Comparativeproteome mapping ofVL and PKDL
Chapter 8: Comparative proteome mapping of the clinical isolates of
Leishmania donovani from Post-kala-azar dermal leishmaniasis and
Visceral leishmaniasis patients using quantitative Stable Isotope
Labeling of Amino acids in Cell-culture (SILAC)
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Comparative pmteome mapping ofVL and PKDL
1.0 Introduction
The human parasitic diseases continue to present a major health problem on a global
scale. Leishmaniasis represents one such disease condition, which is wide spread and
continues to plague mankind. The causative agent of Leishmaniasis is Leishmania, a
member of the trypanosomatid protozoa which is transmitted by the bite of blood sucking
phlebotomine sandfly. The aggressiveness of the individual Leishmania species, their
organ preference and host immune status determine the outcome of its infection. This
infection can range from a solitary, spontaneously healing ulcer (cutaneous leishmaniasis,
CL), to often destructive mucocutaneous disease (MCL) to generalized involvement of
viscera in visceral leishmaniasis (VL). These parasites affect around 2.0 million people
every year, mainly in India, Sudan and Latin American countries (Weniger et al., 2001).
Kala-azar transmission in India is thought to be anthroponotic and Post-kala-azar dermal
Leismaniasis (PKDL) patients are considered to serve as source for new outbreaks.
Post kala-azar dermal leishmaniasis (PKDL) is a complication of visceral
leishmaniasis. It is characterized by a macular, maculopapular and nodular leisons in
which Leishmania parasites are present. PKDL patients are therefore considered as
reservoirs for Leishmania parasites. Most cases occur in the Indian subcontinent (India
Nepal, Bangladesh) and East Africa (Sudan, Ethiopia, Kenya), where Leishmania
donovani is the causative parasite. It is mainly seen in Sudan and India where it develops
as a sequel to VL in 50% and 5-10% of cases, respectively. The interval at which PKDL
follows VL is 0-6 months in Sudan and 2-3 years in India. In Sudan, most cases get self
cured and only severe and chronic cases are treated, whereas, in India PKDL treatment is
always needed. Considerable research has focused mainly on the pathogenesis and
management of PKDL, leaving unexplored important issues such as the parasite genetic
factors responsible for the pathology.
Therefore, the major challenge is to ascertain the factors that enable the parasite to
move from visceral organs to the skin emerging as PKDL. In the course of emergence,
the parasite might express prospective set of genes differentially expressed. Thus, the
comparison of PKDL and VL isolates is of particular relevance in this study.
Quantitative proteomics has traditionally been performed by two-dimensional gel
electrophoresis, but recently, mass spectrometric methods based on stable isotope
150 I P age
Comparative pmteome mapping ofVL and PKDL
quantitation have shown great promise for the simultaneous and automated identification
and quantitation of complex protein mixtures. SILAC is a simple, inexpensive, and
accurate procedure that can be used as a quantitative proteomic approach in any cell
culture system. Quantitative proteomics, employing stable-isotope labeling and high
resolution mass spectrometry, has gained success lately and enables deciphering diverse
biological processes due to its high level of coverage of the proteome, accuracy m
quantification and high-throughput platforms (Julka and Regnier, 2004).
We used high resolution mass spectrometry and labelling techniques for
quantitative proteomics (using stable isotope labeling by amino acids in cell culture or
SILAC (Ong et al., 2002). Briefly, two cell populations are grown in media containing
either a naturally occurring amino acid or a stable isotope labeled analog. Since the stable
isotope analogs are heavier than their naturally occurring counterparts, protein
quantification occurs directly at the level of the peptide mass spectrum using the
difference in signal intensity between the peptides derived from isotope-labeled or
normal amino acid proteins. Here we describe for the first time, a comparative study of
the proteome of a VL and a PKDL isolate using stable isotope labeling by amino acids in
cell culture, for the in vivo incorporation of specific amino acids into all leishmania!
proteins.
2.0 Materials and Methods
2.1 Parasite and culture conditions
Promastigotes of L. donovani clones, AG83 (MHOM/IN/80/AG83) were isolated from a
patient with VL and the strain, NR3A used in the present study was isolated from a
patient with PKDL. Clinical isolates obtained from the VL patient responded to SAG
chemotherapy and was designated as SAG-S (SAG-sensitive) whereas isolate from the
PKDL patient who did not respond to SAG was designated as SAG-R (SAG-resistant).
SAG-sensitive strain, AG83-S and the SAG-R isolate, NR3A-R have been characterized
earlier (Mandai et al., 2010). The interval between the cure ofVL and the onset ofPKDL
was 11 years. The patient had nodular lesions on the back of the neck, lower extremities,
legs, face; macular lesions on the back of the neck and upper arms. These clinical isolates
were maintained in the absence of drug pressure in vitro. Promastigotes were routinely
cultured at 22 "c in modified M-199 medium (Sigma, USA) supplemented with 10%
1511Page
Comparative proteome mapping ofVL and PKDL
heat-inactivated fetal bovine serum (FBS; Gibco/BRL, Life Technologies Scotland, UK)
and 0.13 mg/mL of each penicillin and streptomycin.
2.2 Preparation of cellular lysate of total cellular proteome by Stable Isotope
Labeling of Amino acids in Cell-culture (SILAC) method
The VL isolate AG83-S and SAG-resistant PKDL isolate NR3A-R were grown in custom
synthesized media, M199 which was depleted of L-lysine and L-arginine (Sigma, USA)
supplemented with 10% dialyzed foetal bovine serum (Invitrogen), antibiotics plus L
lysine and L-arginine at 22 °C. A regular sub-culturing till both the strains got adapted to
the medium was done, as reflected by their growth curves. Cell lines were then grown for
six cell divisions in the same medium containing either normal L-lysine and L-arginine or 13C6 L-lysine-HCl and 13C6
15N4 L-arginine-HCI. A replicate sample preparation was also
done by reverse labeling of PKDL and VL isolates with either normal L-lysine and L
arginine or 13C6 L-lysine-HCl and 13C6 15N4 L-arginine-HCI.
2.3 Preparation of protein samples
The cells were harvested and washed thrice with ice-cold 1X PBS to remove the serum
proteins and resuspended in the lysis buffer provided with the Invitrogen kit. The cells
were lysed according to the manufacturer's protocol and total proteins were quantified by
Bradford's method using bovine serum albumin as the standard (Bradford, 1976). Equal
quantity of proteins ( 100 Jlg) from both the strains was mixed and precipitated with ice
cold acetone. Six volumes of acetone were added and each tube was inverted three times
and incubated overnight at -20 °C. Proteins were precipitated by centrifugation at 13000
rpm for 15 mins at 4 °C and acetone supernatant was decanted. Tubes were kept at room
temperature for some time to evaporate the remaining acetone from the precipitated
protein. Sample preparation involved reducing and denaturing the sample and finally
digesting the proteins with trypsin at 3 7 oc for overnight. Then the samples were dried in
speed vac. To simplify the peptide mixture before reversed-phase LC-MS/MS, peptides
were washed and fractionated off line using a strong cation exchange column and finally
analyzed by electrospray ionization tandem (ESI), using reverse-phase LC-MS/MS.
2.4 Strong Cation Exchange (SCX)
The peptide mixture was separated by off-line strong cation exchange (SCX)
chromatography using an HPLC with a UV detector (1200 series, Quaternary pumps,
152 I P a g 0
Comparativeproteome mapping o{VL and PKDL
Agilent). Briefly, the labeled and digested peptide sample (200 Jlg) was resuspended in
SCX running buffer (5 mM ammonium formate, 30% acetonitrile (ACN) with 0.1%
formic acid (Fairlamb and Cerami, 1992) and loaded onto a PolyLC Polysulfethyl A
Zorbax 300SCX column, 5 Jlm (2.1 m m x 150 mm, Agilent). Peptides were eluted with
increasing concentrations of 5 mM ammonium formate, 30% ACN (Solvent C), to 500
mM ammonium formate, 30% ACN (Solvent D), using a gradient: For first 5 min, 100%
C and 0% D; 5-35 mins, 30% C and 70% D, 35-55 mins, 0% C and 100% D, 55-66 mins
100% C and 0% D and final run till 73 mins. HPLC was run on 100% C to re-equilibrate
the column with solvent C. Forty five fractions were collected at a flow rate of 400
,uL!min according to UV trace at 220 nm. Fractions were lyophilized to remove ACN and
stored in -80 °C for further analysis. Peptides were reconstituted in 0.1% formic acid in
the ratio of3:97 ofacetonitri1e: water before subjecting to the 1D Nano LC (1200 Series,
Agilent Technologies) for peptide separation on Enrichment column & RP column. The
LC had been further coupled to Nano ESI-LC/MS system (4000 Q TRAP LC/MS/MS
system, Applied Bisosytems MDS SCIEX) for peptide ionization and detection. The latter
was equipped with a nanoelectrospray ionization source (Nanosource II, Applied
Bisosytems MDS SCIEX) and fitted with a 15 11m fused silica emitter tip (New
Objective, Woburn, MA). RP LC was performed on a 3.5 Jlm (75-Jlm x 150-mm)
ZORBAX 300 SB-C18 Nano LC column (Agilent Technologies, Germany) with a 5 Jlm
(300-J.tm x 5-mm) ZORBAX 300 SB-C18 Nano LC column (Agilent
Technologies,Germany) in place. Samples were loaded onto a trap or guard column in a
volume of 5-10 Jll and were equilibrated for 20 min in 97% solvent A, 3% solvent B at a
flow rate of 10 JlVmin. Solvent A is 0.1% formic acid in water and solvent B is ACN. On
switching in line with the MS, a linear gradient at 300 nL!min from 97-45% solvent A
was developed for 40 min, and in the following 5 min the composition of mobile phase
was decreased to 10% A, before increasing to 97% A for a 20-min equilibration before
the next sample injection. MS data were acquired automatically using Analyst 1.4.2
software (Applied Biosystems MDS SCIEX, Concord, Canada). An EMS survey scan
was conducted over 400-1600 amu, followed by three enhanced product ion scans over
mass range of 140-1600 amu. The three most intense peaks were selected for
fragmentation which satisfied IDA (Information Dependent Acquisition) criteria. A
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Comparative proteome mapping ofVL and PKDL
precursor ion within a 2.5 amu window, once selected for fragmentation, was excluded
from detection for 60 s. Curtain gas was set at 15, nitrogen was used as the collision gas,
and the ionization tip voltage used was 2000 V.
2.5 Database analysis and Realtive Quantification
LC-MS/MS data files from both replicate groups were processed using the Protein Pilot
software (Ver. 3.0) (Applied Biosystems MDS Sciex) as described by Wolf et al. A list of
peptides (i.e., m/z values) that were detected with confidence greater than 95% was
generated by Protein Pilot, saved as an Excel file, and imported into the Analyst method
as a text file. The same data acquisition method (.dam file) was used to acquire the first
and second LC/MS/MS data. Duplicate aliquots of sex fractions were analyzed by the
first and second methods. Peptides were excluded throughout the entire run of a specific
sex fraction based on their m/z value. This procedure allowed identification of
significantly more peptides than a single Le/MS/MS analytical cycle.
The 'Search Effort' parameter 'Thorough ID' which provides a broad search of
various protein modifications and multiple mass cleavage was chosen. The Paragon
algorithm used in Protein Pilot requires no definition of peptide/fragment mass tolerance,
as it iteratively searches for the optimal mass error for a data set. It also examines a
number of common modification forms included in a generic workup set. Searches were
performed against the integrated theoretical combined proteome (L. infantum, L. major
and L. brazilensis) downloaded from Tritrypdb (http://www.tritryp.org). A majority of
average protein ratios reported by Protein Pilot have a p-value (evaluating the statistical
difference between the observed ratios and unity) and EF (error factor) for each protein
ID. The EF term indicates the actual average value lies between (reported ratio/(EF) and
(reported ratio) x (EF) at a 95% confidence. Only those protein matches having a p value
::; 0.05 and a meaningful EF ( <2), and at least two unique peptides identified, were saved
for protein identification and relative quantification. The false positive rates of the
aforementioned filter criteria were all below 5%, estimated by using an individual
reversed (decoy) sequence database of the entire Leishmania proteome. In brief, false
positive rates are calculated by dividing the number of decoy hits by that of hits acquired
in search against forward sequence database. All statistical tests and analyses were
performed using Excel and R programs.
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Comparativeproteome mapping ofVL and PKDL
sex seperation
J U<>ht
!l!K~-Protein identification and Quantitation by LC MSIMS
Figure 1: Strategy followed for the SILAC experiment. Briefly, two cell populations are grown in media
containing either a naturally occurring amino acid or a stable isotope labeled analog, lysed, equal amount of
proteins are mixed and fractionated using strong cation exchange column after trypsin digestion and further
protein identification and quantification is done using ESI-LC MS/MS.
3.0 Results
3.1 Identification of soluble proteins by SILAC
Comparison of protein expression in VL and PKDL isolates has the potential to reveal
proteins that are involved in mediating phenotypic changes caused by them. Briefly, the
VL isolate, AG83 and the PKDL isolate, NR3A were cultured as described in materials
and methods. AG83 and NR3A promastigotes were labeled with heavy and light amino
acids in the first set. Reverse labeling of cells was done to be used as a replicate to
increase accuracy and authenticity of result. Cell lines were then grown for six cell
divisions. It is expected that after six doubling times, each instance of these amino acids
will have been replaced by its isotopically labelled analogue. Protein extracts were
prepared from the log phase promastigotes of both the VL and the PKDL isolates
155 I P a g c
Comparativeproteome mapping ofVL and PKDL
growmg m appropriately supplemented media (Materials and Methods). The
experimental strategy is represented in Figure 1. Equal amount of protein (100 flg) from
both the samples were mixed and subjected to the cation exchange chromatography and
peptide masses obtained from mass-spectrometry analysis were characterized using the
theoretical combined (L. infantum, L. major and L. brazilensis) proteome obtained from
Tritrypdb [http://www.tritryp.org]. These were grouped to identify total 2140 proteins out
of 8184 theoretical proteins. Of the identified proteins, 687 have a known or predicted
function, whereas 1453 are proteins with no known function. The detected proteins
represent 26.1% of the entire Leishmania proteome.
To confirm the SILAC ratios, reverse labelling was also performed and the data
was used as a replicate group. 457 proteins identified with confidence included all the
protein IDs from two individual datasets (SILAC-1/-2, where dataset -2 represents
reverse labelling of the replicate group). Of these, 262 proteins having known function
were further classified into different categories depending on their function, remaining
195 proteins were having no known function (hypothetical proteins). Major metabolic
pathways represented in the data, included glycolysis, gluconeogenesis, ~-oxidation, the
tricarboxylic acid cycle, oxidative phosphorylation, the pentose phosphate pathway,
mitochondrial respiration, purine salvage pathway and amino acid catabolism. Functional
classification for all the 262 proteins identified has been shown in Figure 2. It includes all
of the protein IDs from two individual datasets of SILAC labeling. According to the
functional description, all significantly identified proteins fell into 23 major groups:
metabolic enzymes, oxidation and reduction, signal transduction, transcription, protein
biosynthesis, transcription and translation, and miscellaneous (unclassified) etc. Their
relative distribution is shown in Figure 2. Proteins were grouped according to their
cellular functions. The up- and down- regulated hits are combined in each group. The
'miscellaneous' group consists mostly of proteins of unknown function.
1561 P a g <:
Tr.msposons 2%
Various enzymes
13"
DNA synthesis and Repair 8%
infection 2%
111%
ComporaTil't' pruteo/1/L' mapping of' I Land PKD/_
Oxidative phosphorylation 2%
Calpains 2%
Ribosom.1l Proteins
5"
Figure 2: Pie diagram showing relative distribution of Leishmania proteins identified by using stable
amino acid in cell culture method. Proteins were grouped by their cellular functions. The up- and down
regulated hits are combined in each group. The 'Miscellaneous' group consists mostly of proteins of
unknown function .
Of these, 43 distinct proteins were quantitated having peptides identified at ~95%
confidence. The results of this analysis are presented in Figure 3. Among the strictly
identified proteins, we defined three additional thresholds for selecting proteins that had
significantly altered expression: 1) P value or that protein ratio (S l or S2) given by the
software for each peptide is :S 0.05, which indicates the ratio statistically differs from
unity (>95% confidence); 2) the PKDL:VL ratio is either 2: 1.5 or :::;0 .5 (using the same
cutoff for analyzing SILAC co-quantified pairs); and 3) must be a 'quantifiable entry'
157 I P a g c
Comparative proteome mapping o{VL and PKDL
with EF (error factor) <2. Applying these filtering criteria, a subset of regulated proteins
were identified, and then the total number of protein IDs (with P-value and EF), the
number of proteins with P<0.05, and the number of regulated hits (meeting all the three
requirements) found in two experiments approaches were determined. The proteins which
were 2:1.5 fold were considered to be up regulated whereas the proteins which were :S 0.5
fold were considered to be down regulated. Using these stringent parameters 15 proteins
were found to be up-regulated in the PKDL isolate NR3A (Table 1 ); 10 were found to be
downregulated (Table 2) and 18 remained unchanged (Table 3).
Figure 3: Bar diagram showing global modulation of Leishmania cellular functions based upon distict
peptides quantitated at > 95% confidence when PKDL proteome is compared to VL proteome.
158 I P a g e
S. No. Gene DB ID Name of the Proteins PKDL/VL Putative Peptides SILAC Function identified ratio
mJ 2 Cl () J::' II ' l)
2 Lbr:tv130 \'2.2950 glyceraldehyde 3- 24 3
phosphate dehydrogenase.
Lmjf09.1340 histone H2B 2.30 ' .., 001\6 I
LbrM32 histone H2A. putative 1.78 Transcription ., -
LinJ3:\ j:l
LinJ25 V3.0760 eukaryotic initiation factor 1.43 Translational 3
Sa. putative factors
8 LbrM07 V2.0570 60S ribosomal protein L7a. 3.01 Protein 5
putative synthesis 1 I l 112 1 () e
300
LinJ29 V3.1240 tryparecloxin 1.79 Oxidati\e
stress -I) ]_(ll)
1 J(l
r
l' I
(\ l:
15 Lbrl\11 V2.0200 tubulin .M _),~
. ·---~----·- ~-------·---·--------------~··
Table 2: PKD USlng S lll
]1;1)
Table 3: List of un-changed protems in PKDL Sl method
.\TPa~e alpha ,ubunit
2 LmJ25 \'3.1210 A TPase beta ~ubunit. putati\t:
3 LmJl-t \·3.12411
1 Lbr;-..117\'2.0090 elongation factor ]-alpha Protein 1 3
~--j_·-,-......,--,.--:--c-:-c-:--j_--:-__ -:--:--:--:---:--,.--:-----l--:--:--:-----L..,.b.,._i c_Js-';,-·n:--t_h:--e ._si_s_ -1-----____J 6 [ mfCI l \; IJ:;:oo l'ukanc,uc imt1at1un facwr --2-+ TratbbtJOnal · ~
putati\ e factoh
-7-rinJ13 \'3.0460 40S ribosomal protein S 1 ::'.
putatiw
8 Lbr\11- \'2 (1090 elongation factor
12 LinJ::'5 \'3.0940
13
14 LinJ3Cl V3.2530
b mdmg I
cyclophilin a (Protein folding)
-;uhunn- like protem. eqn 1
\hcat shock 70-related protein I.
I mitochondrial precursor.
putatiYe --~-----------+--
15
16
l.inJ:''\ \ 3 3(1h(i
LinJ2LJ V3.1360 II
. I
hc:n-,Jwck protein h,p-11.
putatJ\ c
AfP-dependent Clp protea,c
subumt.
heat shock protein 100
(liSP I 00 ). putatiw.serinc
_ -~-------- _ __l_l:Jcl'~ 1dasc . .-:p_u_t_a_ti_\·_e _____ _
' 0 7 311
0.65()9
0.92RR
Protein
synthesis
: I I -
I
' ' Iranslattlmal ~ 1 r1
h1cWrs
Cheperone I s
( 'hcpcn >nc
I •
-----~--------- --------~~---
Comparative proteome mapping olVL and PKDL
Figure 4 illustrates the coupling of stable isotopes and mass spectrometry spectra for both
the identification of proteins and the quantification of their relative expression levels as
determined from spectra. In instances where mixtures of light and heavy labeled peptides
were analyzed, the identification of trypsin digested Arg/Lys-containing peptides was
facilitated by the characteristic doublets of peak clusters present in the mass spectra.
From the MS spectra, we were able to confirm these doublets. Using MS/MS, as seen in
Figure 4, the similar fragmentation patterns from L-lysine and L-arginine and 13C6 L
lysine-HCl and 13C6 15N4 L-arginine-HCl peptides can also help to confirm the identity.
The observed ratio of peak intensities of the light and the heavy amino acid has been
shown.
7e5
6e5
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682 684 686 688 690
y3 y4 y3+2 b3 b4
light '\ heavy
696.5400
697 0162
- 697.4567
Peptide ratio= 15
7
.4S4:J 0154
7020105 I 701.5200
03.9855 698.9116 705.4200
694 696 698 700 702 704 706 708 710 712 714 mlz, Da
VSIVATDIFTGNR
y5 y6 y7 y8 y9 y10 y11 '
994.72 92 .73
59 .60 70 48 109 .04
82 .71
y12
185.16 237.24 34E .36 4 3.40 1~93 84 697.62 881.16
I il ,I Ill ,(u 549.00 ,, 760.32 I I
961.5E I Ill I 1111 I II I
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 mlz, Da
Figure 4 [A]: ESI MS/MS spectra showing the fold changes Eukaryotic initiation factor 5a (upregulated)
illustrates the coupling of stable isotopes and mass spectrometry for both the identification of proteins and
the quantification of their relative expression levels using MS spectra (upper panel); MS/MS spectra (lower
panel)
1621 Page
•'
Comparative proteome mapping ofVL and PKDL
1.1e5
1.0e5
9.0e4
8.0e4
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oght
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/ 4.4913
I I I II I I 538 540 542 544 546 548 550 552 554 556 558 560 562 564 566 568 570
mlz, Da
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120.014- y1 y2 y3 y4 y5 y6 y7 y8 b2 b3 y9 2
191 18"
II 219.3 0
386 4 0 432..200 887 60
I ooo I 53 580 645 GOO 275 160 348 I, 758 580
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
light
607. 900
mlz. Da
heavy II~ 613 4044
612.8929 Peptide ratio =Ll
603.4197 613.8794
609.3600 615.4467
Hsp 70 putative
598 GOO 602 604 606 608 610 612 614 616 618 620 622 624 626 628 630 mlz, Da
DAGTIAGLEVLR
I y2
b41 y3
b11+~ y5 yfJ y7 y8 y9 y10
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615.59 62- 45 143 05 L 4~80j '492 9S .74 l(ljr 67
100 200 300 400 500 600 700 800 900 1000 mtz, Da
I
I
1100
Figure 4: ESI MS/MS spectra showing the fold changes [B] KMP-11 (Downregulated) [C] HSP-70,
putative (Not changed) illustrates the coupling of stable isotopes and mass spectrometry for both the
identification of proteins and the quantification of their relative expression levels using MS spectra (upper
panel); MS/MS spectra (lower panel)
163 I Page
Comparativeproteome mapping of'VL and PKDL
3.2 Glycolysis
Biochemical analyses of Leishmania promastigotes have shown that the promastigotes
can use both glucose and amino acids, such as proline, as energy sources. The catabolism
of these substrates appears to involve both glycolysis, compartmentalized in
peroxisome-like organelles called glycosomes, and mitochondrial metabolism with
an active tricarboxylic acid (TCA) cycle and linked electron transport chain. We
observed that the expression of glycosomal glycolytic enzyme glyceraldehyde 3-
phosphate dehydrogenase and cytosolic phosphoglycerate kinase B was ~2.4 fold, ~3 fold
up-regulated respectively in the PKDL isolate, NR3A compared to the SAG-S VL isolate,
AG83-S. (Tablet). In contrast, fructose 1, 6-bisphosphate aldolase and 2, 3-
bisphosphoglycerate independent phosphoglycerate mutase was ~ 1.8 and ~ 1.9 fold
downregulated respectively. We also observed that NADP-dependent alcohol
dehydrogenase involved in acetaldehyde detoxification was ~4 fold downregulated.
3.3 Gluconeogenesis
The enzymes essential for gluconeogenesis i.e. phosphoenolpyruvate carboxykinase and
glycosomal malate dehydrogenase was ~2 and ~3.0 fold down-regulated respectively.
From the present data it appears that in the PKDL isolate, glycolysis is enhanced whereas
gluconeogenesis is downregulated.
3.4 Translational machinery
SILAC identified 10 ribosomal protein subunits, ofwhich 60S ribosomal protein L7a (rp
L7a) was upregulated ~3.0 fold in the PKDL isolate NR3A. Ben-Ishai et al have shown
that ultraviolet irradiation and other DNA-damaging agents specifically induce transient
increases ofrpL7a transcripts. It has been recently reported that the changes in the tissues
of PKDL patients are compatible with the effects of UVB light and it is probable that
UVB appears to be a key factor in the pathogenesis of PKDL (Ismail et al., 2006). Our
present study further supports these studies. Nucleolar protein is an important pool of
dormant proteins, some of which are key regulators of the cell cycle. Nucleolar protein is
also involved in ribosome biogenesis (Andersen et al., 2005). It was -2.8 fold up
regulated. Eukaryotic initiation factor 5a was ~ 1.4 fold upregulated in the PKDL isolate
NR3A compared to AG83-S. Besides its role in protein synthesis, the elongation factor 2
1641Page
Comparative proteome mapping ofVL and PKDL
is implicated in other cellular processes such as signal transduction and apoptosis (Ejiri,
2002; Lamberti et al., 2004).
3.5 Histones
The basic unit of chromatin is the nucleosome, formed by 146 bp of DNA wrapped
around an octamer containing two copies of each of the core histones, H2A, H2B, H3,
and H4. Histones affect chromatin structure, which has been shown to have consequences
for nucleosome assembly, DNA replication and repair and, most notably, gene expression
(Fischle et al., 2003 ). The elevated expression of 3 histone proteins H4 ( -1.5), H2B
(-2.3) and H2A (-1.8) in the PKDL isolate may indicate transcriptional changes.
3.6 Protein Folding Machinery
Glucose-regulated protein 78 which has been characterized in a number of species
including yeasts, rats, and trypanosomes (Pelham, 1986; Normington et al., 1989; Rose et
al., 1989; Tibbetts et al., 1994) was -2.5 fold upregulated in PKDL isolate NR3A
compared to AG83-S. Its functions include translocation of proteins from the cytoplasm
into the endo-plasmic reticulum (ER) and sequestration of glycoprotein precursors in the
ER matrix until they have been glycosylated and/or assembled into multiprotein
complexes (Jensen et al., 2001). Therefore, it will not be a great expectation to propose
that this protein might have a role in phenotypic change (VL to PKDL).
3.7 Signal transduction
IgE-dependent histamine-releasing factor was -1.6 fold upregulated in PKDL isolate
NR3A. IgE-dependent histamine-releasing factor causes histamine release from human
basophils or mast cells and IL-8 secretion from eosinophils. Histamine regulates Th cell
differentiation (Jutel et al., 2001) and possibly drives Th2 responses during the chronic
phase of Leishmania infection (Pos et al., 2004). 14-3-3 protein that is -2.7 fold
downregulated in PKDL isolate NR3A, is part of a conserved family of proteins capable
of binding numerous phosphorylated proteins implicated in several cellular processes
including apoptosis (Dougherty and Morrison, 2004).
3.8 Drug Resistance
Several proteins belonging to this category (such as calpain-like cysteine peptidase,
tryparedoxin, Kinetoplastid membrane protein-11, and HSP 83-1) that were identified in
this study were recognized. We observed upregulation of calpain-like cysteine peptidase,
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Comparative proteome mapping of'VL and PKDL
putative, cysteine peptidase, Clan CA, family C2, putative by ~ 1. 7 fold. Cysteine
peptidases (CPs) play important roles in facilitating the survival and growth of the
parasites in mammals (Mottram et al., 2004). A heat shock protein, HSP83-1 was also
found to be upregulated in our study. Heat shock proteins are the family of proteins
whose function is to protect cell from toxic external stimuli. The association of drug
resistance with the induction of stress proteins has long been established in cancer cells
(Wallner and Li, 1986; Li, 1987). HSP83-1 itself has been implicated in resistance to
antimony, miltefosine and amphotericin B, by counteracting the drug related programmed
cell death (Vergnes et al., 2007). Kinetplastid membrane protein-11 (KMP-11) was ~2.3
fold downregulated in PKDL isolate NR3A. Down regulation ofKMP-11 in Leishmania
infantum axenic antimony resistant amastigotes has been reported earlier (El et al., 2009)
Importantly, this NR3A-R isolate was collected from a PKDL patient who was SAG
unresponsive. This led us to suggest that tryparedoxin, KMP-11 and HSP83-1 might play
an important role in antimony resistant phenotype of PKDL isolate NR3A-R (Mandai et
al., 2010). Glutathione peroxidase-like protein and type II (glutathione peroxidase like)
tryperadoxin peroxidase were ~3.3 and ~2.5 fold downregulated in PKDL isolate
respectively.
3.9 Cell motility
Actin and a tubulin were found to be upregulated in PKDL isolate by ~1.9 and ~1.7 fold
respectively. The microtubules are ubiquitous structural elements of eukaryotic cells that
are found in the cytoskeleton, flagellar, and ciliary axonemes and are essential for a
variety of diverse cellular processes, including formation of mitotic spindles during cell
division, intracellular transport and secretion, regulation of cellular morphology, and
motility of cilia and flagella (Kirschner, 1978).
3.10 Hypothetical proteins
Of the 457 L. donovani proteins identified with high confidence in the present analysis,
~42.6% were from genes annotated as hypothetical, confirming these are bonafide genes.
66 hypothetical proteins were identified as differentially expressed. Of these 66 proteins, \
25 were found to be upregulated, 30 were found to be downregulated and 11 not changed.
This is because the Leishmania genome sequencing project though complete, however,
has a number of genes, which do not match aruiotated sequence within the sequence
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Comparative proteome mapping ol VL and PKDL
database (Ivens et al., 2005). So the majority of genes belong to the unclassified category,
indicating that they have as of yet an unknown function. It will be interesting to study
these genes as they might encode proteins with functions specific to phenotype of interest
and may provide novel targets for therapeutic intervention.
We have used Western blotting to examine a selected group of proteins in both
the VL and the PKDL isolates to determine the correlation between the SILAC/mass
spectrometery analysis and the protein expression. We examined two proteins, heat shock
protein with a SILAC ratio of PKDLNL of 1.0, eukaryotic initiation factor 5a, with a
ratio of 1.5. As shown in Figure 5 no difference in the expression of HSP70 was observed
in VL and PKDL isolate. However, eukaryotic initiation factor 5a, putative was
upregulated in PKDL isolate(~ 1.3 fold). A Western blot using size-fractionated parasite
protein, antiserum could detect a high intensity band of L. donovani eiF5a of~ 19 kDa in
NR3A promastigote extracts compared to AG83 (Figure 5). The fold change in band
intensity is in agreement with the value calculated from the protein pilot software was
~ 1.43 fold up-regulated in PKDL isolate NR3A.
Protein name
Hsp70 Putative {LinJ28 _ V3.3060)
Enkaryotic initiation fuctor5a (LinJ25 _ V3.0760)
Western Foldchange AG83 NRJA
1.00
1.3
SILACmtio
1.1
1.43
Figure 5: Western blot analysis using anti-HSP70 antibody and anti-eiF5a raised against LdHSP70 and
eiF5a in BALB/C mice. Polyclonal antiserum against LdHSP70 and LdeiF5a generated in mouse was used
to detect HSP70 and eiF5a protein to confirm the SILAC ratios.
4.0 Discussion
Post-kala-azar dermal leishmaniasis (PK.DL) is a common problem (Zijlstra et al., 2003;
Dey and Singh, 2007) in kala-azar patients but the actual reason behind the outbreak of
this disease is not yet known. The present study followed changes in the abundance of
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proteins during VL to PKDL manifestation in order to understand, how the parasite
moves itself from visceral organs to skin. At present, the isolation of the parasite from the
same patient having VL and after few years getting PKDL is not possible because it
requires continuous monitoring. Therefore, we compared one VL and one PKDL isolate
isolated from the same geographical region. Using high-coverage, comparative proteomic
analysis of soluble parasite proteins, we collected expression information on 26% of the
L. donovani proteome. Our data reveal changes in the expression level of most proteins
involved in the core metabolic pathways and of many molecules involved in translation,
protein folding, cell motility and signal transduction and drug resistance. This study, for
the first time, enables a comprehensive view of the biogenesis processes underlying
differentiation from VL to PKDL. This is summarized in the Figure 1. Our major
observations include: a) during VL to PKDL manifestation, the glycolysis has increased
where parasites are dependent on glucose as the main source of metabolic energy; b)
concomitantly, PKDL parasites down-regulate gluconeogenesis (producing sugars from
glycerol and amino acids) c) Transcription and translation factors have increased in
PKDL parasites and d) significant changes in the proteins involved in signal transduction
and drug resistance.
Interestingly, we also observed a highly synchronized increase in the abundance
of translation machinery proteins, including ribosomal protein 60S ribosomal protein L 7 a,
translation factor eukaryotic initiation factor Sa, and histones. Of note, the increased
expression ofhistones suggests transcriptional changes during PKDL manifestation.
The proteins that are observed to be strongly modulated have functions which fit
with phenotypic changes of PKDL isolate NR3A that could be a reason for sodium
antimony gluconate (SAG) resistance as characterized earlier (Mandai et al., 2010). A
complex remodelling of the Leishmania proteome may give rise to cells that can tolerate
the cytotoxic effect of antimony. It has been suggested that Sb V inhibits macromolecular
biosynthesis possibly via perturbation of energy metabolism due to inhibition of
glycolysis and fatty acid ~ oxidation (Berman et al., 1985; Berman et al., 1987).
Upregulation of glycolytic pathway and downregulation of gluconeogenesis may
compensate the cytotoxic effect of antimony in case of SAG-R isolate NR3A. Consistent
with this, upregulation oftryparedoxin and HSP83-1 with concomitant downregulation of
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Comparativeproteome mapping ofVL and PKDL
KMP-11 might play an important role in antimony resistant phenotype of PKDL isolate
NR3A-R.
Taken together, our data strongly supports the notion that the specific differences
in the protein expression may be responsible for the underlying differences in the biology
and disease phenotypes of PKDL. Since proteins are the main catalysts, structural
elements, signalling messengers, and molecular machines of biological tissues, proteomic
studies are able to provide substantial clinical relevance. These proteins can be utilized as
biomarkers for tissues, cell types, developmental stages, and disease states as well as
potential targets for drug discovery and interventional approaches. The real potential of
proteomic fingerprinting is in use as a discovery tool for novel biomarkers that can then
be incorporated into simple bedside diagnostics based on affordable technologies such as
immunologically based antigen-detection tests that could be implemented in dipstick or
cassette formats.
This comparative proteomic analysis identified a large and diverse pool of
proteins differentially expressed in PKDL isolate. The current study implicates the
existence of parasite molecules that may facilitate parasite persistence in cured VL
patients, leading to the development of PKDL. It will be significant to understand that the
same parasite species in a different disease presentation may show a different pattern of
gene expression by using more number of clinical isolates.
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