Background
Leishmaniases are among the most important protozoan infections that affect humans in the world. The disease is widespread in 88 endemic countries with 350 million people at risk, 12 million people permanently affected, and an estimated annual incidence of 1.5–2 million cases [
2]. This results in a global morbidity of 2,357 thousands DALYs (Disability Adjusted Life Years: number of healthy years of life lost due to premature death and disability) and a mortality rate of 59,000/year [
3].
A surprisingly broad spectrum of clinical expressions is observed in humans ranging from asymptomatic to cutaneous (CL), diffuse cutaneous, mucocutaneous and visceral (VL) diseases, and an intermediary form known as post-Kala-Azar dermal leishmaniasis.
The various clinical issues of
Leishmania infection depend on a complex host-parasite relationships where both the genetic or immunological status of the host [
4‐
6] and the proper parasite biodiversity in terms of tropism and virulence [
7,
8] appear as determinant factors. A number of parasitic factors have been identified as susceptible to play a role in virulence/protection mechanisms in leishmaniases [
9]. Among these, since its first identification in
Leishmania infantum [
10] several lines of evidence indicate that the A2 gene/protein family could be one of the most eligible candidate factor of virulence in VL infections: i) A2-proteins and mRNA transcripts are developementally expressed at the amastigote intracellular stage while undetectable in the promastigote [
11], ii) Inhibition of A2-expression in
Leishmania donovani using anti-sense RNA or by generation of partial knock-out mutants results in reduced virulence in vivo, iii) by contrast, increased parasite levels are observed in spleen of mice infected with A2-expressing transfected
L. major [
11,
12] iv) A protective immunity can be achieved experimentally in mice by immunization with recombinant A2 protein or DNA vaccination showing that A2 from
L. donovani is highly immunogenic and represents a potential antigen for protection in VL [
13,
14] and more recently in
L. amazomensis infections [
15].
A2 genes were detected by karyotype analysis in
L. donovani,
L. infantum and L. chagasi (Old World and New World VL) and in
L. mexicana and L. amazonensis (New World DCL and MCL, respectively) but not in cutaneous species from the Old World (
L. tropica, L. aethiopica and
L. major) and the New World (
L. brasiliensis, L. guyanensis and
L. panamensis) [
16]. Accordingly, A2-antibodies were found in sera from human and dogs naturally infected with
L. chagasi (VL) [
17] and in patients with VL in Sudan and India and CL due to
L. mexicana, while they were not detected in
L. tropica and
L. brasiliensis infections (CL) [
16].
While long considered absent in the
L. tropica group, we identified by sequencing an A2 gene from crude PCR products of two strains of
L. major (
AF532102,
AF532103) showing that the gene is present in
L. major in a truncated form lacking most of the repeated motives that are present at the 3'end variable region of the VL A2 genes. Moreover, the
L. major A2 gene was found subsequently to be non expressed and was considered to represent a pseudogene [
1].
These observations raised the question of a possible role of the structure/functionality of A2 genes in the cutaneous or visceral tropism of leishmania parasites. As no data were available on the A2 gene of CL Leishmania except for L. major, our objective was to investigate this gene in Old World CL species. We amplified and sequenced A2-genes of additional strains of Old World CL species and in a clonal lineage of a L. infantum mediterranean strain isolated from a VL patient. Our results show that: i) The A2 sequence is extremely conserved both among strains and species of Old World CL Leishmania, ii) The CL A2 gene is a single copy gene of only 153 base pairs (bp) encoding for a protein of 51 amino acids, as opposed to A2 of VL species that are multicopy genes of varying length, ii) The CL A2 gene is functionally transcribed at the promastigote and amastigote stages.
Methods
Parasites
Strains. Six strains of one visceral and four cutaneous Old World
Leishmania species were used for sequencing:
L. infantum and
L. major, L. tropica, L. killicki and
L. aethiopica, respectively (Table
I). Four of the five cutaneous strains were reference strains recommended by the W.H.O. [
18]. In addition two
L. donovani VL strains LEM3467 and LEM3566 were used for PCR analysis. All strains originated from the International
Leishmania Cryobank and Identification Center, Montpellier, France.
Visceral (LV) |
L. infantum
| MHOM/FR/92/ LEM2385 Cl 1 | MON-29 | II | AY255807 | | AAP21103 |
| | | | III | AY255808 | | AAP21104 |
| | | | IV | AY255809 | | AAP21105 |
Cutaneous |
L. major
| IPAP/MA/86/ LEM898 | MON-25 | I.1 | AF5321022
| AY25581 | AAM95954 |
| | MHOM/SU/73/ 5 ASKH1
| MON-4 | I.2 | AY185122 | | AAP21106 |
| | | | | | | AAO27297 |
(LC) |
L. aethiopica
| MHOM/ET/72/ L1001
| MON-14 | I.1 | AY255804 | | AAP21100 |
|
L. killicki
| MHOM/TN/86/ LEM904- CL1
| MON-8 | I.1 | AY255805 | | AAP21101 |
|
L. tropica
| MHOM/SU/74/ K271
| MON-60 | I.1 | AY255806 | | AAP21102 |
Parasite clones. Parasite clonal lineages were obtained from
L. infantum MHOM/FR/92/LEM2385 and from
L. major MHOM/SU/73/5 ASKH strains using a microplate technique as previously described [
19].
Culture and isolation. Promastigotes were cultivated at 27°C in HOSMEM liquid medium [
20] supplemented with hemin 10 μM (Sigma, Saint Quentin Fallavier, France) and 10% fetal calf serum (Gibco, Cergy-Pontoise, France). Parasites were inoculated into 25 ml culture flasks at day 0 (d0) at a final concentration of 10
5 ml
-1. Amastigote organisms were isolated from foot-pad (
L. major) or spleen (
L. infantum) of Balb/c mice inoculated subcutaneously or intraveinously with 10
7 log-phase promastigotes, respectively. Parasites were washed twice in PBS and counted in Malassez chambers.
Washed parasites (100 μl PBS / ≈109 parasites) were lysed by thermal shock in Eppendorf tubes, 1 mn in boiling water – 1 mn in melting ice, three times. DNA extraction was performed using classical phenol/chloroform/isoamylic alcool protocol and precipitation was made using NaCl/ethanol procedure. The DNA was dissolved in 40 μl of sterile water.
PCR and sequencing
Amplification of the parasite DNA matrix (50 ng) was made using L2/R3 primers (5'-TTGGCAATGCGAGCGTCACAGTC / 5'- CAACGCGTACGATAATGCCACA). The L2/R3 primers correspond to the 5' end position 16301 and 16603 of the inverse-complementary strand of the AC010851 sequence, respectively. The PCR was performed in a reaction mixture of 50 μl containing either 1 or 3 mM MgCl2, 200 μM each dNTP, 25 pmol of each primer (Proligo, Paris, France), 1 U Taq polymerase (Eurogentec, Seraing, Belgium). L2/R3-PCR conditions consisted to denaturation for 3 mn at 94°C, followed by 35 amplification cycles at 94°C for 1 mn, 1 mn at 58°C, 1 mn at 72°C, then one cycle at 72°C for 5 mn. Amplification of cDNA from bacterial culture medium (0.5 μl) was made using M13 forward-20 / M13 reverse (Qiagen PCR cloning kit, Qiagen, Courtaboeuf, France) with 1 mM MgCl2. M13 forward-20 / M13 reverse -PCR conditions consisted in a hot-start denaturation for 10 mn 95°C, followed by addition of 1 U Taq polymerase, 30 amplification cycles at 94°C for 30 sec, 30 sec at 48°C, 1 mn at 72°C, then one cycle at 72°C for 5 mn. Five microliters of PCR product was electrophoresed in 2% agarose gel in the presence of ethidium bromide, and visualized under UV light. A 50-bp ladder (Sigma) was used as MW marker.
For sequencing, the two strands of PCR-amplified DNA were purified with QIAquick PCR Purification Kit (Qiagen) and sequenced with the corresponding PCR primer set using the BigDye Terminator Sequencing Kit V3.1 (Applied Biosystems, Courtaboeuf, France) on an automated sequencer 3100 Genetic analyser (Applied Biosystems).
RNA extraction and reverse transcription-PCR (RT-PCR)
L. major IPAP/MA/86/LEM898 total RNA was extracted from promastigote cultures or infected organs (≈106-107 parasites) using Rneasy Plant Mini Kit (Qiagen). To eliminate any remaining DNA the RNA extract mixture (5 μl) was additionnaly treated by Dnase Rnase-free (Eurogentec) for 30 min at 37°C in a final volume of 30 μl. As a negative control, an aliquote of the sample (5 μl) was subsequently digested with Rnase A (Qiagen) 700 mg 1 h at 25°C in a final volume of 16 μl. RT was performed in a total volume of 20 μl by 50-min incubation at 42°C followed by 15 min at 70°C to inactivate the reverse transcriptase. The reaction mixture contained a sixth of the initial volume of the RNA extraction products, and the following final reagent concentrations: 1X hexanucleotide mix (Roche, Meylan France), 500 μM dNTP mix, 40U Rnase inhibitor, 1X first strand buffer, 100 mM dithiothreitol, and 200 U Super Script II (Invitrogen, Cergy Pontoise, France). Two microliters of RT products were PCR-amplified with L2/R3 primer set.
DNA libraries
L2/R3-PCR products from genomic DNA matrix were synthetized as described above except for the MgCl2 concentration (3 mM). PCR products were purified on QIAquick column (Qiagen). Poly-A treatment, insertion of the PCR products into pDrive vector, transformation of E. coli EZ competent cells (Qiagen) and cloning were performed using a PCR Cloning Kit (Qiagen) as described by the supplier. A2-containing genomic clones were screened by digestion of M13-PCR products with Sau3AI endonuclease (BioLabs, Saint Quentin en Yvelines, France) which cuts off A2-gene nucleotidic sequences at position 33–34 (reference : L. major MHOM/IR/-/173; AF532103).
GeneBank accession numbers
Accession numbers for genomic DNA, mRNA and putative protein sequences are given in Table
I.
Discussion
Leishmania A2-genes were first identified in two strains of the
L. donovani complex,
L. infantum LV9 and
L. donovani 1S2D determining visceral infections [
10,
21]. In these VL strains A2 genes were shown to be organized in several clusters each comprising multiple A2 genes of varying length that are tandemly associated with related sequences (A2rel) [
10,
11,
22]. However, these results were obtained from leishmania strains isolated from naturally infected hosts which are known to be most likely composed of multiple parasite populations [
23,
24]. Therefore, the existence of multiple A2 genes remained to be confirmed using a genetically pure parasite clonal lineage. As in the present study three different A2-alleles type II, III and IV were sequenced from the
L. infantum MHOM/FR/92/LEM2385 Clone-1 genomic library, our results provide additional evidence that A2 of VL species is a multiple gene family. Alleles type I, II and III differ only in the number and arangement of the repeated motives at the 3'end variable region of the gene as previously described in VL strains [
10]. However, in the present study we identified A2 sequences showing a limited number of repeats and consequently a length of only 371 to 464 bp contrasting with the previously published A2 genes of about 700–800 bp. The inability to evidence A2 sequences > 1 kb in the present study is most probably due to the limits of the PCR technique performed on crude genomic parasite DNA or to the absence of long A2 sequences in this strain. Thus these results are not contradictory to the previously published data but bring additional information on the variability of the
L. infantum A2 genes.
We previously identified A2 sequences in two strains of
L. major IPAP/MA/86/LEM898 and MHOM/IR/00/173 (
AF532102 and
AF532103, respectively). These sequences were 95% (88 nt/93) identical to the S69693 stage-specific S antigen homolog (A2) of
L. infantum VL [
10] at the 5'-end of the ORF (nucleotides 74 to 167). By contrast, a major deletion of the 3'end variable region of repeated nucleotide motives was observed in
L. major A2 genes.
In the present study, the amplification of Old World Leishmania genomic DNA with L2/R3 A2-gene primer set resolved in a single amplification product on gel electrophoresis, as opposed to the complex pattern observed with L. infantum. Accordingly, a single sequence of 258 nucleotides was obtained by direct sequencing crude DNA products from all CL Leishmania, whatever the strain or species. A common sequence was also isolated from 32 genomic clones of L. major LEM898 and 15 clones of L. tropica, L. aethiopica and L. killicki. The A2 sequence shared by all strains and species of Old World leishmania presented a single polymorphism C/G at position 58 of the ORF. These results show for the first time that the A2 gene of Old World cutaneous Leishmania is unique and highly conserved, contrasting strongly with the multiple A2 sequences of varying length observed in VL isolates.
RT-PCR on mRNA extracts from strain IPAP/MA/86/LEM898 followed by sequencing evidenced that the CL A2 gene is functional. It is noteworthy that this finding does not presume of the expression of the protein at the post-transcriptional level, however it contrasts with the previous suggestion based on the failure to demonstrate A2 gene transcripts in
L. major that the A2-gene of
L. major is a non-expressed pseudogene [
1]. RT-PCR amplification of both promastigote and amastigote mRNAs resulted in a similar signal on gel electrophoresis in the present study, showing that
L. major A2-gene is transcribed at both amastigote and promastigote stages. However, these results do not signify that
L. major A2 are not developmentally expressed since RT-PCR is not quantitative. Actually,
L. infantum promastigotes were reported to express very low levels of A2 [
10,
22].
Acknowledgements
We thank H. Bui, Jean Dausset Foundation, Centre d'Etude du Polymorphisme Humain (C.E.P.H.), Paris, France, for the sequencing.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
YJFG conceived and conducted the study and drafted the manuscript. PM carried out the work at the technical level. FP and JPD supplied and characterized the parasite strains. FD is the Chief Manager of the Laboratory and revised the manuscript and FL directed the biomolecular and bioinformatic analyses and participated to the writing of the manuscript.