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Clonaje y caracterización de la ADN polimerasa θ de Leishmania infantum

AutorFernández-Orgiler, Abel
DirectorLarraga, Vicente
Palabras claveLeishmania
Polimerasas beta
Enzimas de reparación
Fecha de publicación2014
EditorCSIC - Centro de Investigaciones Biológicas (CIB)
Universidad Complutense de Madrid
ResumenLeishmaniasis is a wide spectrum of vector borne diseases with great epidemiological and clinical diversity. It is caused by more than 20 species of the protozoan parasite that belongs to the genus Leishmania (Singh, Kumar et al. 2012). Leishmaniasis affects 88 countries: 72 are developing countries and 13 of them are among the least developed. It is estimated that annual incidence is 1-1,5 million cases of cutaneous leishmaniasis and 0,5 million cases of visceral leishmaniasis. Overall, prevalence is 12 million people and the population at risk is 350 million. Nevertheless, there is probably an even greater difference between the number of cases actually occurring and the number of reported ones because leishmaniasis is not always a notifiable disease (only in 33 of the 88 countries endemic) (Desjeux 2004). The form and severity of the disease greatly depends on the infecting Leishmania species and the immune status of the host. Leishmaniasis has traditionally been classified into three major clinical forms: visceral, cutaneous and mucocutaneous leishmaniasis, which differ in immunopathologies and degree of morbidity and mortality. Cutaneous leishmaniasis, a clinical form consisting of cutaneous ulcers usually localized to the initial site of the sand fly bite, will heal spontaneously in uncomplicated cases within 2 months to a year. In the case of mucocutaneous leishmaniasis, the primary lesion occurs at the initial site of the bite on the skin but the infection also involves the mucosal system of the nasal and buccal cavity, causing degeneration of the cartilaginous and soft tissues. These ulcerations are often quite disfiguring to the lips, nose, hard and soft palates and vocal cords. Death is often due to secondary bacterial infections or malnutrition. Finally, in the case of visceral leishmaniasis, parasites reside within the reticuloendothelial cells of the viscera, including the spleen, lymph nodes, liver and intestine and is usually fatal when untreated (Cunningham 2002).
Leishmania spp. are heteroxenous, digenetic protozoan parasites and as such they live successively in two hosts, the insect vector (the female sandfly of the genus Lutzomyia in the New World and of the genus Phlebotomus in the Old World) and a vertebrate playing the role of reservoir. In female sand flies, Leishmania spp. exist extracellularly in the lumen of the digestive tract where they adopt a flagellated, elongated promastigote form and undergo several differentiation stages. Leishmania spp. are obligate intracellular parasites in the vertebrate host. Indeed, after the bite of an infected sand fly, at least some of the promastigotes injected are rapidly engulfed by resident dermal phagocytic cells or cells rapidly recruited from the epidermis or the blood. During the early stages of infection, a large part of the cells internalizing parasites appears to be macrophages, inside which promastigotes differentiate into amastigotes devoid of the external flagellum. This process takes several days and occurs within organelles named parasitophorous vacuoles. The life cycle is completed when a sand fly takes a blood meal on a parasitized vertebrate. During this process, the vector can be infected by free amastigotes or by infected cells. In the gut of the insect, the amastigotes differentiate rapidly into promastigotes being the cycle closed (Antoine, Prina et al. 2004). To survive successfully and multiply within these two hosts, the parasites must confront and overcome different challenges as the hostile digestive conditions found within the sand fly vector or the oxidative burst of the vertebrate host macrophages (Cunningham 2002). In the last years, the genomes of three Leishmania species (L. major, L. infantum and L. braziliensis) have been sequenced. However it has been possible to assign putative biological function only to 35% of the protein-coding genes based on experimental characterization (3,7%) or sequence similarity to proteins with known function in other organisms (31,5%) (Myler and Fasel 2008).
One of these genes whose function has been inferred from data obtained in other organisms is the gene Linj24.0910 (www.genedb.org) which encodes DNA polymerase theta (Polθ), a nuclear A-family DNA polymerase (Pang, McConnell et al. 2005). In the fruit fly Drosophila melanogaster, first organism where it was described, Polθ (DmPolθ) is characterized by an N-terminal helicase-like domain, a C-terminal DNA polymerase domain, and a large central domain that spans between the two and its function has been related to resistance to DNA interstrand crosslinking agents (Boyd, Sakaguchi et al. 1990; Harris, Mazina et al. 1996). Genes encoding proteins with similarity to Polθ are present in plants, protists, and multicellular eukaryotes, but not in yeast or other fungi. However it remains to be determined whether these proteins are true orthologs, as it appears that there are structural and functional differences between species. The main structural differences observed are: the presence or absence of the helicase domain at the N-terminus (absent in some lower eukaryotes), the length of the central region (between 800 and 1600 amino acids depending on species) and the presence or length of three insert regions within the polymerase domain (the inserts are much shorter in the non-vertebrate family members). Similarly, at the functional level, depending on the organism tested, different activities have been attributed to Polθ such as repair of interstrand crosslinks, translesion synthesis, base excision repair or double strand breaks repair (Yousefzadeh and Wood 2013). The DNA polymerase θ of Leishmania infantum (LiPolθ) is a protein of 1171 amino acids in length and a molecular weight of 125 kDa. LiPolθ lacks helicase domain and the N-terminal part of the protein has not similarity with any other protein different to DNA polymerases theta of trypanosomatids. The C-terminal part of the protein is a DNA polymerase domain where all six conserved motifs of A-family polymerases are present in the primary protein sequence. Among some of these conserved motifs there are also the three characteristic inserts of the DNA polymerases θ. However, the length of these inserts differ from that of higher eukaryotes. Furthermore LiPolθ has two additional inserts exclusively shared with DNA polymerases θ of trypanosomatis. In order to determine the role played by LiPolθ in Leishmania infantum parasite, three specific objectives were developed: - Determination of the subcellular localization of LiPolθ and its expression levels along the parasite cycle. - Biochemical characterization of LiPolθ and study of its potential role in translesion synthesis. - Determination of the physiological role of LiPolθ. To achieve the earliest goal, firstly we obtained a polyclonal antibody anti-LiPolθ. For this purpose, the LinJ24.0910 gene was cloned into the pRSET-C vector, which allows the expression of the protein of interest fused with a 6 histidine tag at its N-terminal end. LiPolθ expressed in non-soluble form in E. coli BL21(DE3)pLysS strain was purified by affinity chromatography and subsequently inoculated in form of 4 doses of 100 μg each in a New Zealand White rabbit. The specificity of the polyclonal antibody obtained was evaluated by Western blot. The polyclonal antibody did not present any cross reactivity with E. coli proteins and in total extracts from Leishmania infantum recognized a single band of 125 kDa (molecular weight expected for LiPolθ).
Once the specificity of the polyclonal antibody anti-LiPolθ was verified, we proceeded to the assessment of the LiPolθ expression levels by Western blot in promastigote and amastigote stages of the parasite. The results showed that LiPolθ is expressed at both stages. At the promastigote form, LiPolθ expression levels are similar along the growth curve of the parasite and no differences between procyclic and metacyclic promastigotes were observed. At the amastigote stage, LiPolθ is expressed at lower levels regarding to the promastigote stage. Subcellular localization of LiPolθ was evaluated by indirect immunofluorescence using polyclonal antibody anti-LiPolθ and Alexa 488 labeled secondary antibody. LiPolθ has mainly a nuclear localization, although a small fraction of the protein is also found in the mitochondria of the parasite. Next, the biochemical characterization of the protein was performed. As a preliminary step, we verified that LiPolθ has an intrinsic DNA polymerase activity. To do this the protein was expressed by a cell-free system (TnT® Quick Coupled Transcription/Translation System) and subsequently tested in a DNA polymerase activity assay with “open” template/primer molecules. After verifying that LiPolθ has an intrinsic DNA polymerase activity, we proceeded to the production of the protein under native conditions on a large scale for further purification and study of its activity. To do this we used two different strategies. On one hand LiPolθ expression fused to a 10 histidine tag at its C-terminus was conducted, in Pichia pastoris yeast. However, although the protein could be expressed in soluble form, the levels obtained were too low. On the other hand we performed the expression of LiPolθ and three truncated forms of the protein (all corresponding to the polymerase domain) fused to the MBP protein at the N-terminus in E. coli. The protein MBP-LiPolθ and the truncated forms (MBP-970, MBP-640 and MBP-358) were expressed in native form and purified by affinity chromatography on amylose resin. Of the 4 protein forms only MBP-970 and MBP-LiPolθ were active, implying that in addition to the polymerase domain part of the central region is required for activity. This result has also been observed for hPolθ where the presence of at least 300 amino acids of the central region of the enzyme appears to be required for its functionality (Prasad, Longley et al. 2009; Hogg, Seki et al. 2011). Of the MBP-LiPolθ and MBP-970 forms, the first one was that showing greater activity, so it was selected to perform the biochemical characterization.
LiPolθ is a template-dependent DNA polymerase that incorporates nucleotides with low processivity, so it should be required for the synthesis of short DNA fragments and not for genome replication. Like most of the DNA polymerases, LiPolθ preferably inserts the nucleotide complementary to the template base following the rules of Watson-Crick base pairing. LiPolθ can carry out the polymerization reaction using both magnesium and manganese cofactors though in the presence of the latter shows an increase in its activity. Under optimum conditions LiPolθ is able to incorporate a nucleotide at blunt ends in a similar way to other A-family DNA polymerases, including hPolθ (Seki, Masutani et al. 2004). LiPolθ possesses a high strand displacement activity in the absence of factors which is independent of the group present at the 5’ end of a gap or the metal used as cofactor. This ability to insert nucleotides coupled to strand displacement is important in the repair of interstrand cross-links and long patch base excision repair (LP-BER). In this regard it is noteworthy that the absence of Polθ in D. melanogaster is associated with hypersensitivity to agents that cause DNA interstrand cross-links (Boyd, Sakaguchi et al. 1990), whereas it is associated with decreased levels of base excision repair, especially of the LP-BER, in chicken DT40 cells (Yoshimura, Kohzaki et al. 2006). Therefore LiPolθ may participate in both pathways. Human DNA polymerase θ (hPolθ) has intrinsic 5′-deoxyribose phosphate lyase activity and it was suggested that it could participate in short nucleotide base excision repair (Prasad, Longley et al. 2009). However this activity was not observed in LiPolθ. Moreover LiPolθ has a high strand displacement activity and consequently it does not seem likely to be involved in that mechanism. Another feature of LiPolθ is its ability to extend mismatched primer termini either directly or via misalignment mechanism. According to the data obtained in different experiments, in the presence of a mismatch, LiPolθ at first tries to generate a correct base pair by dislocation of one of the two DNA strands from which performs the extension, thereby causing the appearance of insertions or deletions. In case that the sequence context does not allow the formation of a correct base pair, LiPolθ performs direct extension, setting the mutation.
Currently, the only Polθ that has been purified and tested in vitro is hPolθ, which has been related to translesion synthesis activity (Seki, Marini et al. 2003; Seki, Masutani et al. 2004). To determine if LiPolθ could have the same function, DNA polymerase assays were performed with open template/primer molecules containing modified bases simulating different lesions. The lesions studied were: 8oxodG, Tg, 6mdG, abasic sites and CPD. The major product of oxidative base damage is 8-oxo-7,8-dihydro-2’-deoxyguanine (8oxodG) (Batra, Beard et al. 2010). This lesion can be generated by the direct oxidation of deoxyguanosine in DNA or by the incorporation of oxidized dGTP (8oxodGTP) into DNA by DNA polymerases (Katafuchi and Nohmi 2010). 8oxodG is a promutagenic lesion due to its ability to form base pairs with dA in addition to dC (Shibutani, Takeshita et al. 1991). We demonstrated the ability of LiPolθ to bypass the oxidative lesion 8oxodG in the template. LiPolθ “copied” the lesion, as it preferentially inserted a correct dCTP opposite 8oxodG with either manganese or magnesium as metal cofactor. In the presence of manganese LiPolθ activity rose and the protein was also able to elongate the 8oxodG:dCMP pair, thus performing an error-free tolerance of this lesion. LiPolθ extends the “correct” 8oxodG.dCMP and the mutagenic 8oxodG.dAMP base pairs with similar efficiency which means that fidelity of the 8oxodG bypass lies primarily at the insertion step. Regarding fidelity at 8oxodGTP incorporation, LiPolθ prefers to insert the oxidized nucleotide opposite template dA rather than template dC at a ratio of 34:1. LiPolθ incorporates 8oxodGTP opposite dAMP with a 20% efficiency compared to normal dTTP incorporation. This value is more related to a Y-family DNA polymerase involved in translesion synthesis than to an A-family replicative DNA polymerase (Katafuchi and Nohmi 2010). Oxidative damage can affect pyrimidines also, being thymine glycol one of the major DNA lesions formed (Aller, Rould et al. 2007). LiPolθ is able to insert a nucleotide opposite to thymine glycol, but direct extension is not possible beyond the lesion. LiPolθ just can extend a thymine glycol base pair via dNTP-stabilized misalignment mechanism, generating single-base deletions.
In vivo experiments with human cells have suggested a combined role of hPolk and hPolζ in translesion synthesis opposite thymine glycol. According to this hypothesis hPolk could act primarily at the insertion step with the subsequent extension step being performed by hPolζ (Yoon, Bhatia et al. 2010). However, Polk is localized to the mitochondria in trypanosomatids (Rajão, Passos-Silva et al. 2009) so it is possible that LiPolθ replaces Polk incorporating a nucleotide opposite thymine glycol. O6-methylguanine, a mutagenic and cytotoxic DNA adduct that can be formed in vivo by alkylating agents, was also studied. LiPolθ can incorporate a residue opposite the lesion and extend from the incorporated nucleotide. However, in a similar way to other DNA polymerases, LiPolθ incorporates dTTP opposite 6mdG more often than dCTP, giving rise to dG to dA transition mutations. Abasic (apurinic/apyrimidinic) sites are noncoding lesions in DNA that arise by hydrolysis of the glycosidic bond connecting purine or pyrimidine base to deoxyribose sugar. Abasic sites are one of the most common DNA lesions. These noncoding lesions can occur because of specific DNA glycosylases removing altered bases or by labilization of the glycosidic bond resulting from chemical modifications of bases (Randall, Eritja et al. 1987). When an abasic lesion is copied, dATP is predominantly incorporated by DNA polymerases opposite the lesion. Preferential incorporation of dATP opposite abasic lesions is referred to as the “A-rule” (Strauss 2002). LiPolθ like hPolθ inserts dATP and dGTP primarily opposite an abasic site, independently of the next template base. LiPolθ is able to perform direct extension of nucleotide incorporated. However, LiPolθ shows much lower efficiency compared to hPolθ which has been reported to insert dATP against an abasic site with 22% of the efficiency of a normal template and continuing extension as avidly as with a normally paired base (Seki, Masutani et al. 2004). It has been suggested that inserts 2 and 3 may be relevant for overcoming abasic sites by hPolθ (Hogg, Seki et al. 2011). These inserts are present in LiPolθ, however the length of the insert 3 is much shorter than in the case of hPolθ, which could explain the different behavior between the two DNA polymerases in the synthesis opposite an abasic site. LiPolθ activity, like that of hPolθ, will be error free as long as the abasic site has been generated as a result of an intermediate step in the base excision repair of dUTP incorporated. Conversely, if the abasic site arose from spontaneous hydrolysis, insertion of dATP will be mutagenic.
Finally the cyclobutane pyrimidine dimers (CPD) lesion was also evaluated. CPD are the most prevalent form of the lesion induced by UV radiation, and if not removed, lead to mutations (Gonzalez-Púmariega, Vernhes et al. 2009). LiPolθ shows a low efficiency at the incorporation step opposite thymines of a CPD in a similar way to other A-family polymerases like hPolθ (Seki, Masutani et al. 2004), hPolν (Takata, Shimizu et al. 2006) or T7 DNA polymerase (Li, Dutta et al. 2004). This is in contrast with the high efficiency showed by Polη, a DNA polymerase specialized in CPD bypass (Kusumoto, Masutani et al. 2002). After incorporation opposite the dimer, the extension step is performed by LiPolθ with similar efficiency to that on non-damaged template.
To achieve the third objective (the determination of the physiological role of LiPolθ) we generated a cell line of Leishmania that overproduced the protein by transfecting the parasites with the plasmid vector pTEX containing the gene encoding LiPolθ. Overexpression of LiPolθ did not alter the growth curve of the parasite. Transfected parasites overexpressing LiPolθ were used to evaluate in vivo the impact of this enzyme in damage tolerance through a comparative study of resistance to different genotoxic agents among wild-type and transfected parasites. The resistance of each of the cell lines was evaluated measuring the percentage of viable promastigotes (determined by MTT assay) after 72 hours of incubation in the presence of different concentrations of the genotoxic agents. The transfected cell line showed greater resistance to the oxidative agent H2O2, as well as to mitomycin C and cisplatin compounds which generate interstrand cross-links. Increased resistance to H2O2 is consistent with previous in vitro results where it was found that LiPolθ can perform translesion synthesis through 8oxodG (main product of oxidative damage) in an errorfree mode. Similarly the increased resistance to cisplatin and mitomycin C, is consistent with the results obtained in other organisms such as Drosophila melanogaster, Caenorhabditis elegans or Mus musculus, where the absence of Polθ is related to an increased sensitivity to agents that cause interstrand cross-links (Boyd, Sakaguchi et al. 1990; Muzzini, Plevani et al. 2008; Li, Gao et al. 2011). By contrast, the transfected parasites showed a lower tolerance to ultraviolet radiation. Based on the low efficiency exhibited by LiPolθ in translesion synthesis through CPD (main product of UV radiation) a possible explanation is that LiPolθ as its over-expressed, competes with LiPolη for DNA, thus preventing repair of the lesion and causing the lower survival rate observed. Finally, in the presence of the alkylating agents tested (MMS, EMS and ENU), wild-type and transfected parasites showed no difference in survival rate. The similar response observed against MMS, is in agreement with previous studies conducted with chicken DT40 cells and larvae of D. melanogaster where knock out for Polθ showed no differences in sensitivity to this compound (Boyd, Sakaguchi et al. 1990; Yoshimura, Kohzaki et al. 2006). In the same way, the absence of a differential response against ENU is consistent with the low fidelity of LiPolθ observed in in vitro assays in which LiPolθ shows a preference for dTTP misincorporation opposite 6mdG. Because 6mdG bypass by LiPolθ would imply the emergence of mutations, it seems unlikely to be one of the roles played by LiPolθ in vivo.
Due to the ability of LiPolθ to perform translesion synthesis through 8oxodG in an error-free mode as well as the increased resistance to hydrogen peroxide showed by transfected parasites overexpressing LiPolθ we assessed whether LiPolθ could have a role during the first steps of Leishmania infection where oxidative stress is the main defense mechanism that phagocytic cells use to kill the parasites. To test this, in vitro infections of macrophages were carried out with metacyclic promastigotes of wildtype and transfected cell lines. Transfected promastigotes showed an infection rate three times higher than wild-type promastigotes (33% vs. 11% respectively). In summary, according to the results obtained in this study, LiPolθ is involved in resistance to oxidative damage, which is relevant in the infective process of the mononuclear phagocytic system cells.
Descripción180 p.-63 fig.-2 tab.
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