Experimental study of the function of the excreted/secreted LeishmaniaLmSIR2 protein by heterologous expression in eukaryotic cell line
© Sereno et al; licensee BioMed Central Ltd. 2005
Received: 12 October 2004
Accepted: 24 January 2005
Published: 24 January 2005
In yeast and Caenorhabditis elegans, Silent Information Regulator (SIR2) proteins have been shown to be involved in ageing regulation. In Leishmania, the LmSIR2rp was originally isolated from the excreted/secreted material of the Leishmania parasites. Among the function(s) of this protein in Leishmania biology, we have documented its implication in parasite survival, and in particular in Leishmania amastigotes. In this paper we question the role of the excreted/secreted form of the protein. In particular we wonder if the Leishmania Sir2 homologue is involved in some aspect of its biological function(s), in various components and pathways, which could promote the host cell survival. To test this hypothesis we have mimicked an intracellular release of the protein through constitutive expression in mouse L929 fibrosarcoma cells.
Our results demonstrate that the LmSIR2 protein was properly expressed by fibroblasts and that LmSIR2 is localized both in the cytoplasm and the nucleus of all the transformed cell clones. Unexpectedly, we found that cells expressing LmSIR2 presents reduced saturation cell density ranging from 40% to 60% and expressed an acidic (pH6.0) β-galactosidase activity, which is known to be a senescence biomarker. As a consequence, we observed that LmSIR2 positive fibroblasts were more permissive towards Leihmania infection.
LmSIR2 is able to substantially interfere with the host cell physiology. Thus, it is tempting to speculate that these modifications could help Leishmania to survive for a long period in a cell with reduced capacity to multiply or respond to immunologic stimuli. The potential implications of our finding during the in vivo infection process are discussed.
Leishmania is an intracellular pathogen that causes Leishmaniasis, which remain an important medical problem in several countries. Protozoan parasites of the genus Leishmania result in a spectrum of human disease that range from self-healing cutaneous ulcers to potentially fatal visceral infection, depending primarily upon the species of parasite involved [1, 2]. Leishmania live as either extracellular flagellated promastigotes in the digestive tracts of their sand fly vectors or as nonflagellated amastigotes within macrophages where they survive and replicate within phagolysosome. Leishmania are known to export large range of proteins and glycoconjugates including lipophosphoglycan .
In a previous paper we have characterized a Leishmania major gene encoding a protein carrying extensive homology to SIR2 of yeast that we consequently termed LmSIR2rp . It belongs to a large family of closely related NAD-dependent deacetylase named Hst proteins (Homologous of Sir two) or sirTuins, present in both prokaryotic and eukaryotic species . Post-translational modifications of histones, like acetylation by histone acetylase (HAT) and deacetylation by histone deacetylase (HDAC), within the context of chromatin has been shown to regulate gene expression [6, 7]. To date, three classes of HDACs are characterized in eukaryotes, the class I and II HDACs, and the class III defined by the sir2 family. In eukaryotic species some members of the Sir2 family are much more closely related to the core domain of the yHst2p protein than to the core domain of ySir2p itself . These are: the SIR2 family members from Schizosaccharomyces pombe, C albicans, Leishmania, chicken, human (hSirT2p, hSirT3p) and the closely related mouse protein (MmSirT2p and MmSirT3p) . This group has been designated Hst2-like, as it forms an independent branch within the Sir2 family tree. Five members of this group (LmSIR2rp), like it yeast (yHst2) human (SIR2L) and mouse (MmSIR2L2 and MmSIR2L3), are cytoplasmic [4, 9, 8, 10, 11]. The implication of SIR2 proteins bearing nuclear localization, in aging process has been extensively studied. In yeast, the deletion of SIR2 shortens lifespan and over expression extends lifespan . This phenotype of life extension has been also observed in Caenorhabditis elegans carrying extra copies of the SIR2 orthologue, Sir-2.1 [review in ]. In mammalian cell, SIRT1 promote cell survival . SIRT1 is able to deacetylate FOXO3 and/or FOXO4, thus attenuating FOXO-induced apoptosis and potentiating FOXO-induced cell-cycle arrest. Thus SIRT1 might increase longevity by shifting FOXO dependent responses away from cell death and toward cell survival . Also, the unusual NAD-requirement for the Sir2 deacetylase may link metabolic rate to silencing and life span .
We have shown that the cytosolic LmSIR2rp protein, when over expressed in Leishmania amastigotes, was able to promote parasite survival in in vitro culture systems . Further, we also observed that LmSIR2rp can be detected in the excreted/secreted material of Leishmania parasites when using radiolabelled immunoprecipitation technique, suggesting therefore that fraction of LmSIR2 is actively excreted by parasite (personal observations). We then initially surmised that this protein could also be involved in some aspect of host cell-parasite interplay, particularly in pathways that could contribute to cell survival. To test this hypothesis, the leishmania LmSIR2 gene was cloned in a mammalian shuttle vector and the expression of the protein was performed in fibroblasts. Unexpectedly, expression of the Leishmania Sir2 protein leads to a cell growth arrest phenotypes, which correlates, with the expression by the cells of an acidic (pH 6.0) β-galactosidase activity, known to be a senescence biomarker. Furthermore, fibroblasts expressing LmSIR2 were more permissive towards Leishmania infection than control ones. Altogether our results may suggest that the excreted forms of LmSIR2 could participate in the perturbation observed during chronic disease.
Materials and Methods
Molecular construct and transfection procedure
LmSIR2 was amplified using sense GAA TTC GAT ATG ACA GGG TCT CCG and antisense CTC GAG CAG TCA CCA TGT TGG CAG primers. The 1119 bp PCR amplified genomic fragment subcloned into pCR2.1 was digested with EcoRI and inserted into the EcoRI digested and dephosphorylated pcDNA3.1 shuttle vector (Amersham). Restriction analysis of the recombinant plasmids using EcoRI and XhoI allowed the identification of recombinant vector carrying the LmSIR2 gene in sense orientation. Large-scale preparations of pcDNA-LmSIR2 and pcDNA empty vectors were performed using the QIAGEN plasmid maxi kit. Transfected cells were selected in RPMI 1640 medium containing 10% FBS and 400 μg/ml G418. Each G418-resistant clone, isolated by limiting dilution, were propagated and screened by immunofluorescence. Four clones were isolated and named Cl2, Cl9, Cl10 and Cl11.
Production of monoclonal antibodies (mAb) against the fusion protein
LmSIR2 recombinant protein containing 6 X histidine residues at its N-terminal (hisLmSIR2) was previously obtained after having subcloned the encoding gene in the pQE-expression vector . Mice of BALB/c strain were injected intraperitoneally with 50 mg of hisLmSIR2 in saline solution emulsified with 0.1 ml of Freud's Complete Adjuvant. After 2 weeks, a booster injection of the same preparation was given. The antibody response of the animals was tested 1 month later. The same dose of antigen was injected intravenously 3 days before the hybridization experiment.
The fusion procedure and the screening of mAb were carried out as previously described . The class of mAb was determined by agarose double diffusion of supernatants against antisera specific for immunoglobulin subclasses obtained from cliniscience (Montrouge, France). Several hybridoma clones producing mAb reacting in an ELISA test with LmSIR2 protein were obtained; only one mAb of the IgG1 isotype (IIIG4) reacted in Western blot against the fusion as well as the native LmSIR2 protein.
Growth kinetic and 3H-thymidine incorporation
L929 cells were inoculated at 400 cells/cm2, after various period of incubation they were collected by using Trypsin-EDTA (0.025%). The mean number of viable cells was determined at 100-× magnification after staining with trypan blue. In order to evaluate the rate of 3H-thymidine incorporation, cells were seeded in 96 wells plates at 1000 cells/cm2, after various periods of incubation 1 μCi of 3H-thymidine was added to each well. Cells were further incubated for 4 hours before reading the level of incorporated radioactivity.
Giemsa staining and Immunofluorescence analysis
L929 cells were seeded in 16-well Labteck chambers for 48 hrs. Cells were then washed with PBS and fixed for 30 minutes with 4% paraformaldehyde at 4°C. After two washes with PBS, cells were permeabilized with 0.01 M PBS containing 0.5% TritonX-100 and 2% BSA for 30 min at 4°C. Slides were then washed and incubated for 1 hour with mAb IIIG4 in 0.01 M PBS containing 2% BSA. After two washes, slides were incubated for 45 min with fluoresceine-conjugated rabbit anti-mouse IgG (Diagnostic Pasteur, Marnes la Coquette, France) diluted 1:100 in PBS containing Evan's blue (final dilution in PBS: 1: 10.000) and mounted in glycerol PBS (1:1).
Giemsa staining of the cell culture was performed as follows: cells were inoculated at 400 cells/cm2 in 25 cm2 flasks, after 7 days of growth they were fixed with methanol and stained with Giemsa and examined observed on inverted microscope (40× magnification) after adding a solution of PBS 0,01 M-Glycerol (1/1).
Total RNA was isolated from wild type and transformed cells using the Rneasy Mini Kit following the manufacturer's instructions. One microgram of RNA was reverse transcribed to cDNA with an oligonucleotide [poly (dT)12–18] using the Superscript II RNase H-reverse transcriptase. PCR amplifications were performed using 1–2 μl of reverse-transcription of each product and 20 pmol of each primer, using Taq DNA polymerase. Each PCR cycle consisted of a denaturation step (94°C, 1 min), an annealing step (55°C, 1 min) and an elongation step (72°C 1 min). For the last cycle, the elongation step was extended to 10 min at 72°C. Reactions were carried out for 25–35 cycles in a thermocycler (PTC-100™, MJ Research, Inc.). PCR products were analyzed on 1.5% agarose gel and visualized with ethidium bromide. As an internal control, a housekeeping gene, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript, was amplified. LmSIR2 primers were: Sense GAA TTC GAT ATG ACA GGG TCT CCG and antisense CTC GAG CAG TCA CCA TGT TGG CAG. GAPDH primers were: Sense GTC TTC ACC ACC ATG GAG and antisense CCA AAG TTG TCA TGG ATG ACC.
β-galactosidase pH6 analysis
Acidic (pH 6.0) β-galactosidase was assayed according to Dimri et al. . Briefly, stationary-phase cells were washed three times in phosphate buffered saline (0.1 M PBS, pH 7.4), fixed in 2% formaldehyde/0,2% glutaraldehyde for 5 min at room temperature, washed three times with PBS, then incubated at 37°C without CO2 for 12 h with freshly prepared β-Gal staining solution pH 6.0, containing the 5-bromo-4-chloro-3-indoyl-b-D-galactosidase (X-Gal) chromogenic substrate which produces blue 5-bromo-4-chloro-3-indole upon hydrolysis. Cell preparation was stored at 4°C in 70% glycerol upon analysis.
Results and Discussion
Expression of LmSIR2 by L929 fibroblasts
When L929 cell culture, of either clone 1, clone 2, clone 9 or clone 10, were stained with Giemsa, a high rate of cells with unusual shape and size associated with the presence of multiple nuclei was revealed (Figure 3 D) as compared to cells carrying the empty pcDNA vector (Figure 3C). The rate of multinucleated cells was determined by counting the number of cells bearing more than 2 nucleus per 10 champs at 10-× magnification. In fact all the clone cultures present a 10 to 15-fold increase in giant multinucleated cells as compared to cultures of L-929 fibroblast transformed with the empty vector (Figure 3 C). Moreover, majority of the cells if not all, present an enlarged and flattened morphology (Figure 3 D).
Growth kinetic parameters of L929 cell expressing LmSIR2
Expression of senescence biomarker
Permissivity of fibroblast expressing LmSIR2 towards Leishmania amazoensis
Our study demonstrates that LmSIR2 is able to down regulate the proliferation of fibroblasts and to induce a senescence-like state in L929 fibroblast cell line. The most interesting finding is that a parasitic protein is able to modify the physiology of the host cell making them more permissive toward Leishmania infection. These results suggest that LmSIR2 has dual function according to the cell type in which it acts: in Leishmania parasites, LmSIR2 is able to promote cell survival while in the host fibroblast it induces a senescence phenotype. This effect could be beneficial for Leishmania since senescent cells are no longer capable of dividing yet remaining metabolically active. They could thus represent safe target for parasite. It is documented that senescent fibroblasts present an inherent higher capacity to resist to apoptotic death . In this view, it will be of interest to determine if fibroblasts that express LmSIR2 were more resistant to apoptotic stimuli. It is known that infected macrophages are usually more resistant to apoptosis, however it is not know if such mechanism operate in infected fibroblast. Thus, the implication of LmSIR2 in a general mechanism leading to apoptosis resistance of both macrophages and fibroblasts await further investigations. The in vivo relevance of our observation is rather difficult to appreciate because; (1) the level of the protein produced in fibroblasts transformed with the pcDNA is certainly far higher than the level achievable in vivo, and (2) the presence of excreted parasite material inside the fibroblast cytosol has yet not been reported. However, an action of the excreted form of LmSIR2 on he host cell could no be ruled out. Two series of observations support this possibility: (1) it has been shown that the Leishmania EF1-a could be translocated from the parasitophorous vesicle into the macrophage cytosol ; (2) the ultrastructural examinations of skin biopsies obtained from patients with cutaneous leishmaniasis showed that intracellular amastigotes could be frequently identified in vacuoles and in the cytosol of the host cell .
In conclusion, although the effect of the protein, when delivered extracellularly, on the host cells have not being examined in the present study, our data support the notion that the excreted form of LmSIR2 is able to interfere with the host cell physiology, when delivered intracellularly. Unexpectedly the expression of the protein is associated with the appearance of a senescence biomarker. Relevance of such finding during the in vivo infection process awaits further investigations
This work was supported in part by a grant from IRD and INSERM. AMA was supported by a DSF IRD (France) grant. BV was supported by an ANRS (France) grant.
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