Leishmania are a group of protozoan parasites which cause

Transcription

1 DISCUSSION

2 Leishmania are a group of protozoan parasites which cause Leishmaniasis, a disease that is devastating and often fatal to humans. It is transmitted by blood sucking Phlebotomine sandflies. The parasite exists in two forms : as an extracellular flagellate promastigote form in the digestive tract of the vector and as a nonflagellate intracellular amastigote form in the macrophages of the susceptible vertebrate host. The genome of Leishmania is a fertile ground for investigators interested in gene amplification and chromosomal rearrangement. The gene sequences that are amplified seem to play a role in drug resistance and perhaps other responses that are important for the survival of the parasite. In eukaryotes, circular, non mitochondrial extrachromosomal elements have been found in the fungus Saccharomyces (81) and Dictyostelium (82) and the amoeba Naegleria (83). Naturally occurring extrachromosomal DNA elements have been reported in some wild type Leishmania sp~cies examined. These elements are found in these species in two forms, as extrachromosomal DNA and as integrated DNA in a chromosome. The extrachromosomal forms are found to be supercoiled DNA molecules consisting of large \nverted repeats and are homologous to chromosomal sequence. The inverted repeat structures may reflect a common mechanism of excision and circularization to form the amplified DNA's in cells. In Leishmania, DNA amplification occurs after their selection for drug resistance and some times spontaneously in wild type population. Various types of amplifications observed in Leishmania have been discussed in detail in the introduction and the review of literature section. Two types of gene amplifications which occur in the field isolates are the H and LDl gene loci amplification which are not due to intentional selective pressure. All these DNAs are amplified from endogenous chromosomalsequences. H and R DNAs have different source chromosomes from each other and from those of G, D and LD I DNAs that map to the large chromosome. The LD I DNA is amplified in several isolates of Leishmania. It is distinctive since it can occur in at least three 85

3 different genomic organizations. It can be amplified as an independent circular (53) or small linear chromosome or be duplicated by gene conversion. Other rearrangements have been observed but not yet characterized. LD 1 DNA was originally observed as a multicopy 250 Kb linear chromosome in one isolate (36), but was subsequently defined as the 27.2 Kb DNA sequence which occurs as a 54.4 Kb multicopy circular inverted dimeric repeat in L. ilifantum/itmap263. The LD 1 sequence occurs in all Leishmania species, in Crithidia fascicu/ata, Ttypanosoma cruzi and T. lewisi, but is absent or diverged in the African Trypanosomes (50). The entire LDl sequence has been sequenced as a series of genomic clones. Transcript mapping studies identified nine putative mrnas that map to adjacent positions on one LD l strand (53). Analysis of the sequence showed that one of these genes (orfb) encodes a ribosomal protein L37 (56). Another eight likely protein coding genes which are amplified in LSB 7.1 have been identified. One (orfc) encodes a protein homologous to SjhB which is widely conserved and has a role in control of cell growth in E. coli (Personal communication, 1993) and is a cause of virulence in E1winia carotovora. There are epidermal growth factor and ATP/GTP binding motifs in the protein predicted by orfs A and I, respectively, but their function is not known. The protein predicted for orf H is s:mall, basic and may be another ribosomal protein or a nucleic acid binding protein. Otherwise the function of proteins from orf A, H and I are unknown. The orfg protein is very interesting and important since it has potential. membrane spanning domains. It is probable that orfg encodes integral membrane proteins and is most likely being involved in the formation of aqueous channels through the lipid membrane, suggesting a likely role as a transporter. It is related to the ESAG 10 of African trypanosomes whose function is not known (58). The LD1 DNA is amplified in about 20% of L. donovani isolates, from Visceral Leishmaniasis patients, especially in those from India. This 1s an extremely high occurrence of. amplified genes and suggests that gene amplification provides a survival advantage to the parasite. Thus LD l has been identified as important set of genes. 86

4 This thesis deals with the characterization of LD I status in different strains of Leishmania with a special emphasis on the Indian isolates. The relationship between LDl locus expression levels and resistance to drugs has also been worked out. Characterization of selected LD I locus genes, ortf and orfl has been done by making recombinant proteins and raising specific antibodies against these proteins. The anti-ortf antibodies have been used to deterlnine the subcellular localization, developmental regulation of ortf protein and characterizing it in different isolates. A correlationship between gene copy nur11ber, transcript levels and protein expression has been worked out in different isolates. An attempt has also been made to identify the relevant antigen i.e orff by ELISA in this case with patients sera. Results of the ELISA studies show that the recombinant orff protein has a great value as a serodiagnostic antigen in Indian Leishmaniasis. The LD I sequence was found to occur within a ~ 2.2 Mb chromosome in all Leishmania isolates examined. The sequence was amplified, either as a small linear chromosome ( Kb) in strains LSB 52.1, LSB 51.2, LSB 60. I or as a circular chromosome in LSB 7.1, LSB Two Indian isolates of L. donovani, strains UR6 and AG83, showed the presence of LDl on 2.2 Mb chror~wsome only. Another novel form of LD l amplification is reported in strain LSB 51. I. Southern blot analysis of pulse field gel separated chromosomes showed hybridization of orff probe EKI to the 2.2 Mb chromosome and a 1.2 Mb chromosome in LSB 51. I. This duplication of the LD I orff/orfg sequence within the 1.2 Mb chromosome in strain 51.1 also involved orfg sequence (52). This duplication of LDI ortf/orfg sequence in LSB 51. I was reported to involve homologous recombination between short (12-13 bp) regions within the LD I locus on the 2.2 Mb chromosome and the rrna gene locus on the 1.2 Mb chromosome (52). A genomic rearrangement in L. donovani LSB 51.1, resulted in duplication of a 3.9 Kb segment of LDI containing two genes (orff and orfg) and a 1.3 Kb segment (X) into the rrna gene repeat region of the 1.2 Mb chromosome (Plate- 43) (52). This duplication of orff. orfg and X sequence to 1.2 Mb chromosome occurred by replacing one 87

5 or more rrna repeats, apparently at the 3' penultimate rrna repeat. This duplication was a phenomena of gene conversion which was facilitated by homologous recombination (52). A 13 bp sequence upstream of orff is repeated downstream of the putative rrna promoter and a 12 bp sequence at the 3' end of X is repeated 3' of the 28 S rrna gene, which forms the 5' and 3' boundaries respectiv.ely of the duplication. A ::::::I 0 Kb sequence between orfg and X is deleted from the duplicated DNA, agam apparently by homologous recombination between a 13 bp sequence of the 3' of the orfg and same sequence at the 5' boundary of X (52). An Indian field isolate, S-LAL showed occurrence of ortf sequence of LD I on 2.2 Mb chromosome only. Gene amplification is a likely hallmark of genes which are important for drug resistance and hence are advantageous to the parasite for various reasons. In order to determine if there is any correlation between gene amplification observed for LD I locus in different Leishmania! strains and drug resistance, the response of all the Leishmania! strains to Pentostam and Glucantime was worked out.. The measurement of growth with increasing concentration of these drugs sh?wed a range of IC 50 values (Table 3) with a possible correlation with LD I an1plification, particularly against pentostam. The Indian patient's isolate S-LAL was found to be resistant to Glucantime. Though Glucantime resistance does not correlate with LD I gene amplification in S-LAL, however, ortf protein product is found to be three times higher than AG83. In order to further check if any possible correlation exists between drug resistance and LD I amplification, pulse field gel electrophoresis and southern analysis was done using laboratory raised L. donovani lines resistant to Pentostam (P-UR6) and a-dfmo (DR-UR6); whose IC 50 values were 3.2 times and 80 times higher respectively than their wild counterparts. No amplification of LD I was found in these two resistant lines. Additional experiments employing resistance to higher concentrations of Pentosfam may be required to further confirm the correlation bcfwccn LD I amplification and drug resistance. As far as resistance to O'-DFMO is concerned it is known that in Leishmania the 88

6 amplification of the target DNA which is in this case is ornithine decarboxylase is observed (47). So it is not surprising to find that LDI is not amplified in this resistant strain. LD 1 is a 27.2 Kb locus in all leishmania! strai.ns and it amplifies in several strains. The survival advantage of this parasite may be linked to the amplification of LD I. Does LD I amplification contribute to the virulence of the parasite? This was checked by infecting the J774A.l macrophage cell line with different Leishmania! strains in vitro. Significant but slight increase in the infectivity was observed in strains containing amplified LD I or amplifying at least part of it over the strains containing LDI only on 2.2 Mb chromosome. As LDl is a rnultigene sequence and transcribes polycistronically, its amplification must increase the transcript levels of the genes of LDI. This was checked by Northern hybridization with EK I probe (Results Section 5). A 1.1 Kb transcript was found in all strains of Leishmania and the transcript level well correlates with the gene copy number of orff except in LSB 51.1 (Table 3). In LSB 51.1, highest level of ortf transcript was found due to the gene conversion of ortf into a rrna locus of 1.2 Mb chromosome under putative rrna promoter (52). On the other hand, Northern hybridization with ~Kl2 probe (containing orfl only) showed that the orfl transcript level is lower in LSB 51.1 than the other strains which amplified LD 1, because in LSB 51.1, gene conversion does not include orfl (52) under putative rrna promoter. So orfl is present in LSB 51.1 only as a chromosomal copy on the 2.2 Mb chromosome and thus this strain has comparatively less transcripts of orfl than other strains which amplify LDl. The common feature of all LD I gene amplifications is greater expression of orff at the RNA level. Hence, it was of interest to study orff and to characterize it. The work in this thesis is related to the characterization of orff and ortl. To express and characterize orff of LD I locus, it was cloned in bacterial expression vector pet-17b. One splice leader addition site was found 89

7 in the edna clone of ortf which was flanked by two ATG sites. To clone the orff, PCR primers were designed on the shorter open reading frame (Result-7.2.1) and the ortf sequence was amplified from the B-30 probe which contains orfc, D, E, F and part of orfg. The amplified sequence was cloned m pet-17b and the ortf fusion protein was expressed from the positive clone. The induced orff fusion protein was purified and rabbits were immunized to raise antibodies. Western analysis with purified anti-ortf antibody showed orff protein expression in all Leishmania! strains tested. The protein expression pattern also correlates with the amplification pattern of LD 1 in different strains. The protein expression is higher in all the strains which either amplify LDI as a circular or small linear chromosome over AG83 which contains only the 2.2 Mb chromosomal loci of LD I. However, the Indian patient isolate (S-LAL) showed higher expression of ortf protein, though it did not amplify LDI. Highest amount of ortf protein IS Jound in LSB 51.1 which includes ortf in gene conversion into the I.2 Mb chromosome in proximity of a putative rrna promoter. Amplification and gene conversion of LD I from its 2.2 Mb locus correlates with its transcription and translation products, at least, in reference to orff and <Jrfl. The expression level of orff protein varies in two morphological forms of the parasite (promastigotes and amastigotes). The orff protein expresses more in L.donovani (AG83) amastigotes as compared to its promastigote counterpart, but on the other hand L. major promastigotes express more ortf protein than the amastigote form. So differential expression pattern of ortf protein was found in promastigotes and amastigotes of different strains of the parasites. The expression of orff protein also varies along the growth curve of the :. promastigotes. In all the strains, mid-log-phase promastigotes showed highest expression of ortf protein hinting at the probability that it may be a developmentally regulated protein. 90

8 . When the nuclear and cytoplosmic fractions of the promastigotes were separated, the presence of ortf protein was found in nuclear fraction. This result was further supported by the immunofluorescence microscopy analysis where orff protein was found to express in the nucleus of the promastigotes. OrfF protein represents 0.284% 0.084%, 0.121% and 0.061% of the total cellular protein of promastigotes of strains LSB 51.1, LSB 7.I, LSB 52.1 and AG 83 respectively. This result was obtained by comparing the band intensities of native and fusion ortf proteins. Though, these calibrations allow a conservative estimate, amplification of LDI in different strains of Leishmania proportionally increases its gene product (in reference of at least orff) in comparison with its LD I non amplified counterparts. To characterize the orfl of LDI locus, PFGE followed by genomic Southern analysis was done. Orfl amplification correlates with LD I amplification status in all strains tested. Northern analysis with SK12 probe (which contains only the orfl sequence) shows a significant correlationship between LD I amplification and orfl m-rna expression. Strains with no amplification of LD I show less abundance of orfl transcripts than the strains amplifying LDI. LSB 51.1 sh9wed least abundance of orfl transcripts among the strains checked. This is because gene conversion to 1.2 Mb chromosome does not include orfl and thus LSB 51.1 has only2.2 Mb location of orfl. LSB 52.1 which amplifies LDl on small linear chromosome showed highest expression of orfl transcripts among the strains "Checked. In this strain orfl protein product was also found to express higher in comparison to other strains which amplify LDI either on small linear chromosome or as circular amplified molecules and those which do not amplify LD I. This result again hints at the positive correlation between LD I amplification and its transcriptional and translational products. The orfl of LDI was cloned from... psk12 into the bacterial expression vector, prset-a. The polyclonal antisera raised against purified orfl antigen showed strong reactivity with the fusion protein (orfl). When western was done 91

9 on cell lys;ates of different strains of Leishmania, maximum expression of ortl protein was found in LSB 52.1, whereas other strains showed very weak expression of ortl protein. In LSB 52.1, the transcript levels and protein products of ortl are higher than the other strains checked. The diagnosis of VL uptill now has relied chiefly on finding the parasites in the sple:nic or bone marrow aspirates. These surgical procedures are rather traumatic and sometimes dangerous (2). Hence the development of les,s painful means for diagnosis have become all the more urgent especially in view of the recent unexpected outbreak of VL in Asia and Africa (16). The method employed includes ELISA to identify relevant antigens with patients' sera (86,87) and circulating antigens in patients with the aid of monoclonoal antibodies (88) or to detect Leishmania specific antibodies in patient sera with whole cell soluble lysates and purified antigens. Recently, a recombinant antigen (rk39) which is a Kinesin related gene was discovered (89). This gene encodes a protein with a repetitive epitope, consisting of 39 aminoacid: reisdues and is conserved largely in Leishmania species that cause YL. Exami'nation of sera from the VL patients by ELISA using this recombinant K39 (rk39) antigen revealed a seropositive rate of nearly 100% regardless of the patients' origin from Brazil or from Sudan. In contrast there was virtually no qetectable anti-rk39 in patients with South American Mucocutaneous or Cutaneous Leishmaniasis. Immunodiagnostic potential of ortf recombinant antigen was worked out in order to throw light on the possible funtions of this recombinant antigen. The orff recombinant protein reacted with YL patients sera collected from Sudan. The normal human sera did not react to ortf fusion protein on western blot. The titration curve was obtained by ELISA with varying amounts of soluble antigen and ortf fusion protein with constant amount of sera (1: 10 diluted) from a parasitologicaiiy confirmed Visceral Leishmaniasis patient. The ortf fusion protein was observed to be II times more sensitive than the soluble antigen at an OD level of 1.0 (Piate-28). Thus, I 0 ng of ortf fusion protein was needed instead of II 0 ng of soluble antigen to produce an OD of

10 Sera collected from clinically confirmed Visceral Leishmaniasis patients were checked by OAT (reciprocal titer 500) and 90% of them were DAT positive4 When the same samples were checked for orff positivity, the ELISA results were comparable to DAT results. In this case ELISA was done on 5 ng of orff fusion protein with 1:20 diluted sera. DAT showed very high degree of specificity and sensitivity towards kala-azar. More than 97% of parasitologically confirmed Kala-azar sera was considered to be seropositive by DAT on the basis of ;::: 800 reciprocal titer (77). DAT result and orff positive results were comparable in the samples studied and thus orff fusion protein may be used as a marker for serodiagnostic study of Visceral Leishmaniasis or Kala-azar. The serodiagnostic data with orff against VL serum samples collected from the endemic area shows that the infants are more susceptible ( ~ 60%) and also that males are more affected (55%) than the females (Table II), which supports the previous work (84,85). When ELISA was done on 500 ng of SA with J: 100 diluted sera, the SA positive resutt was not consistent with DAT result. Guimaraes et a/ (198 J) (78) and Harith eta/ (1987) (19) reported discrepancies in results with ELISA when done with crude antigens or soluble antigens of Leishmania. On the other hand J 0 confirmed Cutaneous Leishmaniasis sera did not produce positive response in ELISA when done o~_ 5 ng of orff fusion protein (Plate-3I) as compared to SA. So orff fusion protein was showing DAT compatible result in case of Visceral Leishmaniasis sera, but CL sera produced discrepancies. Additional work is necessary to fully evaluate the immunodiagnostic potentials of.this antigen for Visceral Leishmaniasis. Further studies are necessary to correlate the LD I status in the field isolates with the drug resistance pattern. A positive correlation will suggest that LD I gene products contribute to resistance and will help in the development of novel diagnostics for the detection of drug resistance in Leishmaniasis. 93

11 Plate- 43: Physical maps of the LDl locus on the 2.2 Mb chromosome, the rrna gene locus, and FGx duplication on the 1.2-Mb chromosome. LDl genes (orfa-orfl), as well as the 18S and 28S rrna genes are boxed, and the duplicated LDI. ~anking sequence (x) is indicated by the thick box. The thick line within the genomic map indicates the 27.2 Kb LDI sequence. Flags denote the inferred locations of rrna Pol I promoters, and verticla bars indicate the region of 64-bp repeats in the rrna gene external spacers. Triangles (numbered 1 to 9) indicate the locations of short sequence homologies which are involved in the recombination event(s) denoted by vertical lines between maps. The arrows indicate the putative sequence of events resulting in the duplication of the 15 Kb LDI sequence into the rrna gene locus and subsequent (or concomitant) deletion of a 10 Kb segment of this d:uplication. Restriction enzyme abbreviations: B, BamHI; E, EcoRHI; N, Norl (Reporduced from Molecular and Cellular Biology vol-15, Page ).

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