Supplementary Information(SI)
SI Material and methods
Cloning, expression and purification of LdISP1and LdISP2
L. donovani genomic DNA was isolated from ~1×108 cultured promastigotes.LdISP1 and LdISP2 genes were amplified using primers, as described in table numberS1(1,23,4). LdISP1 and LdISP2genes were sub-cloned in TOPO-TA vector, the insert were released by using restriction enzymes BamHI/HindIII and BamHI/NdeI respectively. The inserts were again ligatedinto similarly digested pET-28a+ and pET-15b cloning vectors respectively. For protein expression, recombinant constructs were transformed into E.coli (BL-21) competent cells. Expression of the recombinant LdISP fused proteins in BL21 (DE3) cells were optimized (0.75mM IPTG) to get maximum expression in the soluble fraction. The proteins were purified with Ni2+/NTA affinity column. The integrity and purification of the rLdISPs proteins were confirmed by 15% SDS-PAGE analysis and coomassie brilliant blue R-250 staining.
Sandfly maintenance
Laboratory breeding of P. argentipes was initiated using male and female sandfliescollected from the endemic natural habitats according to Kumar et al. [22].During insect breeding process the larvae transformed into pupae and then adults emerged. The emerged adult sandflies consisting of both males and females were kept starved for 24 to 48 hrs. After that they were offered a blood meal on anaesthetized rabbit for 2 hrs. Unfed male and unfed female sandflies were provided with 30% sucrose solution after blood feeding with the help of cotton wool and kept in round plastic container bottom lined with plaster of paris. These pots were placed in a tray on a wet lint cloth and were kept at standard lab condition at 28°C to 30°C temperature and 75-80% relative humidity in insectariums.
Sand fly midgut lysate preparation
Midguts were isolated from the uninfected sand flies as well as the sand flies infected with WT, ISPKD, ISPOE Leishmania parasites for different experiments. The midgut lysate was prepared according to Telleriaet al. (2015) for the different experimental set up[23]. Briefly, midguts werefirstly isolated and collected as 10 guts/100 µl in PBS. Isolated midguts were ground in 10mMTris-HCl, pH-8 for enzymatic assay or zymography, whereas for SDS-PAGE it was ground in 125 mMTris-HCl, pH-6.8 and 0.1% sodium dodecyl sulfate (SDS). The ground midguts were frozen and thawed 3 times and centrifuged at 14,000 g for 30 minutes at 4 °C. The supernatant was stored at -80°C.
Generation of ISP1 & ISP2 knocked down and overexpressedL.donovani cell lines
For the knocked downand overexpression of LdISP1 and LdISP2, the respective genes were amplified by PCR using genomic DNA ofL. donovani (Ld) parasite and primers as described in table number S1.For the knocked down, the amplified products of LdISP1 and LdISP2 were cloned in antisense orientation in SmaI and BamHI sites of pLGFPN vector. Whereas, forthe overexpression of LdISP1 and LdISP2,sense cloning wasdone in the same vector. The recombinant construct made for KD and OE such asLdISP1+pLGFPN (KD),LdISP1+pLGFPN(OE), LdISP2+pLGFPN (KD),LdISP2+pLGFPN(OE), and pLGFPN were transformed in DH5α cells for its stability. The construct along with empty vector pLGFPN were transfected in L.donovani. For transfection,the late log phase promastigotes were washed with transfectionbuffer (21 mM HEPES, pH 7.5, 137 mMNaCl, 5 mMKCl,0.7 mM Na2HPO4, 6 mM glucose). Transfection of theLeishmania parasiteswith recombinant construct and pLGFPN vectorwere carried out in 0.2 cm electroporationcuvette using a GenePulsar (Bio-Rad). Transfectants were allowed to recover for 24 hrsand then were selected for resistance to G418 at 5, 10, 20, 50,100,200 µg/mL.ISP1 and ISP2 knocked down cell lines wereabbreviated as ISP1KD and ISP2KDrespectively where as ISP1 and ISP2 overexpressed cell lineswere abbreviated as ISP1OE and ISP2OE respectively.
Morphological analysis of the parasites
Sand flies were infected with ISP1KD, ISP1OE and WT Leishmaniaparasites by artificial blood feeding mechanism and after infection sandflies were incubated for 6-8 days at standard lab conditions. The whole gut of the sand flies was isolated and collected in M199 media (10guts/50µl M199). Undamaged 20 isolated guts from each group were selected for morphological analysis. Different forms of the Leishmania promastigotes inside the sandfly midgut were identified and their numbers were counted under bright field microscopy at 100 X magnification.Promastigotes forms were categorized on the basis of flagellar length and cell body size ratio according to Killick-Kendrick et al.(1974) [34][Amasigote: Ovoid body form, no flagellum; Procyclic: Body length 6·5–11·5 µm, flagellum<Body length (body width variable); Nectomonad:Body length>12 µm (body width and flagellar length variable);Leptomonad: Body length 6·5–11·5 µm, flagellum>body length(body width variable); Haptomonad: Disc-like expansion of flagellar tip (body form and flagellar length variable); Metacyclic: Body length 6·5 –11·5 µm, flagellum> bodylength].Image-J software was used to measure cell body and flagellar length.
SI Results
ISPs of L. donovanishowed sequence similarity to the bacterial ecotin
In the present study we have identified two ISP like proteins named LdISP1and LdISP2 in L. donovani. The protein sequence of LdISP1 was retrieved from the NCBI protein database [PDB: XP_003859560.1] andLdISP2 was sequenced in our laboratory (sequence submitted to NCBI). Multiple sequence alignment of LdISP1 and LdISP2 were performed by Clustal Omega against the well annotated sequences of E. coli ecotin retrieved from GenBank [GenBank accession AAA16410.1]. The sequence of LdISP1 and LdISP2 encode the predicted protein of ~16.5 kDa and ~17.5 kDa respectively which is similar in size as that of E. coli ecotin (16.1 kDa) [32]. The sequence alignment of LdISP1 and LdISP2 with ecotin showed that the percent identity between ecotin and LdISP1 was 35%, whereas that of ecotin and LdISP2 was 38%. The percent identity between LdISP1 and LdISP2 was only 40%. The C-terminal domain of LdISP1 and LdISP2 do not share significant sequence identity with ecotin. Moreover, they have shorter N-terminus sequence compared to ecotin (SI Fig. S1). P1 residue binds to the active site of target proteases in a substrate-like manner mimicking the interactions of a canonical P1 substrate residue. From the sequence analysis of ecotin, LdISP1 and LdISP2 it is evident that the primary binding sites of LdISP2included the inhibition site consisting of P1 reactive methionine which is absent in LdISP1. Further, the sequence alignment also showed that the amino acids present in the secondary binding sites of the aligned sequence are highly conserved and consist of identical and similar amino acids. However, no significant sequence similarities were observed in the dimerization site of LdISP1 and LdISP2 when compared to ecotin.
MD simulation inferred strong interaction of ISP1 and ISP2 with trypsin and chymotrypsin
Molecular dynamics (MD) simulation was performed to study the interaction of ISPs (ISP1ISP2) with trypsin and chymotrypsin.The interacting complex (ISPs with trypsin and chymotrypsin) was simulated to investigate its stability in explicit solvent condition. The Root Mean Square Fluctuation (RMSF) plot of system depicted the mean fluctuation in the structure (residue) throughout the simulation.The atoms of ISP1-trypsin complex showed flexibility at different loci (Fig. 1 Panel-A; b.i), and the major fluctuations were observed from 750-1100 atoms or 100-140 residues of ISP1. Similar fashion of fluctuations has also been observed with ISP2-trypsin complex (Fig. 1 Panel-A; b.ii). These fluctuations are the part of active site composed of loop and β sheets. On the other hand RMSF of ISP1-chymotrypsin showed major fluctuation from 2700-3300 atoms or 39-165 residues (Fig. 1 Panel-B; b.i), and ISP2-chymotrypsin had core fluctuation from 1200-1500 atoms or 13-54 residues (Fig. 1 Panel-B; b.ii) encompassing active site residues, where the interaction with trypsin and chymotrypsin has been reported in docking study.
The distance between all possible amino acid residue pairs and polar contacts between ISPs-trypsin (Fig. 1 Panel-A; c.i,ii) and ISPs-chymotrypsin (Fig. 1 Panel-B; c.i,ii) (3D complex) were investigated by analyzing two dimension matrix. The contact map was generated through coordinate files at regular interval of MD simulation. The map described contact points of interaction among the ISPs and interacting proteins (trypsin/chymotrypsin), despite of dissimilarity among them. The mode of interaction of ISPs with trypsin and chymotrypsin were also investigated by analyzing the polar interaction within the proteins and also between protein and water. The hydrogen bonds formed between the protein atoms in the complex state and protein atoms (in complex state) with the water molecules remained constant throughout the simulations (Fig. 1 Panel-A; d.i,ii & Panel-B;d.i,ii). Interestingly, the solvent accessible surface area (SASA) of ISPs-trypsin/chymotrypsin complexes depicted a decrease of 10-30 nm.S-2.N-1(Fig. 1 Panel-A; e.i,ii & Panel-B; e.i,ii), theseconfirmed the hydrophobic bonding as the main force of interaction within the complex responsible for compact packing of protein complex.Therefore, all these MD simulation data inferred strong interaction of ISP1 and ISP2 with trypsin and chymotrypsin.
rLdISP2 showed more inhibitory effect on trypsin and chymotrypsin than rLdISP1
In this study, midguts isolated at different time intervals were used to measure trypsin and chymotrypsin activities(Fig. 3a & 3b). The activities increased with increasing time intervals up to 36 hrsand after that we observed a decrease in the activities at 48, 60 and 72 hrs. Maximum trypsin and chymotrypsin activity was observed at 36 hrs ABF. Therefore, we have used the midgut extract at 36 hrs ABF to study the inhibitory effect of rLdISPs. The inhibitory effect of rLdISP1 and rLdISP2 on trypsin and chymotrypsin, present in the sand fly midgut were analyzed through overlay-zymography (Fig. 3c). The activity of these proteases on their chromogenic substrates was measured by substrate cleavage followed by colour production. The enzyme activities were represented by appearance of pink coloured bands at ~23 and ~26 kDa for trypsin and chymotrypsin respectively (Fig. 3c; lane 2 & 7). In presence of protease inhibitor, the colour production was either substantially decreased or absent.
Significant reduction in trypsin and chymotrypsin activities was observed in presence of rLdISP2 (Fig. 3c; lane6 & 11). However, less remarkable reduction in activity was observed in presence of rLdISP1 (Fig. 3c; lane 5 & 10). Simultaneously, significant reduction in colour was also observed for both trypsin and chymotrypsin in presence of PMSF (serine protease inhibitor, 0.5mM), benzamidine (trypsin inhibitor, 0.5 mM) and TPCK (chymotrypsin inhibitor, 0.5 mM) (Fig. 3c; lane 3, 8 & 4, 9).
Altered population of nectomonad, leptomonad and metacyclic promastigote forms inside sandfly infected with ISP1KD and ISP1OE Leishmania parasites.
The lifecycle of Leishmaniapromastigotes consist of different morphological forms inside the sandfly.The number of different promastigote forms of the parasites were measuredin WT, ISP1KD and ISP1OE Leishmania parasite infected sandflies at the late stage of infection.Nectomonad, leptomonad and metacyclic promastigote forms of the parasites were mostly found inside sandfly in all the cases. However, the percentage of populationdiffered in each group of infection. In case of infection with ISP1KD Leishmania parasites, nectomonad population was found to be increased by ~1.3-fold (P = 0.0320), whereas leptomonad and metacyclic populationwas decreased by ~1.8-fold (P = 0.0425) and ~1.4-fold (P = 0.302) respectively when compared to infection with WT parasites. Simulteneously, in case of infection with ISP1OELeishmaniaparasites, nectomonad population was decreased by ~1.2 fold (0.0373), whereas leptomonad and metacyclic form was increased by ~1.25-fold (0.0220) and ~1.4-fold (0.0435) respectively when compared to infection with WT parasites (SI Fig.S5).
SI Table
Table S1.List of primers used in the experiments.
No / Name / Primer sequences / R.E site1. / ISP1F / 5'-GTCGGATCCATGTCATACTGCAAGATCGAGGC-3' / BamHI
2. / ISP1R / 5'-GTCAAGCTTACTCCGTGACCGCCTGCATC-3' / HindIII
3. / ISP2F / 5'-GTCGGATCCATGCCCGCAGGGATGTCCGG-3' / BamHI
4. / ISP2R / 5'-GTCCATATGTCACATCTCCCTAGCGTTGATG-3' / NdeI
5. / SemiQ-ISP1F / 5'-AGCATTGAGCAGCACACAGT-3'
6. / SemiQISP1R / 5'-GGCTTCTGTACGGGAACAAG-3'
7. / SemiQISP2F / 5'-CGTGACACTGACGACGATG-3'
8. / SemiQISP2R / 5'-TCCCTAGCGTTGATGCTCTT-3'
9 / ISP1F(OE) / 5'-GTCCCCGGGATGTCATACTGCAAGATCGAG-3' / SmaI
10 / ISP1R(OE) / 5'-GTCGGATCCTTACTCCGTGACCGCCTGC-3' / BamHI
11 / ISP2F(OE) / 5'-GTCCCCGGGATGCCCGCAGGGATGTCCG-3' / SmaI
12 / ISP2R(OE) / 5'-GTCGGATCCTCACATCTCCCTTGCCTTGGTG-3' / BamHI
13 / ISP1R(KD) / 5'-GTCCCCGGGTTACTCCGTGACCGCCTGC-3' / SmaI
14 / ISP2R(KD) / 5'-GTCCCCGGGTCACATCTCCCTTGCCTTGGTG-3' / SmaI
SI Figures & figure legends
Figure S1.Sequence alignment of ecotin with L. donovani ISP1 and ISP2. The predicted amino acid sequence of LdISP1 and LdISP2 was aligned with E. coli ecotin using Clustal Omega software (version 2.0).The primary P1 reactive site methionine of E. coli ecotin is marked by an asterisk. The E. coli ecotin primary binding site, inhibition sites, secondary binding sites deduced from the trypsin-ecotin complex (Yang [41]), are shown in box. Identical amino acids conserved in at least the aligned sequences are indicated by stars. Similar amino acids between the aligned sequences are shown as (:). E. coli ecotin (GenBank: AAA16410.1], L. donovani ISP1 (PDB: XP_003859560.1), L. donovani ISP2 (unpublished data).
Figure S2.Cloning, expression and purification of LdISP1 and LdISP2. LdISP1(a-c)and LdISP2 (d-f) genes were cloned in expression vector pET28a+ and pET15b, respectively, expressed in E. coli (BL-21) cells and purified by Ni2+/NTA affinity column. aPCR amplification of LdISP1.Lane M: marker;Lanes 1, 2: PCR amplicon (441bp) of LdISP1. bRestriction enzyme digestion of recombinant LdISP1- pET-28a+ vector with BamHI and HindIII.Lane 1: undigested vector; Lane 2: double enzyme digested vector; Lane 3: single enzyme (BamH1) digested vector;Lane M: marker. cExpression and purification of LdISP1.Lane 1: whole cell lysate of cells (no insert);Lane 2: whole cell lysate of uninduced cells; Lane 3: whole cell lysate of induced cells with 0.75 mM IPTG at 37 °C; Lane 4: supernatant of induced cells;Lane 5: purified eluted fraction of rLdISP1.dPCR amplification of LdISP2.Lane M: marker;Lanes 1, 2: PCR amplicon (477bp) of LdISP2. eRestriction enzyme digestion of recombinant vector ISP2-pET-15b with BamH1 and NdeI. Lane1: undigested vector; Lane 2: single enzyme (BamHI) digested vector; Lane 3: double enzyme digested vector; Lane M: marker. f Expression and purification LdISP2.Lane 1: whole cell lysate of uninduced cell (no insert);Lane 2: whole cell lysate of the uninduced cell;Lane 3: whole cell lysate of cells induced with 0.75 mM IPTG at 37 °C;Lane 4: supernatant of induced cells;Lane 5: purified eluted fraction of rLdISP2.
Figure S3.Measurement oftrypsin and chymotrypsin activity in the presence of different concentration of rLdISP1 and rLdISP2.Concentration of rLdISP1 and rLdISP2 were standardized for measurement of trypsin and chymotrypsin activity and to observe their inhibitory effect. aTrypsin activity in presence of rLdISP1 (0.1 nM-10 µM). bTrypsin activity in presence of rLdISP2 (0.1 nM-1 µM). cChymotrypsin activity in presence of rLdISP1 (0.1 nM-10 µM). d Chymotrypsin activity in presence of rLdISP2 (0.1 nM-1 µM). eSand fly midgut trypsin activity in presence of rLdISP1 (0.1 nM-10 µM). f Sand fly midgut chymotrypsin activity in presence of rLdISP2 (0.1 nM-10 µM).
Figure S4.Generation of construct for LdISP1 & LdISP2 KD and OE.Antisense and sense cloning of LdISP1 and LdISP2 genes in pLGFPN expression vector were performed for KD and OE of LdISP1 and LdISP2 respectively. aPCR amplification of LdISP1.Lane M: marker;Lanes 1, 2: PCR amplicon for ISP1KD and ISP1OE (441bp), respectively.b PCR amplification of LdISP2.Lane M: marker; Lanes 1, 2: PCR amplicon for ISP2KD and ISP2OE (477 bp), respectively. cGeleluted product of R.E (SmaI andBamHI) digested pLGFPN vector and PCR products of LdISPs.Lane1: pLGFPNvector; Lanes 2, 3, 4, 5: eluted digested amplicon for ISP1KD, ISP1OE, ISP2KD, ISP2OE, respectively.d Plasmid PCR of the transformed colony.Lanes 1, 2, 3, 4: PCR amplicon for ISP1KD, ISP1OE, ISP2KD and ISP2OE respectively. Lane M: marker.
Figure S5.Morphological forms of L. donovani promastigote inside P. argentipes during the late stage of infection. Sand flies were infected with either WT, ISP1KD or ISP1OE Leishmania parasites. Fully engorged sand flies were kept in standard laboratory conditions for 6–8 days and after that whole gut of the infected sand fly of each group was sampled. The morphology of the parasite was determined as described in material and methods (Additional file 1: Text). The percentage of nectomonad, leptomonad and metacyclic forms in each infected group was determined. Approximately 150 parasites were analyzed for each group. Image-J software was used to measure cell body and flagellar length and one of the representative picture images was provided. Result was expressed as the arithmetic mean ± SD of 20 sand flies dissected for each group from two independent experiments.