes have a relatively low number of acquired genes by bacterial standards. Stolz et al. (2002) supposed that there was a transfer of the nitrate reductase gene from a bacterium to a fungus with the enzyme having 85% identity in the two groups. Some other cases of such kind of transfer have also been reported thereafter (Screen and Leger, 2000). Direct introduction of DNA from bacteria to mammalian cells has been reported by Schaffner (1980). He found that plasmid harbouring tandem copies of SV40 virus genome could be transferred from pathogenic E. coli to mammalian cells by incubating the cell culture with a bacteria suspension. However, spontaneous transfer was found to occur at a very low frequency, x 109 bacteria yielding one infection per 107 monkey cells. There are several possible mechanisms by which the eukaryotes acquired the prokaryotic genes. In ancient, the horizontal gene transfer events occurred based on “you are what you eat” principle (Doolittle, 1998). The genome of phagocytic unicellular eukaryotes, which prefer α- proteobacteria or all proteobacteria as their food, could be modified by food-derived genes over a long period of time when an incidental transformation or DNA uptake occurred. As we know, the mitochondria present in all eukaryotes and the chloroplasts in plants are descendants of free –living eubacteria (α-proteobacteria) acquired by the host cell through endosymbiosis. The lysis of organelles (even if infrequent) would inevitably have provided a stable source of prokaryotic genes for a plausible introduction into the nuclear of eukaryotes. Therefore, there had been ample time for such undoubtedly rare but inevitably event to take place so that organellar genes, which were prokaryotic in origin, might have been transferred into the nuclear genomes. In a relatively recent event, the plant, 41 fungi and animals (the multicellular eukaryotes) might acquire bacterial genes through the mechanisms of the transposons, retroposones and retrovirus. Although many mechanisms have been proposed (including the ones discussed above), there are few solid evidences to support these hypotheses. The gene transfer between Agrobacterium and many muticellular eukaryotes, including plant, mammal, yeast and fungi, is the only best understood example of interkingdom gene transfer. As such, further investigation on the interaction between the Agrobacterium and eukaryotes might shed light on the exact mechanism of HGT involving eukaryotes. 1.3.3. Horizontal gene transfer between eukaryotic cells Horizontal transfer of genetic information between mammalian cells was not known until very recently. Holmgren et al. (1999; 2002) demonstrated that DNA could be transferred from apoptotic cells to recipient cells after phagocytosis and the DNA would be lost unless it is conferred with a strong selective advantage to the recipient cell. Cocultivation of cell lines containing intergrated copies of Epstein-Barr Virus (EBV) resulted in rapid uptake and transfer of EBV-DNA as well as genomic DNA to the nucleus of phagocytotic cell. These results suggest that lateral gene transfer of DNA between eukaryotic cells may also play important roles in normal physiology and evolution. 1.3.4. Bacteria as gene delivery vectors for mammalian cells As discussed above, bacteria can transfer some of its genes to a very broad eukaryotic recipient cells, including yeast, plant and mammals (Sprague, 1991). Most recently, several laboratories have reported that some pathogens or bacteria harboring 42 pathogen genes were used as gene delivery vectors for mammalian cells. Two independent research groups (Sizemore et al., 1995; Courvalin, 1995) demonstrated that mammalian cells could transiently express genes delivered intracelluarly by Shigella flexneri. A diamininopimelated autxotroph (dap-) mutant of Shigella, in which cell wall synthesis was impaired, was used as the bacterial vector. The mutant bacteria would lyse if they invad mammalian cells due to the impaired integrity of their cell wall. This process is known as abortive or suicidal invasion and the bacteria have to be lysed for DNA delivery to occur. The Shigella mutant strain (dap-) harbored plasmid pCMVβ, which contains the E. coli β-galactosidase gene under the control of a eukaryotic promoter, was incubated with cultured BHK cells for 90 min. After two days, the expression of β-galactosidase in BHK cells could be detected. Using similar strategies, some other attenuated pathogen strains such as L. monocytogenes and Salmonella have also been used as bacterial vectors to deliver foreign genes into the mammalian cells. In addition, E. coli strains harboring virulence genes of different pathogens (Shigella flexneri or Yersinia pseudotuberculosis) have also been used (Grillot-Courvalian et al., 1998). These results showed that these artificially invasive strains could also deliver foreign gene into mammalian cells at different levels. Studies on microbial pathogenesis will help us in understanding the mechanism of bacteria-based DNA delivery into the mammalian cells. Being a successful bacterial vector needs to satisfy two requirements. Firstly, bacteria must keep the ability to enter host cells. Secondly, bacteria must survive in the host cells in order to escape from vacuoles and release the DNA in the cytosol. The bacterial species to be used as the vectors must have the ability to invade the 43 mammalian cells in order to satisfy the first requirement mentioned above. There are two major strategies that bacteria could employ to enter the mammalian cells: (1) some bacterial genera, such as Salmonella or Shigella, might secrete a set of invasion proteins when in contact with the host cells. This process will induce signaling events in the host cells such as cytoskeletal rearrangement, membrane ruffling and bacteria uptake by micropinocytosis; (2) some bacterial genera, such as Yersinia or Listeria, can bind to a particular ligand on the host cell surface and be directly uptaken by host cell by a zipper-like mechanism. After invasion, many pathogens such as Listeria and Shigella can rapidly escape from vacuoles due to the release of some proteins that could lyse the vacuole membrane. The use of bacteria as a gene delivery vector has been mainly exploited for DNA vaccination because the delivery of DNA into the cytosol by intracellular bacteria may constitute a means of protecting this DNA from being degraded by cytosolic nucleases. All the abovementioned bacteria used as DNA delivery vectors are human pathogens (Salmonella and Shigella) or bacteria that harbored pathogen virulence genes. Therefore, safety concern should be taken in serious consideration, if these bacteria were to be used as gene delivery vector in vivo. 1.4. Objectives of this study A. tumefaciens is often used as a vector to generate transgenic plants, while little is known about its interaction with mammalian cells. Although there are some evidences, which suggest Agrobacterium can transfer its gene into mammalian cells (Kunik et al., 2001), the mechanism of such transfer needs to be understood thoroughly. The efficiency of Agrobacterium mediated DNA transfer into 44 mammalian cells is quilt low, which is about 10-5 , when compared to that of plant cells. Additionally, the vir gene induction is unnecessary in this process. Thus, the aim and intention of this study is to elucidate the mechanism of Agrobacteriummediated DNA transfer into mammalian cells and find out the mammalian cells and Agrobacterium factors which might be involved in this process. 45 [...]... from A tumefaciens into mammalian cells Kunik et al (20 01) first found that the host range of Agrobacterium could be expanded to mammalian cells They have confirmed that Agrobacterium could transport its T-DNA into human cells and integrate it into their genomes The efficiency of Agrobacterium- dependent stable transfection is about 1. 6 ± 3 × 10 -5 cells In stably transformed HeLa cells, the integration... cerevisiae is very similar to T-DNA transfer into plant cells (Bundock et al., 19 95; Piers et al., 19 96) The Ti plasmidencoded vir genes that are required for T-DNA transfer into plant cells are also required for T-DNA transfer into S cerevisiae and vir gene induction is also necessary The frequency of A tumefaciens -mediated transformation of S cerevisiae is approximately 10 -3 10 -6 transformants per recovered... encode enzymes catalyzing the synthesis of auxin and cytokinin respectively The gene ons (or 6a) controls octopine and nopaline export from plant cells, while tml (or 6b) increases the sensitivity of plant cells to phytohormones (Clarence, 19 91; Sheng and Citovsky, 19 96; Winans et al., 19 86; 19 89) 1. 1 .1. 8 Summary of the functions of Ti-plasmid encoded virulence genes After sensing the particular plant... and iaaH) infers that some of these genetic materials were possibly introduced into 21 Agrobacterium by horizontal gene transfer from an eukaryotic organism, although no direct evidence has been obtained so far 1. 1.2 Roles of chromosomal virulence genes of A tumefaciens Some Agrobacterium chromosomal virulence (chv) genes have also been shown to play important roles in tumorigenesis (Gelvin, 2000; Zhu... are transferred from Agrobacterium into the plant cells through this type IV secretion system, although the precise role of pili in DNA transfer is still not clear in any conjugal transfer system 1. 1 .1. 4 VirF The 23-kDa VirF protein is encoded by a gene only present in the vir region of octopine-type Ti plasmid and absent in nopaline-type Ti plasmid (Melchers et al., 19 90; Schrammeijer et al., 19 98)... surface, transfer of T-DNA from the bacteria to plant cells across the plant cell wall and membrane, nuclear translocation of the single-stranded T-DNA molecule; and stable integration of T-DNA into the plant genome 1. 1.3 .1 The plant gene expression in response to Agrobacterium transformation Although Agrobacterium does not induce the hypersensitive response in plant, it can trigger changes in the gene. .. transfer from A tumefaciens into other eukaryotic cells The host range of A tumefaciens is not restricted to plant species It has been proven that A tumefaciens can also transfer its T-DNA into yeasts such as Saccharomyces cerevsiae, fungi such as Kluyveromyces lactis, as well as some mammalian cells 1. 2 .1 T-DNA transfer from A tumefaciens into yeast T-DNA transfer from A tumefaciens into S cerevisiae is... al., 19 88; Ward et al., 19 88 ; 19 90; Kuldau et al., 19 90; Shirasu et al., 19 90) These proteins are thought to be located in or transported to the Agrobacterium inner membrane The proteins VirB2 through VirB 11 are absolutely required for gene transfer and the efficient assembly of extracellular T pili, while VirB1 is an efficiency factor for T-complex transmembrane assembly (Berger and Christie, 19 94;... recombination 1. 2.2 T-DNA transfer from A tumefaciens into fungi de Groot et al (19 98) demonstrated that the host range of Agrobacterium includes the filamentous fungi The mechanism of this transfer is not fully understood, but it has been proven that this transfer is depended on the induction of the bacterial virulence genes, which leads to the processing of the T-complex and the establishment of VirB pillus... additional studies 1. 1 .1. 7 Other genes on Ti plasmid There are some other gene loci on the Ti plasmid besides vir genes Some of them confer ancillary functions in tumor formation, such as inter-bacterial conjugation genes and vegetative replication genes Inter-bacterial conjugation genes include oriT, traAFB and trbB, which control the conjugative transfer of Ti plasmid Vegetative replication genes, including . of Vir proteins as well as Chv proteins that are involved in the T-DNA transfer will be described in detail. 1. 1 .1. Roles of Ti-plasmid encoded virulence genes 1. 1 .1. 1. VirA/VirG, member of. cells to phytohormones (Clarence, 19 91; Sheng and Citovsky, 19 96; Winans et al., 19 86; 19 89). 1. 1 .1. 8. Summary of the functions of Ti-plasmid encoded virulence genes After sensing the particular. development of Agrobacterium as a plant genetic vector has been one of the most important technical developments in the past 25 years. 1 . Fig. 1. 1. Schematic diagram of the Agrobacterium