healthmedicinet

Kinetoplastid genome phylogeny

We compared the genomes of Leishmania species to Leptomonas in order to obtained information on kinetoplastid phylogeny (Fig. 2). Crithidia was taken as an outgroup. We observed that the L. donovani isolate from India was placed at the crown in comparison to Leptomonas.

Fig. 2. Kinetoplastid genome phylogeny. A cladogram depicting kinetoplastid phylogenetic relationships
among genome of Crithidia fasciculate, Leishmania aethiopica, Leishmania amazonensis, Leishmania arabica
, Leishmania braziliensis, Leishmania enriettii, Leishmania gerbilli, Leishmania donovani,
Leishmania major, Leishmania Mexicana, L. sp. MAR LEM2494, Leishmania tarentolae,
Leishmania tropica , Leishmania turanica and Leptomonas seymouri

Leishmania donovani and Leptomonas farnesyltransferase

In Leishmania donovani, ?-subunit of farnesyltransferase (LdFTase alpha) was located on chromosome 29 (LdBPK_292070)
with accession number XP_003862625.1. In Leptomonas ?-subunit of FTase (Leptomonas FTase alpha) was identified from the contig (contig_2652) of Leptomonas genome. Using ClustalW, percent identity of the ?-subunit of LdFTase and Leptomonas FTase with L. major (XP_003722277.1) and L. infantum (XP_001466722.1) was summarized in Fig. 3a and it can be seen that the region of sequence identity was found extensively at
the C terminus (Additional file 1: Figure S1).

Fig. 3. a Percent identity of the ? and ?-subunit of FTase enzyme in Leishmania species and its sister Leptomonas.b Percent identity of the ? and ?-subunit of GGTase-II enzyme in Leishmania species and its sister Leptomonas

?-subunit of FTase in Leishmania donovani (LdFTase beta) was located on chromosome 26 (LdBPK_261450) with accession number
XP_003861732.1. In Leptomonas, ?-subunit of FTase (Leptomonas FTase beta) was identified from the contig (contig_1135) of Leptomonas genome. Using ClustalW, percent identity of the ?-subunit of LdFTase and Leptomonas FTase with L. major (XP_001684151.1) and L. infantum (XP_001470492.1) was summarized in Fig. 3a. In comparison with the ?-subunit of FTase among L. major, L. infantum and L. donovani the ?-subunit showed more sequence identity (Additional file 1: Figure S2). It is also reported that the ?-subunit of prenyltransferases are more
conserved than ?-subunit 11]. The ?-subunit appears to have invariable domain architecture throughout evolution
and its invariable nature is due to functional rather than structural constraints.

Leishmania donovani and Leptomonas geranylgeranyltransferase

Using ClustalW, percent identity of the ?-subunit of LdGGTase-II (XM_001468149) with
L. major (XM_001685808) and L. infantum (XP_001468186) was summarized in Fig. 3b. ?-subunit of this enzyme was totally conserved between the two visceral species
L. donovani and L. infantum but quite variable between the cutaneous L. major and visceral species (Additional file 1: Figure S3). Non conservative amino acids alanine and serine at positions 82, 88,
214, 440 and 235, of the visceral species were replaced by valine and threonine in
the cutaneous species respectively (Additional file 1: Figure S3). It has been reported by Peterson et al. that parasites harboring a pair
of point mutations from Ala-16 to Val-16 and from Ser-108 to Thr-108 are resistant
to cycloguanil 12]. A similar substitution in ?-subunit of GGTase-II could be responsible for its survival
and multiplication within the host cell. No sequence identity was observed to ?-subunit
of GGTase-II in Leptomonas genome with L. major (XM_001685808), L. infantum (XP_001468186) and LdGGTase-II (XM_001468149).

In our study, the ?-subunit of GGTase-II of L. donovani was totally conserved in both the Leishmania species responsible for the visceral manifestation of the disease viz. L. infantum (XP_001468743) and L. donovani (XP_003864545). The carboxy terminal of this enzyme was completely conserved between
the visceral (L. infantum, XP_001468743) and cutaneous species (L. major, XP_001686510) of Leishmania (Additional file 1: Figure S4) however in the N-terminal, differences in the cutaneous species were
observed. Non conservative amino acids aspartate and glycine at positions 57 and 61
of the visceral species were substituted in the cutaneous species (Additional file
1: Figure S4). The susceptibility to infection by Leishmania NRAMP1 gene has been similarly reported 13] to be substitution in non conservative amino acid aspartate and glycine. A similar
substitution in ?-subunit of GGTase-II maybe due to the different requirement for
mode of protein trafficking.

Percent identity of the ?-subunit of GGTase-II in Leptomonas to Old World L. major (XP_001686510) and L. infantum (XP_001468743) is shown (Figs. 3b and Fig. 4). New World, L. panamensis (XP_010698581) and L. braziliensis (XP_001564646), showed 81 % identity with Leptomonas whereas in L. donovani identity with only Old World was observed.

Fig. 4. Multiple sequence alignment of GGTase-II (?-subunit) of Leishmania donovani (accession number XP_003864545) with its sister Leptomonas (contig_5507) showing conserved regions (red colored) based on percentage identity

The evolutionary relationship between FTase and GGTase-II of Leishmania and Leptomonas was reconstructed by using Neighbor-Joining method (Fig. 5). In our study, ?-subunit of FTase between Leishmania and Leptomonas were in close phylogeny. But as regards to the holoenzyme, ?-subunit was placed very
differently to the ?-subunit of these enzymes. We concluded that ?-subunit of GGTase-II
is almost similar to the corresponding ?-subunit of FTase in both kinetoplastids.
?-subunit of GGTase-II is very similar to the corresponding ?-subunit of FTase in
both kinetoplastids.

Fig. 5. We reconstructed the evolutionary relationships between FTase and GGTase-II of Leishmania and Leptomonas by using Neighbor-Joining method

Rab sequence mining in L. major and L. infantum

Among the prenylation proteins, Rabs are small GTP-binding proteins which are the
largest family within the Ras superfamily. These act as membrane associated molecular
switches and regulate vesicular trafficking 14]–16]. The number of Rab proteins and gene complexity in an organism has been correlated
with multicellularity of organism. A multicellular organism will require/have higher
number of Rabs encoded by its genome 17]. Plasmodium falciparum has been shown to encode 11 Rabs, Toxoplasma gondii encodes 15 Rabs, Leishmania encodes 12 Rabs, Drosophila melanogaster encodes 29 Rabs, and Homo sapiens encodes??60 Rab proteins, which correlates with their multicelluarity and organism
structural complexity.

Until now, despite the importance of the intracellular vesicle transport in Leishmania pathogenicity, very few publications are available regarding number, diversity, complexity
and functions of Leishmania Rab gene family 18]–20]. Therefore, it was of interest for us to carry out the phylogenetic profiling of
the Rab repertoire in the lower eukaryote Leishmania. The exhaustive mining of Leishmania Rab proteins in GeneDB (www.genedb.org) and NCBI (http://www.ncbi.nlm.nih.gov/) database revealed many full length proteins, some were annotated as Rabs of various
families, but most were annotated as putative Rabs or Rab like proteins, in this aspect
the L. major Rabs are better annotated compared to L. infantum Rabs. Our search retrieved 14 completely annotated Rabs in L. major whereas 18 Rabs are reported in L. major by Berriman et al. 21]. Similarly in L. infantum, we found 13 proteins annotated as Rabs and few other proteins annotated as putative
Rab or Rab like proteins. The details of Rabs of both L. infantum and L. major, along with name, accession number, gene ID and chromosomal location in Additional
file 2: Table S1.

Rab sequence mining in Leishmania and Leptomonas

From our whole genome sequence of L. donovani and Leptomonas some Rabs were annotated as Rabs of various families, but most were annotated as
putative Rabs or Rab like proteins. We retrieved 13 completely annotated Rabs in L. donovani (Additional file 1: Figure S5). Similarly in Leptomonas, we found 11 proteins annotated as Rabs (Additional file 1: Figure S6). The details of Rabs of both L. donovani and Leptomonas are presented in Additional file 2: Table S2 and S3 respectively. A pair wise comparison of the Rab proteins retrieved
from the L. major (Fig. 6a) and L. infantum (Fig. 6b), L. donovani (Additional file 2: Table S4A) and Leptomonas (Additional file 2: Table S4B) were calculated and phylogenetic tree was constructed using the Rabs
orthologues sequences of L. major, L. infantum, L. donovani and Leptomonas (Additional file 1: Figure S7).

Fig. 6. Heat-map depicting percent identity between aL. major and bL. infantum

To visualize the synteny in Rab genes between Leishmania donovani and Leptomonas, we constructed a Circos plot linking the homologous Rab genes (Additional file 1: Figure S8) The Rab genes of Leptomonas are represented on the left side and Rab genes of Leishmania donovani are represented on the right side. Rab4, Rab7 GTP and Rab18 showed extensive synteny
in both the species. Rab1 and Rab7 of Leishmania donovani have no similarity with any genes in Leptomonas. Whereas few genes in Leishmania donovani are partially similar such as Rab28, Rab14, Rab11B, Rab11, Rab6, Rab5, Rab2A and
GTP binding Rab1 to Leptomonas.

Thirteen annotated Rabs of L. major and L. infantum were exactly the same as in L. donovani, only Rab 21 was present in L. major but was not identified in either of the visceral Leishmania. What does Rab 21 do? The explanation for this has been addressed in the Phylogeny
section. In Leptomonas 11 Rabs could be annotated by us, all of them same as found in Leishmania.

Phylogenetic analysis of Rab GTPase

An important role in evolution is played by small GTPases belonging to the Ras superfamily.
Rab proteins form the largest branch of the Ras superfamily. This family is much diversified
8]. Each Rab protein has a distinct subcellular location and is responsible for a specific
transport step 22], 23]. Rab proteins are key regulators of intracellular vesicular transport and membrane
trafficking in exocytic and endocytic pathways 14]–16]. The evolution of Rab proteins has been analyzed extensively in Trypanosomes 24] but not in Leishmania. We believe that molecular phylogenetic analysis of Rab proteins would provide a
unique opportunity for finding the relationship between evolution of Leishmania and Leptomonas. Leishmania is believed to have evolved from Leptomonas where life cycle changed from monogenetic to digenetic 1]. Our study is the first extensive data mining and phylogenetic construction of clinical
isolates of Leishmania and Leptomonas which is based on annotation of Rabs obtained from our whole genome sequencing 1].

The orthologues which correspond to genes separated by divergence of the Leishmania Rab superfamily in comparison with Leptomonas is shown in Fig. 7a. We found that the core Rab repertoire associated with basic endocytosis and exocytosis
is fully conserved 25], the endomembrane system appears to be very ancient and maintained same for Leishmania and Leptomonas, whilst gene duplication has facilitated the building of additional, lineage-specific
complexity 24].

Fig. 7. a Phylogenetic relationship between Rab sequences of Leishmania and Leptomonas which is based on multiple sequence alignment (ClustalW algorithm in MEGALIGN with
1000 bootstraps). A dotted line on a phenogram indicates a negative branch length,
a common result of averaging and Seed 111 indicates “Random Seed: This option makes
bootstrapping consistent with the Clustal interface. The random seed is the value
that initializes the pseudo-random number generator. Choosing the same seed causes
the generator to produce exactly the same sequence of numbers for a given set of distances
and hence the same result. Choosing a different seed value produces an alternative
sequence of numbers which should introduce some variation in the result, but it should
be similar. The highly conserved values show which parts of the tree should be given
greater confidence”. PrePS analysis (PrePS- Prenylation Prediction Suite, http://mendel.imp.ac.at/sat/PrePS/index.html) between Rab 2A and Rab11B of Leishmania showed that b Rab11B lacks the cysteine residue at C terminus of CAAX signal sequences which is
essential for prenylation. c Rab 2A has cysteine residue at C terminus of CAAX signal sequences

Out of 20 Rabs reported to be present in the last eukaryotic common ancestor (LECA)
26], five Rabs are said to be present in any well characterized genome. The core set
includes a conserved mechanism for ER to Golgi transport (Rab1 and Rab2), trafficking
through the early endosome (Rab5), recycling (Rab4 and Rab11), delivery to the late
endosome/lysosome (Rab7) and retrograde transport through the Golgi complex (Rab6)
24]. Inspection of the tree in Fig. 7a reveals that major groups of Rab proteins in the Leishmania genome can be broadly related to Leptomonas Rabs by homology thus indicating similar function and/or subcellular localization.
This co-segregation of Rab GTPases according to common functions rather than to taxonomic
relationships indicates a conserved mechanism of Rab interaction with regulators/effectors
across evolution 23]. Orthologues of Rabs in Leishmania and Leptomonas e.g. Rab2, Rab4, Rab5, Rab6, Rab7, Rab18 and Rab28 co-segregated with a strong bootstrap
support. This observation suggests that strict phylogeny of function in the Rab superfamily
has been maintained and steps required for the basic vesicular transport were almost
same in both these kinetoplastids. These core set of Rabs also appear to be orthologues
of mammalian Rabs 17]. Remarkably high level sequence conservation is retained between these two kinetoplastids
and higher eukaryote orthologues. Parasites (T. bruci, T. curci and L. major) have?~?20 Rabs gene and most of which are clearly orthologues with metazoan Rabs
21]. Rab28 is ancient Rab protein that arose before the speciation event separating trypanosomes
from the mammalian lineage 24] and is maintained in the kinetoplastid Leishmania and its sister species Leptomonas. Rab8 is lost in kinetoplastids 26].

Non-essentiality of the different Rab isoforms was observed in both the kinetoplastid
genomes but which is observed to be a major contribution in the Rab repertoire of
other organisms. We found isoform of Rab2 (2A) and its orthologues with Leptomonas in the crown position of phylogenetic tree. Isoform of Rab11 (11B) in Leishmania and Leptomonas takes basal position in the tree. PrePS analysis (PrePS- Prenylation Prediction Suite,
http://mendel.imp.ac.at/sat/PrePS/index.html) of these protein showed that Rab11B lacks the cysteine residue at C terminus of
CAAX signal sequences which is essential for prenylation (Fig. 7b). On blastp analysis with the well annotated human Rab family, this Rab11B of L. donovani, showed 58 % identity with Rab like protein 2A isoform X7 (XP_006712281.1). Therefore
we determined that the Rab11B as annotated in Leishmania and Leptomonas genome is actually not Rab11B but an ancient isoform of Rab2A. Maybe this protein
has conserved GTPase activity and as it evolved to Rab2A (crown position) it acquired
specific prenylation activity (Fig. 7c). Alternatively spliced transcript variants encoding different isoforms have been
reported. Evolutionary distance of the lineage requires experimental evidence to further
support these sequence data. It has been established by Diniz et al. 27]: that Rab2 and Rab11B (which we now identify to be isoform of Rab2A) are actin interacting
proteins (AIP) in L. infantum and L. major.

Rab5 is an early endosome marker as established by Real et al. 28], 29]: in Leishmania major and Leishmania amazonensis. From our study we found only Rab5 annotation in the genomes of both the kinetoplatids.
In L. donovani Rab5 has been shown to be is involved in hemoglobin endocytosis 20]. Two isoforms of Rab5 (Rab5A and Rab5B) were reported by them. The strain used by
Singh et al. 20]: was UR6 which is a very long term laboratory cultivated avirulent strain and has
been established to be L. tropica30] whereas our study of Rab annotations is based on whole genome sequences of recently
cultivated clinical isolates of L. donovani and Leptomonas. On blastp analysis of the Rab5 of both L. donovani and Leptomonas with human (taxid 9606, accession number NP_002859.1) showed 45 % and 50 % identity
to isoform 5B respectively and Rab5b may act as a marker for early endosomes in L. donovani31]. No isoform A in the genome sequence of these two kinetoplastids was present thereby
indicating only Rab5 orthologues.

Rab1 GTPase and Rab7 GTPase are actually true Rab1 and Rab7 respectively on the basis
of bastp analysis of human Rab family and these were identified as orthologues in
both L. donovani and Leptomonas. Rab7 and Rab9 are two related subfamilies, which show overlapping localization to
late endosomes 23]. Rab 9 was absent in the kinetoplastid genomes. Rab9 protein has not yet been reported
in unicellular eukaryotes, plants and fungi 23]. Rab7 subfamily belongs to the ancestral set of Rab and arose before the radiation
of eukaryotes. Rab7b isoforms are found only in representatives of amphibians, birds
and mammals 23]. The Rab1 and Rab7 identified only in Leishmania were branched together which suggests paralogous gene duplications. This is a major
force in evolution which provides new gene function. Retroposition or lack of introns,
as observed in kinetoplastids, is a major contributor for these phenomena. However
on blastp analysis with human Rab family, these showed identity 43 and 37 % to Rab
13 and Rab 43 respectively.

RAB11 was found to be a useful marker to track flagellar pocket division and to follow
mitochondrial DNA (kinetoplast) segregation. Leishmania major antibody raised against T. brucei RAB11 which also cross-reacts with the L. major homologous 32]. Rab14, appears to regulate endo- lysosomal fusion in Dictyostelium33] primarily found on the contractile vacuole network of membranes, an organelle important
in osmotic regulation and, in lesser amounts, in the endo-lysosomal pathway 34].

Rabs in intracellular parasites like Leishmania are associated with pathogenesis, as vesicle transport is essential for extracellular
nutrient acquisition, release of virulence factors, microbicidal resistance and evasion
of host immune responses 19], these requirements being different in the monogenetic ancestor Leptomonas hence the difference in phylogeny between Leishmania and Leptomonas for some Rabs like Rab 11 and Rab 14.

In our study Rab 4 and 14 in Leishmania are paralogues and possibly associated with endosomal recycling. Rabs sharing similar
functions and/or locations are more closely related than Rab proteins with distinct
functions 35]. Rab3 and Rab27, two examples of tissue-restricted Rabs 22] with specialized functions are rightly not detected in our genomes. Rabs are involved
in resistance to chemotherapeutic drugs 36]. Rab4 interacts with P-gp, (P-glycoprotein), a large transmembrane protein localized
at the plasma membrane that extrudes anticancer drugs and thus decreases their concentration,
and toxicity inside the cells 37]. Rab8 has also been reported to be involved in resistance 38].

Flow cytometry experiments showed that sensitivity to doxorubicin was associated with
increased drug accumulation in cells expressing the RAB6A variant. WTH3 gene’s product
is homologous to the Rab6 and Rab6c genes 38]. WTH3 is a house-keeping gene and its product is capable of binding to GTP molecules.
WTH3 37] and Rab5 39] both are important genes involved in the cellular multi drug resistance (MDR) phenotype
development. It is a well established fact that the H locus of Leishmania codes for a short chain dehydrogenase (SDR) gene that is involved in antilfolate
resistance 40]. We found SDR to be present in Rab4, Rab6 and Rab18 of Leishmania but not in the corresponding Rab genes of Leptomonas. A study in our lab has provided evidence supporting the important role played in
drug resistance by the Rab6 gene of Leishmania having SDR at C terminus of gene (manuscript in communication).

Structure activity relationship of L. donovani GGTase-II with specific inhibitor

Since most prenylated proteins in cells are modified by geranylgeranyl moiety rather
than farnesyl 41] it offers Rab prenylation as a lucrative target for chemotherapeutic intervention
in Leishmania cell. Impairment of Rabs (a GGTase-II substrate) geranylgeranylation by isoprenoid
pathway inhibitors (lovastatin, zoledronate and DGBP) have been demonstrated 42]. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate.
NE10790 inhibited incorporation of [(14)C] mevalonic acid into Rab but not into H-Ras
or Rap1, proteins that are modified by FTase and GGTase I, respectively 43]. NE10790 therefore appears to be the first specific inhibitor of Rab prenylation.

The active site architecture of GGTase-II (?-subunit) of L. donovani was compared with other organisms by alignment. The protein sequences of GGTase-II
(?-subunit) of Leishmania donovani were aligned with the GGTase-II (?-subunit) sequences of Homo sapiens (NP_004573.2), Plasmodium falciparum (ACT90629.1), Trypanosoma brucei (AAX80948.1) and Drosophila melanogaster (AAF51183.1). Sequence similarity was seen within the kinetoplastid Trypanosoma brucei 65 %, Mycobacterium tuberculosis which is also an intracellular parasite like Leishmania, however, did not show any presence of GGTase-II enzyme on data mining. Available
literature indicated that Mycobacterium is predicted to express secreted effectors containing CAAX motifs that may be prenylated
by host protein prenyltransferases 44]. While Drosophila melanogaster, Homo sapiens and Plasmodium falciparum were showed 52, 48 and 29 % sequence similarity to GGTase-II (?-subunit) of Leishmania donovani respectively (Fig. 8).

Fig. 8. The active site architecture of GGTase-II (?-subunit) of L. donovani was compared with other organisms by alignment. Multiple sequence alignment of GGTase-II
(?-subunit) of Leishmania donovani (accession number XP_003864545) with GGTase-II (?-subunit) of Homo sapiens (NP_004573.2), Plasmodium falciparum (ACT90629.1), Trypanosoma brucei (AAX80948.1) and Drosophila melanogaster (AAF51183.1) showing conserved regions (red colored) based on percentage identity

Template for GGTase-II (?-subunit) of Leishmania donovani was selected on the basis of sequence similarity and lowest E-value. Blastp analysis
against PDB database, GGTase-II (?-subunit) of Rattus norvegicus shows 48 % identity and minimum E-value with GGTase-II (?-subunit) of Leishmania donovani and this template was further used for molecular modeling studies.

We have ascertained that the ?-subunit of GGTase-II in both Leishmania and Leptomonas lie in close proximity done through Neighbor-Joining method (Fig. 5). Therefore the ?-subunit emerges as the excellent target for compounds inhibiting
parasite activity in clinical cases of co-infections. The crystal structure of Rab
escort protein-1 in complex with geranylgeranyltransferase-II (Fig. 9a) and isoprenoid from Rattus showed 48 % of sequence similarity (Fig. 9b) with ?-subunit of Leishmania GGTase-II thus selected as the template for homology modeling 45]. Ten models were generated with Modeller. The validation of the resulting models
was performed using the SAVS server and the best validated model was selected 46]. In the selected model, the majority of the residues (89.4 %) occupy the most favored
region of Ramachandran Plot generated by PROCHECK and 9.9, 0.7 and 0.0 % residues
lie in additional allowed region (yellow), generously allowed (light yellow) and disallowed
(white) region respectively (Fig. 10a). The wiring diagram of ?-subunit of LdGGTase-II was performed using the PDBsum server
(http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/); a schematic diagram showed the protein’s secondary structure elements (alpha-helices
and beta-sheets) together with various structural motifs such as beta- and gamma-turns,
and beta-hairpins. Helices are labeled as H1, H2, etc. (Fig. 10b). The superimposition of the modeled protein (L. donovani RabGGTase) with template 1LTX shows an RMSD of 0.205 Å (Fig. 10c). The farnesyl binding site was selected as the active site for docking of the inhibitor.
The NE10790 has a docking score of ?6.10 kcal/mol. The inhibitor is involved in formation
of three hydrogen bonds with Asn37 and one hydrogen bond with Trp41 as shown by binding
mode (Fig. 10d).

Fig. 9. a Showed interaction between Rab Escort protein-1 (REP- 1) and geranylgeranyl transferase-II
enzyme of Leishmania donovani (LdGGTase-II). REP- 1 is displayed in surface representation and colored in grey.
? and ?-subunits of LdGGTase-II are displayed in ribbon representation and colored
in purple and magenta respectively, whereas arrow indicated active site of ?-subunit of LdGGTase-II. b Sequence alignment of the template (PDB ID 11tx_b) and the protein (?-subunit of
LdGGTase-II). Residues highlighted in red correspond to identical/conserved residues, while residues in red text are similar in these two proteins

Fig. 10. a Ramachandran plot of the homology-modeled structure of Leishmania donovani Rabgeranylgeranyltransferase. The different colored areas indicate “disallowed” (white), “generously allowed” (light yellow), “additional allowed” (yellow), and “most favored” (red) regions. b The wiring diagram of ?-subunit of LdGGTase-II was performed using the PDBsum server
(http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/). c Superimposed structures of L. donovani RabGGTase (cyan) with template 1LTX (magenta). d The binding mode of inhibitor NE10790 (Pink) in the docked complex of L. donovani RabGGTase (Cyan)