Chapter 8: Comparative proteome mapping of the clinical...

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

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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

153 I P a g c


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.

1541 P a g ('


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


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 downregulated 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

~5e5

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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

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"-._heavy -557.4275 I KMP-11

553.3 03 ' 559.4116

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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

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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

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100 200 300 400 500 600 700 800 900 1000 mtz, Da

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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


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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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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Chapter 8: Comparative proteome mapping of the clinical...

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