Proteomic Analysis of Metacyclogenesis in Leishmania

e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183

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journal homepage: http://www.elsevier.com/locate/euprot

Proteomic analysis of metacyclogenesis in

Leishmania infantum wild-type and PTR1 null mutant

1 1

Wilfried Moreira , Danielle Légaré , Gina Racine, Gaétan Roy, ∗

Marc Ouellette

Centre de Recherche en Infectiologie du CHU de Québec, Département de Microbiologie, Université Laval, Québec, QC, Canada

a r a

t i b s

c l e i n f o t r a c t

Article history: The parasite Leishmania exhibits a dimorphic life cycle with promastigotes found in the

Received 23 May 2014 insect vector and amastigotes residing within mammalian macrophages. In the insect, pro-

Received in revised form mastigotes undergo metacyclogenesis with two main stages, the procyclic and metacyclic

14 July 2014 stages. Reduced pterins, provided by the pteridine reductase PTR1, are important factors

Accepted 17 July 2014 controlling metacyclogenesis. We applied here the liquid-based free-ﬂow electrophore-

Available online 8 August 2014 sis technique for the analysis of L. infantum wild-type and PTR1 null-mutant procyclic

and metacyclic proteomes. Signiﬁcant modulations in energy metabolism, antioxidant and

Keywords: stress-related defences, genetic information processing, protein degradation and motility

Leishmania were observed between procyclic and metacyclic forms, in addition to numerous hypothet-

Reduced pterins ical proteins.

Metacyclogenesis Association (EuPA). This is an open access article under the CC BY-NC-ND license

Free-ﬂow electrophoresis (http://creativecommons.org/licenses/by-nc-nd/3.0/).

sequential development involves the rapid division of non-

1. Introduction

infective procyclic promastigotes that ultimately leads to the

appearance of non-dividing but highly infective metacyclic

Leishmania species are affecting 12 million people world-

promastigotes in the anterior parts of the digestive tract

wide with 1.5–2 million new cases each year. These protozoa

[1,2]. This cellular differentiation referred as metacyclogen-

have a dimorphic life cycle and are found as ﬂagellated

esis is a key step for infectivity [2–4]. Indeed, it has been

promastigotes within the sand ﬂy midgut, and as intracel-

shown that promastigote populations recovered from labo-

lular non-motile amastigotes within macrophages of their

ratory infected sand ﬂies displayed a signiﬁcant increase in

vertebrate hosts. In the sand ﬂy, promastigotes maturate

infectivity in BALB/c mice as they develop temporally within

and progress through a series of two distinct stages. This

the ﬂies [2]. Also, promastigotes recovered from the stationary

∗

Corresponding author at: Centre de Recherche en Infectiologie, CHU de Québec, 2705 Boul. Laurier, Québec, QC, Canada G1V 4G2.

Tel.: +1 418 525 4444x48481; fax: +1 418 654 2715.

E-mail address: [email protected] (M. Ouellette).

These authors contributed equally.

http://dx.doi.org/10.1016/j.euprot.2014.07.003

article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

172 e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183

phase of growth were found to be more infective in vitro

2. Material and methods

than promastigotes isolated from the logarithmic phase [1–4].

The infectivity of stationary phase promastigotes has been

2.1. Cell culture, lectin-mediated puriﬁcation and

attributed among other things to changes in their surface

characterization of metacyclics and procyclics enriched

carbohydrates, including the major glycoconjugate lipophos-

parasites

phoglycan (LPG) coating molecule [4]. Changes in the structure

of LPG allow the parasite to detach from the mucosa and

Promastigotes of L. infantum wild-type (MHOM/MA/67/ITMAP-

migrate to the proboscis for injection in the host tissue during −/−

263) and L. infantum PTR1 [18] were grown in SDM79

the blood meal. 9

medium as described previously [25] and parasites (10 cells)

Metacyclogenesis is also accompanied by a number of

in their exponential phase and stationary phase of growth

morphological changes. In particular, procyclics possess a

were pelleted by centrifugation (3000 rpm, 5 min). Pellets were

ﬂagella approximately one body length whereas metacyclics

washed twice in Hepes–NaCl buffer (21 mM Hepes, 137 mM

are shorter cells of reduced body width and ﬂagella longer

NaCl, 5 mM KCl, 0.7 mM Na2HPO4·H2O, and 6 mM dextrose,

than one body length. Clear biochemical and molecular differ-

pH 7.05). Metacyclic parasites were isolated by PNA agglu-

ences have already been established during metacyclogenesis.

tination (100 ␮g/ml, Sigma–Aldrich) as previously described

These include a lower amount of total protein content asso-

[2,26,27] from stationary phase populations whereas pro-

ciated to a decrease in protein synthesis in metacyclics

cyclics were collected from exponential phase cultures.

compared to procyclics, as well as an increase in protease

Enriched metacyclic and procyclic samples were visualized

activities [1]. Secretory acid phosphatase and nuclease activ-

by light microscopy (400×, result not shown) and were further

ities have also been reported to be decreased in metacyclic

characterized by real-time RTPCR to conﬁrm procyclic and

parasites [1].

metacyclic enrichment. Brieﬂy, total RNAs from procyclic and

Reduced pterins (tetrahydrobiopterin, THB) have been

metacyclic parasites were isolated using the Qiagen RNeasy

identiﬁed as an important factor controlling metacyclogenesis

Mini Kit (Qiagen) according to the manufacturer’s instructions.

as a decrease in the THB level seems to stimulate meta-

At least 5 independent RNA preparations from the WT and

cyclogenesis [5]. Leishmania is an auxotroph for pterins but

DKO PTR1 procyclic and metacyclic parasites were prepared

it possesses the biopterin transporter BT1 to supply for its

and used in real-time RTPCR assays. Contaminating DNA was

need in pterins [6–8]. After BT1-mediated uptake, pterins are

digested with Turbo DNA-free Kit (Ambion). The quality and

reduced into their active forms by the enzyme pteridine reduc-

integrity of the starting RNA material was assessed with an

tase 1 (PTR1) [9–11]. Lack of PTR1 leads to reduced levels of

Agilent Technologies 2100 BioAnalyzer using the RNA 6000

intracellular THB, growth arrest and induction of metacycloge-

Nano LabCHIP kit (Agilent). The cDNAs were generated from

nesis. One of the earliest known cellular functions of THB was

total RNAs using a Oligo (dT)12–18 following the protocol of

as a growth factor for the parasite Crithidia fasciculata [12], and

the manufacturer for Superscript II (Invitrogen). Real-time

many studies have indicated that THB is indeed an essential

quantitative RTPCR assays were carried out in a BioRad Cycler

growth factor for Leishmania [13–16], although its exact role is

using SYBR Green I (Molecular Probes). The reactions were

still to be discovered (reviewed in [17]). THB was also shown to

carried out in a ﬁnal volume of 25 ␮l containing speciﬁc

react readily with oxygen, superoxide, H2O2, and peroxynitrite

primers, and iQ SYBR Green Supermix (Bio-Rad). Mixtures

and it was demonstrated that it protects cells against oxidative ◦

were initially incubated at 95 C for 5 min and then cycled 35

damages [18,19] (and reviewed in [20,21]). PTR1 and reduced ◦ ◦ ◦

times at 95 C, 60 C for 15 s and 72 C for 20 s. All real-time

pterins thus play important roles in Leishmania differentiation

RTPCR data were normalized with the real-time ampliﬁcation

and life cycle in which the infective metacyclic form encoun-

of GAPDH. The expression data are shown relative to the

ters an impressive oxidative stress as it meets with the host

data for the wild-type strain. Primer SHERP (LinJ23.1210) For-

immune system.

ward: 5 -GGACCAGATGAACAACGCCGCGG-3 ; Primer SHERP

Few studies have investigated the proteome changes dur-

Reverse: 5 -ACGAGCCGCCGCTTATCTTGTCC-3 ; Primer GAPDH

ing the metacyclogenesis process in Leishmania [22,23] and

(LinJ30.3000) Forward: 5 -GATCACGGTGGAGGCTGTG-3 ;

none have assessed the role of reduced pterins in this cell

Primer GAPDH Reverse: 5 -CCCTTGATGTGGCCCTCGG-3 .

cycle transition. In this study, we enriched for metacyclic and

procyclic parasites from wild-type and PTR1 null mutant Leish-

2.2. Protein sample preparation

mania infantum strains [18]. Their respective proteins were

subjected to free-ﬂow electrophoresis (FFE), a liquid based

Protein extractions were prepared from procyclic and meta-

IEF separation technique. We identiﬁed more than 260 indi-

cyclic parasites by incubating cell pellets in 300 ␮l of 2D

vidual proteins differentially expressed. Important changes

lysis buffer (7 M urea, 2 M thiourea, 3% (w/v) Chaps, 20 mM

in energy metabolism, antioxidant, stress-related defenses

DTT, 5 mM TCEP, 50 mM Tris-base, 0.5% IPG buffer pH 4–7,

and proteins involved in motility were observed. A signiﬁ-

and 0.25% IPG buffer pH 3–10) supplemented with 10 ␮l of

cant modulation in the expression of proteins involved in

protease inhibitor cocktail (2 mM AEBSF, 14 ␮M E-64, 130 ␮M

genetic information processing was also detected, support-

Bestatin, 0.9 ␮M Leupeptin, 0.3 ␮M Aprotinin, and 1 mM EDTA)

ing the notion that changes in the proteome during parasite’s

(Sigma-Aldrich) for 2 h at room temperature with frequent

differentiation and life cycle seem to be controlled by post-

vortexing. The supernatants were collected after centrifuga-

transcriptional mechanisms, including modulation of mRNA

tion (10,000 rpm, 2 min) and protein contents were determined

stability, regulation of translation and post-translational mod-

using a 2-D Quant Kit (GE Healthcare). iﬁcations [24].

e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183 173

2.3. Free-ﬂow electrophoresis (FFE)

Continuous liquid-based isoelectro-focusing (IEF) was per-

formed on a BD FFE System (BD Diagnostics) essentially as

described in [28]. Brieﬂy, the Leishmania protein extracts (3 mg)

were fractionated under denaturing conditions in a pH gra-

dient ranging from 3 to 10 using standard electrophoretic

◦

conditions (10 C, 750 V, media ﬂow rate of 60 ml/h and sam-

ple ﬂow rate of 100 ␮l/h) [28]. Four independent biological

replicates for each stage were processed. Individual fractions

were pooled to cover 3 pH ranges: 4.5–5.8 (fractions 27–33),

5.9–6.9 (fractions 34–40) and 7.0–8.0 (fractions 41–48). Proteins

were concentrated using Amicon Ultra-15 columns (Millipore)

and desalted by three successive centrifugation/washing steps

with 1 ml of 2D lysis buffer followed by resuspension in the

same buffer. Protein quantiﬁcation was performed with a 2-D Fig. 1 – Experimental design of proteome comparisons.

Quant Kit (GE Healthcare). Four 2D-gels corresponding to four biological replicates

were analyzed for each condition (1–4) described here.

2.4. 2D gel electrophoresis Ranges of pH covered in the ﬁrst dimension were: 4.5–5.5,

5–6 and 5.5–6.7. 1, comparison between L. infantum WT

Total protein extracts from procyclic and metacyclic cells procyclic and metacyclic parasites; 2, comparison between

−/−

were concentrated by precipitation using a 2-D Clean-up Kit L. infantum PTR1 procyclic and metacyclic parasites; 3,

(GE Healthcare) and resolubilized in 2D lysis buffer whereas subtractive analysis of non-common protein spots between

pooled FFE fractions were not further processed. The 24 cm comparisons 1 and 2; 4, comparison between L. infantum

−/−

IPG strips have been actively rehydrated at 30 V for 10 h 30 min. WT and L. infantum PTR1 metacyclic forms.

Tw o hundred micrograms of each sample (total cell extracts or

pooled FFE fractions) were loaded onto 24-cm IPG strips of pH

ranges 4.5–5.5, 5.0–6.0 and 5.5–6.7 (GE Healthcare). No paper identiﬁcation that were differentially expressed when com-

−/−

bridge was used. The ﬁrst dimension was performed with the paring metacyclic forms of WT and of PTR1 cells.

Ettan IPGphorII isoelectric focusing system (GE Healthcare) at

◦

20 C for a total of 100,000 V with a maximum current of 50 mA 2.6. In-gel protein digestion

as recommended by the manufacturer. IPG strip equilibration

and second-dimension SDS-PAGE were done as described pre- Gel plugs containing proteins of interest were excised from

viously [29]. the gels with a ProXcision robot (PerkinElmer Life Science),

deposited into a 96-well plate (Corning) and washed once with

2.5. Gel staining, imaging, and analysis distilled water. In-gel protein digestion was performed at the

Proteomics Platform of the Eastern Quebec Genomics Cen-

Proteins were visualized with SYPRO Ruby Protein Gel Stain tre (http://proteomique.crchul.ulaval.ca/en/index.html) on a

(Invitrogen) as described previously [30]. Gels were digitalized MassPrep liquid handling station (Waters Corporation) accord-

on a ProXPRESS 2D Proteomic Imaging system (PerkinElmer ing to the manufacturer’s speciﬁcations as described pre-

Life Sciences) and images were statistically analyzed using viously [28–31]. Digestion products were extracted from gel

the Progenesis PG240 software (Nonlinear Dynamics, ver- plugs using 1% formic acid, 2% acetonitrile followed by 1%

sion 2006) in which spots were automatically detected and formic acid, 50% acetonitrile. Peptides were lyophilized in a

␮

matched across gels. The data were inspected visually and speed vacuum and resuspended in 8 l of 0.1% formic acid.

edited manually when required. Spots with differences in Four microliters of each peptide preparation were used for

normalized spot volume (% volume) greater than 1.5-fold in mass spectrometry analysis.

the four biological replicates were considered as signiﬁcantly

differentially expressed. Signiﬁcance levels of any measured 2.7. Mass spectrometry

differences were determined by the Progenesis software using

the Student’s two-tailed t test. P-values < 0.05 were considered Peptide MS/MS spectra were obtained by capillary liquid

as statistically signiﬁcant. Overall, four proteome comparisons chromatography coupled to an LTQ linear ion trap mass

were performed (see experimental scheme in Fig. 1). In condi- spectrometer equipped with a nanoelectrospray ion source

tion 1, we contrasted the expression of procyclic vs. metacyclic (Thermo Fisher Scientiﬁc). Peptides were loaded onto a

␮

promastigote proteins derived from L. infantum WT cells. The reversed-phase column (PicoFrit 15- m tip, BioBasic C18,

× ␮

same type of analysis was carried with condition 2 (Fig. 1) 10 cm 75 m; New Objective) and eluted with a linear gra-

but involving the PTR1 null mutant. We also searched for dient from 2% to 50% acetonitrile in 0.1% formic acid at a

proteins that differed between comparisons 1 and 2 and for ﬂow rate of 200 nl/min. Mass spectra were acquired using a

which the expression was nonetheless signiﬁcantly altered data-dependent acquisition mode (Thermo Fisher Scientiﬁc

during the metacyclogenesis process (comparison 3 in Fig. 1). Xcalibur software, version 2.0) in which each full scan mass

Finally in comparison 4 (Fig. 1), we selected spots for MS/MS spectrum was followed by collision-induced dissociation of

174 e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183

the seven most intense ions. The dynamic exclusion func- 5.8 (fractions 27–33), between 5.9 and 6.9 (fractions 34–40) and

tion was enabled (30 ns exclusion), and the relative collisional between 7 and 8.0 (fractions 41–48) were pooled. After a desalt-

fragmentation energy was set to 35%. ing and concentration step, the extent of enrichment by FFE for

each pooled fractions was conﬁrmed by 2D gel electrophore-

sis using IPG strips covering three overlapping pH ranges (e.g.

2.8. Interpretation of tandem mass spectra and

4.5–5.5, 5.0–6.0 and 5.5–6.7) by comparing FFE-enriched pro-

protein identiﬁcation

teins to whole cell extracts (total proteins). As exempliﬁed for

the pH range 5.0–6.0 in Supplementary Material Fig. S2, the

MS/MS spectra were analyzed using MASCOT (Matrix Sci-

FFE procedure improved proteome coverage (compare A with

ence, version 2.2.0) and searched in the GeneDB Leishpep

C and B with D in Fig. S2).

database (http://www.genedb.org/Homepage) assuming a the-

oretical digestion with trypsin. A mass tolerance of 2.0 Da for

3.2. Metacyclogenesis in L. infantum

peptides and 0.5 Da for fragments were chosen, with 2 trypsin

missed cleavages allowed. Carbamidomethylation of cysteine

The FFE-fractions extracted from 4 independent biological

and partial oxidation of methionine were considered in the

−/−

replicates derived from either WT or PTR1 cells in two dif-

searches. The Scaffold software (Proteome Software, version

ferent life stages were analyzed by 2D gel electrophoresis.

2 01 01) was used to validate MS/MS-based peptide and pro-

Globally, the number of protein spots detected by the Progen-

tein identiﬁcations. Peptide identiﬁcations were accepted if

esis software over the three pH ranges were 5323 and 5984 for

they reached greater than 95% probability as speciﬁed by the

L. infantum WT procyclic and metacyclic stages respectively

Peptide Prophet algorithm [32]. Protein identiﬁcations were

versus 5006 procyclics and 5207 metacyclics for L. infantum

accepted if they reached greater than 95% probability and

−/−

PTR1 . Based on Scaffold’s parameters (see Section 2), some

contained at least three unique peptides as speciﬁed by the

of the detected spots were either uniquely or differentially

Protein Prophet algorithm [33]. Proteins that contained similar

expressed in one or more comparisons according to our exper-

peptides and could not be differentiated according to MS/MS

imental scheme (Fig. 1). In condition 1, we contrasted the

spectra alone were grouped to satisfy the principle of parsi-

expression of procyclic vs. metacyclic promastigote proteins

mony. The assignment of functional classes was performed

derived from L. infantum WT cells. The same type of anal-

using the GeneDB (http://www.genedb.org/Homepage) and

ysis was carried with condition 2 but involving the PTR1

TritrypDB (http://tritrypdb.org/tritrypdb/) web databases. Data

null-mutant. Common proteins between conditions 1 and 2

integration into biochemical networks were done according to

allowed the identiﬁcation of proteins differentially expressed

the two main databases KEGG (http://www.genome.jp/kegg/)

in both cell types between procyclic to metacyclic forms, inde-

and LeishCyc (http://leishcyc.bio21.unimelb.edu.au/).

pendent of the genetic background. We also searched for

proteins that differed between comparisons 1 and 2 and for

3. Results and discussion which the expression was nonetheless signiﬁcantly altered

when contrasting these two main life stages of the metacy-

3.1. Enrichment of procyclic and metacyclic proteins by clogenesis process (comparison 3). Finally in comparison 4,

free-ﬂow electrophoresis we sent spots for MS/MS identiﬁcation that were differentially

expressed when comparing only the metacyclic forms of WT

−/−

Before undertaking the proteomic analysis, we ﬁrst conﬁrmed and of PTR1 cells.

that our PNA-based enrichment strategy was indeed enrich- The number of spots sent for mass spectrometry analysis

+ −

ing for procyclic (PNA )/metacyclic (PNA ) parasite forms. This and the number of MS/MS protein identiﬁcations to each of the

was achieved visually by light microscopy (result not shown) 4 comparisons (experimental scheme, Fig. 1) are summarized

by comparing the length ratio between the ﬂagellum and cell in Table 1. In total, 136 spots were gel excised, trypsin digested

body [2,34] and by performing real-time RTPCR on parasite and sent in mass spectrometry for identiﬁcation (Table 1).

RNA preparations using SHERP as a metacyclic overexpressed We discarded 12 spots because of either the low quality of

marker [35] and GAPDH as a control. In accordance to optical the peptide mass spectra generated by MS/MS or because the

microscopy analyses, real-time RT-PCR results indeed con- identiﬁed proteins had less than 3 assigned peptides. Over-

ﬁrmed that PNA-based enrichment from L. infantum parasites all, 124 protein spots passed the quality criteria (Table 1) but,

lead to metacyclic parasites with 5-fold increased expression more than one proteins have been identiﬁed in each single

of SHERP in wild-type cells and 11-fold increased in meta- spot. In order to determine which of the identiﬁed protein

−/−

cyclic PTR1 parasites compared to the procyclic stages was responsible for the difference in the expression level of

(Supplementary Material, Fig. S1). Thus, by morphology and a particular spot, we took into account and selected the pro-

metacyclic-speciﬁc SHERP gene expression criteria similar to tein with the highest number of unique peptides and assigned

what have been reported previously [2,34,36–39], our PNA and spectra. Indeed, the most abundant protein in a given spot

−

PNA populations were considered to be respectively procyclic often gives rise to more unique peptides and assigned spectra

and metacyclic enriched forms. [40]. Thus in total, 467 protein hits were recovered (Table 1)

Procyclic and metacyclic proteins were then extracted from (listed in Tables S1–S4) corresponding to 263 individual pro-

L. infantum WT and PTR1 null mutant (four independent bio- teins (Tables S1–S4). The majority of the proteins identiﬁed

logical replicates each) and fractionated by FFE in IEF mode were differentially expressed between the two developmen-

∼

into about 70 3 ml fractions each. The pH of each fraction tal stages of both strains (comparisons 1, 2 and 3 in Fig. 1).

was measured and fractions with pH values between 4.5 and Proteins identiﬁed in comparison 4 (Fig. 1) cannot be linked

e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183 175

Table 1 – Global data on proteome analyses.

Number of spots Number of spots Number of Number of proteins differentially expressed in

analyzed with 3 peptides identiﬁcations

Pro WT Meta WT Pro KO Meta KO

Comparison 1

pH 4.5–5.5 5 5 25 12 13

pH 5–6 12 12 22 0 22

pH 5.5–6.7 3 3 6 0 6

Subtotal 20 20 53 12 41

Comparison 2

pH 4.5–5.5 5 3 8 2 6

pH 5–6 10 10 48 0 48

pH 5.5–6.7 3 3 7 1 6

Subtotal 18 16 63 3 60

Comparison 3

pH 4.5–5.5 14 14 43 6 1 31 5

pH 5–6 25 18 43 12 15 0 16

pH 5.5–6.7 10 10 57 6 12 4 35

Subtotal 49 42 143 24 28 35 56

Comparison 4

pH 4.5–5.5 10 8 30 26 4

pH 5–6 25 24 103 24 79

pH 5.5–6.7 14 14 75 57 18

Subtotal 49 46 208 107 101

Total 136 124 467 36 176 38 217

Summary of proteome analysis for the three covered pH ranges, including number of spots analyzed and number of identiﬁcations for each

comparison. Pro WT, procyclics of L. infantum WT parasites; Meta WT, metacyclics of L. infantum WT parasites; Pro KO, procyclics of L. infantum

−/− −/−

PTR1 parasites; Meta KO, metacyclics of L. infantum PTR1 parasites.

to metacyclogenesis because procyclic expression levels were many novel proteins never reported before to be potentially

not taken into account but they may relate to the different involved in the metacyclogenesis process.

genetic background of the compared cells.

These proteins were tentatively classiﬁed in 3.3. Carbohydrate and energy-related metabolic

metabolic pathways according to the curated LeishCyc pathways

([41]; http://leishcyc.bio21.unimelb.edu.au/) and KEGG

(http://www.genome.jp/kegg/) metabolic web databases. Several enzymes part of the carbohydrate and energetic

The identiﬁed proteins were linked to the carbohydrate and metabolic pathways were shown to be differentially expressed

energy-related metabolic pathways (Fig. 2); stress-related in metacyclic promastigotes compared to procyclic cells (Table

processes (Fig. 3); mevalonate interconnected pathways S1 and Fig. 2). The glycolysis pathway and interconnected

including amino catabolism and ubiquinone biosynthesis pyruvate/malate metabolisms were found activated in meta-

(Fig. 4); protein degradation/modiﬁcation (Table S1); genetic cyclics. Indeed, 7 enzymes (enzymes 4, 8, 9 (cytosolic), 12,

information processing (Table S2); and morphology and motil- 18, 19, 20 in Fig. 2; see LinJ.36.1320, LinJ.14.1240, LinJ.34.0150,

ity related processes (Table S3). In addition, 71 hypothetical LinJ.35.5450, LinJ.15.1070, LinJ.12.0580 and LinJ.25.1790 in Table

proteins of unknown function were also identiﬁed (Table S4). S1 for MS/MS data) of these pathways were found either

As reported previously, there are extensive post-translational uniquely expressed or upregulated at the metacyclic stage

modiﬁcations in Leishmania and related parasites [42–44], of the WT and/or PTR1 null mutant. Downstream of these

including protein fragmentation [28] and proteolysis which pathways, two enzymes part of the TCA cycle (enzymes

were shown to be highly dynamic processes in Leishmania as 9 (mitochondrial) and 32 in Fig. 2; see LinJ.34.0150 and

experimentally validated with the tubulins [29], heat shock LinJ.01.0050 in Table S1) and two entries into the cycle

proteins [45] and elongation factor-1 [28]. Despite the use (enzymes 18 and 20 in Fig. 2; see LinJ.15.1070 and LinJ.25.1790

of protease inhibitors and of carrying protein preparation in Table S1) were clearly up-regulated in metacyclic forms. It is

◦

at 4 C, several of the proteins identiﬁed in our study corre- thus possible that the higher expression of proteins involved

sponded to protein fragments or to different isoforms with in glycolysis or that are part of the Krebs’ cycle can generate

post-translational modiﬁcations (compare experimental vs. sufﬁcient ATP necessary for the intracellular remodeling and

theoretical molecular weight in Tables S1–S4). The majority of morphological differentiation of metacyclic parasites.

proteins identiﬁed in previous small scale proteomic analyses The pentose phosphate pathway (PPP), which generates

related to metacyclogenesis [22,23] were also identiﬁed here, NADPH and pentose (see Fig. 2), two main sources of energy

but the IEF-FFE strategy has translated in the identiﬁcation of and important building blocks of bioactive molecules, was also

176 e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183

Fig. 2 – Schematic of the core metabolome between procyclic and metacyclic developmental forms of L. infantum. This

model is a modiﬁcation of the schemes presented in [44,80,81]. Green arrows represent metacyclic down-regulated enzymes

or unique procyclic enzymes. Red arrows represent metacyclic unique or up-regulated enzymes. Black arrows represent

enzymes that have been detected only in comparison 4 (Fig. 1). Gray arrows indicate undetected enzymes. The pentose

phosphate pathway (PPP) is shaded in light gray. Glycosomes, acidocalcisomes, mitochondrion and cytosol compartments

are depicted. 1, hexokinase; 2, glucose 6-phosphate isomerase; 3, phosphofructokinase; 4, fructose bisphosphate aldolase;

5, phosphoglycerate kinase; 6, glyoxalase I; 7, glucosamine-6-phosphate isomerase; 8, enolase; 9, malate dehydrogenase;

10, phosphoenolpyruvate carboxykinase; 11, pyruvate phosphate dikinase; 12, pyruvate kinase; 13, adenylate kinase; 14,

F0F1 ATP synthase; 15, 2-oxoglutarate dehydrogenase; 16, succinyl-COA ligase; 17, succinate dehydrogenase; 18, glutamate

dehydrogenase; 19, alanine aminotransferase; 20, pyruvate dehydrogenase (enzymatic complex); 21, malic enzyme; 22,

3-ketoacyl-COA thiolase; 23, ubiquinone biosynthesis protein-like; 24, GDP-mannose pyrophosphorylase; 25, acetyl-COA

synthetase; 26, glucose-6-phosphate dehydrogenase; 27, 6-phosphogluconate dehydrogenase; 28, ribokinase; 29,

transketolase; 30, ribulokinase; 31, myo-inositol-1-phosphate synthase; 32, putative carboxylase; 33, putative

methylmalonyl-coenzyme A mutase; 34, putative acidocalcisomal exopolyphosphatase; 35, acidocalcisomal

pyrophosphatase; 36, transaldolase; 37, UTP-␣-D-glucose-1-P uridylyltransferase also named UDP-glucose

pyrophosphorylase; 38, sucrose-P-synthase like; 39, glutamine synthetase; UQ, ubiquinone pool; C, cytochrome c; I, II, III

and IV, complexes of the mitochondrial respiratory chain; MVA, mevalonate pathway.

e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183 177

Fig. 3 – Antioxidants and related pathways between procyclic and metacyclic developmental forms in Leishmania infantum.

This schematic is an expansion of the schemes presented in [82,83]. The main proteins involved in antioxidant and related

pathways in L. infantum are shown. Pathways indicated in red have been identiﬁed as overexpressed in metacyclic

parasites. Pathways indicated in green have been identiﬁed as overexpressed in procyclic parasites. Black arrows are

enzymatic reactions that were only detected in comparison 4 (Fig. 1). Gray arrows represent undetected enzymes. T(SH)2,

reduced trypanothione; TS2, oxidized trypanothione; TpXox, oxidized tryparedoxin; TpXR, reduced tryparedoxin; TR,

− − • •−

trypanothione reductase; H2O2, hydrogen peroxide; ONOO , peroxynitrite; NO2 , nitrite; NO , nitric oxide; O2 , anion

superoxide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NADP , oxidized nicotinamide adenine

dinucleotide phosphate; NDP, nucleoside diphosphate; dNDP, deoxynucleoside diphosphate. 1, trypanothione synthase; 2,

trypanothione reductase; 3, tryparedoxin peroxidase; 4, ascorbate peroxidase; 5, Fe-superoxide dismutase; 6,

glucose-6-phosphate-dehydrogenase; 7, glyoxylase system; 8, ribonucleotide reductase; 9, glutamate dehydrogenase; 10,

transaldolase; 11, peroxidoxin; 12, tryparedoxin.

activated in metacyclic compared to procyclic cells (enzymes 3.4. Antioxidant and stress-related responses

26, 28 and 30 in Fig. 2; LinJ.34.0080, LinJ.27.0430 and LinJ.36.0060

in Table S1). Overexpression of enzymes 26, 28 and 30 (Fig. 2) Mitochondria are the major source of ROS, which may in

are possibly necessary in metacyclics to fuel an activated gly- turn damage these organelles. Our proteomic analysis indi-

colysis with fructose-6P and glyceraldehyde-3P (see Fig. 2) cated that several proteins involved in oxidant response were

and to increase the production of NADPH needed to coun- increased in metacyclics (red arrows in Fig. 3). Indeed, the try-

teract the negative impact of reactive oxygen species (ROS) panothione synthase (enzyme 1 in Fig. 3; LinJ.27.1770 in Table

molecules that metacyclics may encounter (see next section S1), parts of the tryparedoxin redox system (enzymes 3 and

and Fig. 3). Finally, no obvious or signiﬁcant differences have 11 in Fig. 3; respectively LinJ.15.1100 and LinJ.23.0050 in Table

−/−

been observed between wild-type and PTR1 cells in this S1) and a marked oxidative phosphorylation/cytochrome-

carbohydrate and energy-related metabolic pathway (Fig. 2) in related response (LinJ.07.0210, LinJ.12.0620 and LinJ.26.1690 in

any of the 4 comparisons (Fig. 1). Table S1, three proteins present in the mitochondrial inner

178 e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183

Fig. 4 – Leucine catabolism, mevalonate pathway and ubiquinone biosynthesis between procyclic and metacyclic

developmental forms in Leishmania infantum. Pathways indicated in red have been identiﬁed as overexpressed in

metacyclic parasites. Pathways indicated in green have been identiﬁed as overexpressed in procyclic parasites. Black arrows

are enzymatic reactions that were only detected in comparison 4 (Fig. 1). Gray arrows represent undetected enzymes.

Philanthropic pathways are indicated in boxes. 1, putative 2-oxoisovalerate dehydrogenase beta-subunit mitochondrial

precursor; 2, putative sterol-24-c methyltransferase; 3, HMG-COA synthase; 4, HMG-COA lyase; 5, putative 2-oxoisovalerate

dehydrogenase beta-subunit mitochondrial precursor. Note that enzyme 5 is the same as enzyme 1. The leucine

degradation pathway leading to mevalonate (MVA) is being shown apart from the amino acids degradation pathway to

highlight the major role of leucine in the production of MVA in Leishmania, a precursor of sterols; 6, 3-ketoacyl-COA

thiolase-like protein; 7, putative carboxylase; 8, putative methylmalonyl-coenzyme A mutase; 9, putative ubiquinone

biosynthesis protein-like protein; 10, see Fig. 2 for the complete glycolysis/pyruvate pathways in which several enzymes

were overexpressed in metacyclic parasites as depicted here by the 2 red arrows.

membrane schematized in Fig. 2) were upregulated in meta- also identiﬁed an ascorbate-dependent peroxidase (enzyme

cyclic compared to procyclic L. infantum promastigotes. This 4 in Fig. 3; LinJ.34.0070 in Table S1) that was 1.72-fold upre-

is consistent with our observations described above that gulated in metacyclics of WT cells compared to metacyclics

−/−

the pentose phosphate (PPP) metabolism was upregulated in of the PTR1 null mutant, although this enzyme was only

metacyclics, since the PPP has been previously proposed to detected in comparison 4 and cannot be formally involved

have a protective role against oxidative stress [46]. We have in the metacyclogenesis process. Nonetheless, ascorbate

e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183 179

peroxidase in Leishmania has already been shown to be central during the stationary phase of promastigotes cultures (Table

to the redox defense system of this parasite [47,48]. S1). Notably, two lysosomal cysteine peptidases, CPA and CPB,

have been previously implicated in the development of this

3.5. The mevalonate and related pathways parasite [57] and we have indeed found two cysteine pepti-

dases (LinJ.27.0510 and LinJ.29.0860 in Table S1) in our dataset

The mevalonate (MVA) pathway is present in Leishmania and being upregulated at the metacyclic stage in both WT and

−/−

is preceding the isoprenoid biosynthesis pathway leading to PTR1 null mutant. The oligopeptidase B enzyme (OPB)

the synthesis of sterols (Fig. 4). We have identiﬁed a putative (LinJ.09.0820 in Table S1) was also identiﬁed as unique in meta-

sterol 24-c-methyltransferase (enzyme 2 in Fig. 4; LinJ.36.2510 cyclic WT parasites but not detected in metacyclics of the PTR1

in Table S1) that was found to be uniquely expressed in meta- null mutant (comparison 4, Fig. 1). Although OPB has been

−/−

cyclic promastigotes both in WT and PTR1 cells suggesting detected only in comparison 4 and cannot be directly involved

a change in membrane sterols during metacyclogenesis. Inter- in the metacyclogenesis process, it was previously reported

estingly in Leishmania, the MVA-isoprenoid pathway is linked that a L. major null mutant of OPB [58] showed a small deﬁ-

to ubiquinone [49–51] and heme biosynthesis [52] and major ciency in its ability to undergo differentiation to metacyclic

precursors for the MVA pathway are leucine [53,54], some promastigotes. In addition [59], OPB was also shown recently

branched amino acids as well as a number of products from to act as a regulator of the Leishmania enolase (metacyclic over-

glycolysis (Fig. 4). Aromatic amino acids can also be inter- expressed enzyme 8 in Fig. 2; LinJ.14.1240 in Table S1), which

connected with the isoprenoid route (post-mevalonate) and has a classical role in carbohydrate metabolism. The leish-

their degradation will eventually produce para-aminobenzoic manolysin (GP63) metalloprotease (LinJ.10.0490 in Table S1),

acid (pABA) which will in turn serve as a precursor for the most abundant surface protein of Leishmania promastig-

the generation of the ubiquinone ring that will condense otes, was detected in our study as upregulated in metacyclic

−/−

with long chain phenyl phosphate molecules (most likely PTR1 parasites (condition 3, Fig. 1).

farnesyl diphosphate) to lead to ubiquinone (Fig. 4). A num- Our results are consistent with others [42,60,61] that

ber of enzymes from these interconnected pathways have suggested an important change in the phosphorylation state

been detected as being differentially expressed when con- of many proteins during metacyclogenesis. Indeed, one

trasting procyclic to metacyclic forms. These observations kinase (LinJ.31.3070 in Table S1) was shown to be uniquely

might be predictive for an increase in the activities linked expressed at the metacyclic stage in the PTR1 null mutant

with the MVA pathway during the parasite stage transition. strain, and two phosphatases (LinJ.15.0170 and LinJ.20.1610

It has been shown that Leishmania utilizes leucine (instead in Table S1) were pinpointed in our analysis, uniquely

of acetate as most eukaryotes) as the main precursor for expressed respectively at the procyclic and metacyclic stage,

sterol biosynthesis through the MVA-isoprenoid route (Fig. 4). irrelevant of the genetic background of the strains. Other

A uniquely expressed beta-subunit of the 2-oxoisovalerate post-translational modiﬁcations seem also to operate during

dehydrogenase (enzyme 1 in Fig. 4; LinJ.35.0050 in Table S1) the parasite differentiation as exempliﬁed by the aminoacy-

suggests indeed an increase in the catabolism of this amino lases LinJ.20.1740 detected as unique in metacyclic parasites

acid during metacyclogenesis. We also noted a tendency for of both cell types (Table S1). Ubiquitin-mediated degradation

branched-amino acid degradation (enzymes 1, 5 and 6 in of proteins via the proteasome has also been linked with

Fig. 4; LinJ.35.0050 (note that enzymes 1 and 5 are the same Leishmania differentiation [62–64], and an impressive number

in Fig. 4) and LinJ.23.0860 in Table S1) that lead to an over- of proteasome-related proteins including regulatory subunits

production of acetyl-COA, another precursor of the MVA route were upregulated mostly in conditions related to metacyclo-

in Leishmania (Fig. 4) but also a fuel molecule of the TCA cycle genesis (LinJ.03.0520, LinJ.06.0140, LinJ.13.0990, LinJ.21.0840,

(Fig. 2). Acetyl-COA can enter the MVA pathway by the action of LinJ.27.1370, LinJ.29.0120, LinJ.34.0670, LinJ.34.4390 and

the hydroxymethylglutaryl-coenzyme A synthase, leading to LinJ.35.0770 in Table S2). Also in line with the proteome

hydroxymethylglutaryl-COA (HMG-COA) which fuels the iso- turnover operating during metacyclogenesis, a peptide of the

prenoid pathway [55]. Interestingly, an hypothetical protein polyubiquitin protein (LinJ.09.0950, Table S2) was also unique

−/−

with a hydroxymethylglutaryl-coenzyme A synthase (HMGS) in metacyclics of PTR1 cells.

domain was upregulated in our comparative analysis (enzyme Another intriguing ﬁnding of Leishmania metacyclic pro-

3 in Fig. 4; LinJ.24.2200 in Table S4). These observations are con- mastigotes is a depletion of RNA and a down regulation of the

sistent with the MVA pathway having a role during the parasite translation machinery [65]. Supporting this notion, our pro-

differentiation, a suggestion that will require experimental teomic analysis revealed an upregulation of several enzymes

validation. related to the RNA metabolism and post-transcriptional

gene expression (Table S2). Several of these changes were

−/−

3.6. Protein modiﬁcation and turnover detected in the PTR1 mutant cells. The genome of Leish-

mania encodes 29 RNA helicases [66] and three members

During differentiation, Leishmania proceeds to an important were detected in our study (LinJ.21.1820, LinJ.32.0410 and

protein turnover, mostly achieved by the deployment of a vast LinJ.35.0370 in Table S2). Since two of them (LinJ.21.1820

repertoire of proteolytic and proteasome-related enzymes at and LinJ.35.0370) have been identiﬁed only in comparison 4

the metacyclic stage of the parasite [22,56]. The genomes (Fig. 1), their role in metacyclogenesis requires further analy-

of Leishmania spp. contain more than 150 peptidases [56] sis. RNA helicases have been described as regulatory factors

(MEROPS database at http://merops.sanger.ac.uk/) and we dis- for cellular growth and differentiation [67,68]. Most RNA heli-

covered that several of them were differentially expressed cases belong to the DEAD-box family whose prototype is the

180 e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183

eukaryotic eIF-4A translation initiation factor [69,70]. We have both parasites. The up-regulation of these two ARL proteins

indeed identiﬁed many translation initiation factors differen- in metacyclic promastigotes is in agreement with the high

tially expressed when contrasting procyclic and metacyclic motility observed in this stage. Proteins such as coronins are

proteomes (LinJ.17.0090, LinJ.17.1390, LinJ.36.0200) including also thought to be involved in Leishmania intraﬂagellar traf-

eIF-4A (LinJ.01.0790) although some others were only detected ﬁcking and motility because of their actin binding capacity.

in comparison 4 (Fig. 1) (LinJ.18.0740 and LinJ.36.4070 in Table The coronin family comprises two groups of evolutionary

S2) and one (LinJ.08.0570 in Table S2) overproduced in the pro- conserved WD-repeat proteins [79] and three hypothetical

cyclic forms of WT cells. Tw o peptide chain/translation release proteins containing a number of WD-repeat motifs (WD40)

factors (LinJ.25.1510 and LinJ.27.1610 in Table S2) were also pin- (LinJ.06.0030, LinJ.25.1660 and LinJ.28.1210 in Table S4) have

pointed in our proteomic analysis. LinJ.27.1610 is increased been identiﬁed in metacyclic cells but their putative role in

4.68-fold in procyclic cells in the PTR1 null mutant suggest- metacyclogenesis and/or pterin metabolism will need further

ing that this protein may have a more important role in the investigations since they were detected only in comparison 4

procyclics rather than the metacyclic forms. (Fig. 1).

3.7. Motility and morphology-related proteins

3.8. Pterin metabolism

Several cytoskeleton and microtubules associated proteins are

shaping the morphology of the parasite Leishmania during pro-

As expected, PTR1 (LinJ.23.0310 in Table S1) was only

mastigote differentiation. This is supported by our analysis as

present in the metacyclic form of the WT but not in the

exempliﬁed by the i/6 autoantigen-like protein (LinJ.22.1310

PTR1 null mutant, showing the resolution power of our

in Table S3) that is probably involved in cross-linking of

proteomic approach. PTR1 belongs to the short-chain dehy-

microtubule ﬁlaments by other microtubule associated pro-

drogenases/reductases (SDRs) family of NAD(P)(H)-dependent

teins such as Gb4 (LinJ.26.1950 in Table S3), CAP5.5 protein

oxidoreductases. Sequence identities are low between mem-

(LinJ.31.0450 in Table S3) and the rib72 protein-like protein

bers, with 6 members encoded in the genome of L. infantum,

(LinJ.36.6660, Table S3). Alpha- and beta-tubulins are the most

including PTR1. Interestingly, we found a short chain dehy-

abundant ﬂagellar proteins in Leishmania and we detected

drogenase (LinJ.35.1240 in Table S1) that was overproduced in

several isoforms (e.g. ␣-tubulins LinJ.13.0330 and LinJ.13.1450

metacyclic PTR1 null mutant compared to metacyclic WT cells

and ␤-tubulins LinJ.08.1280, LinJ.08.1290 and LinJ.21.2240 in

(comparison 4 in Fig. 1).

Table S3) corresponding to these two main ﬂagellar compo-

nents. Extensive post-translational modiﬁcations have been

described for tubulins [22] which may explain the relative high

number of isoforms detected in our proteomic analysis. Sev- 4. Conclusion

eral of these isoforms were either unique or overproduced in

−/−

metacyclic parasites in both WT and PTR1 cells although This report represents a detailed proteomic analysis of the two

some were also speciﬁc or differentially expressed in the pro- main developmental stages of the Leishmania metacyclogene-

cyclic stage (Table S3). sis process, the procyclic and metacyclic stages, in a context of

The trypanosomatid ﬂagellum is also marked by a special a cell expressing or not PTR1. Several structural proteins and

structure called the paraﬂagellar rod (PFR) which is absent metabolic enzymes participating in at least six main metabolic

from amastigotes [71]. Interestingly, parts of the PFR were or cellular processes were shown to be particularly altered

previously found overproduced in metacyclic cells following when contrasting both forms. Also, numerous hypothetical

metacyclogenesis in Leishmania major [22]. Three PFR proteins proteins have been identiﬁed, some being highly upregulated

were identiﬁed in comparison 4 (Fig. 1) as being upregu- in metacyclic parasites (Table S4) while the expression pro-

lated in metacyclics PTR1 null mutant, namely LinJ.05.0040 ﬁles of others (e.g. LinJ.09.0890, LinJ.19.1470, LinJ.21.1830 and

(PAR4), LinJ.16.1510 (PFR2C), and LinJ.29.1880 (PFR1D) (Table LinJ.28.1210 in Table S4) were found exclusively in metacyclics

S3). Myosin-like proteins are now known to be components of of the PTR1 null mutant strain. These ﬁndings offer new

the ﬂagellum and PFR structures [71,72]. A myosin heavy chain grounds for the investigation of novel hypothetical proteins

kinase A like protein (LinJ.36.4780 in Table S3) was detected, potentially playing a role in metacyclogenesis and/or pterin

but the only peptides corresponding to this protein were found metabolism.

in the procyclic stage of the WT parasite (Table S3). Interest-

ingly, a MAP kinase (LinJ.10.0200 in Table S3) whose substrates

are probably kinesins [73–75] (see unique metacyclic peptides

from kinesins LinJ.14.1600 and LinJ.30.0350 in Table S3) was Biological signiﬁcance

also upregulated in the metacyclic stage of both WT cells and

the PTR1 null mutant. This proteomic study offers new grounds for the investiga-

A number of proteins including ADP-ribosylation factor tion of novel hypothetical proteins potentially playing a role

proteins (ARL, also known as small G-proteins [76–78]) play in metacyclogenesis and/or pterin metabolism. Also, it pro-

a pivotal role in intraflagellar protein trafficking, flagellum vides information about the metabolic pathways modulated

integrity and assembly. We have identiﬁed isoforms of two when contrasting procyclic to metacyclic stages. This knowl-

ARL proteins (LinJ.29.0950 and LinJ.30.2380 in Table S3) that edge may lead to the development of new strategies to block

seem to be uniquely expressed in the metacyclic forms of parasite differentiation.

e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183 181

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