Journal of Proteomics 232 (2021) 104077

Contents lists available at ScienceDirect

Journal of Proteomics
journal homepage: www.elsevier.com/locate/jprot

Quantitative analysis of proteins secreted by Leishmania (Viannia)

braziliensis strains associated to distinct clinical manifestations of American
Tegumentary Leishmaniasis
Andrés Rodríguez-Vega a, 1, 2, Monica Losada-Barragán b, 2, Luiz Ricardo Berbert c, 3,
Camila Mesquita-Rodrigues d, Ana Cristina Souza Bombaça e, Rubem Menna-Barreto e,
Priscila Aquino f, Paulo C. Carvalho g, Gabriel Padrón a, 4, Jose Batista de Jesus d, h,
Patricia Cuervo a, *
a
Laboratório de Pesquisa em Leishmanioses, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil
b
Grupo de Investigación en Biología Celular y Funcional e Ingeniería de Biomoléculas, Universidad Antonio Nariño, Bogotá, Colombia
c
Laboratório de Pesquisas sobre o Timo, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil
d
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil
e
Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil
f
Instituto Leônidas e Maria Deane, Fiocruz, Manaus, AM, Brazil
g
Laboratory for Structural and Computational Proteomics, Fiocruz-Paraná, PR, Brazil
h
Universidade Federal de São João Del Rei, São João del Rei, MG, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The role of Leishmania braziliensis in the development of different clinical forms of American Tegumentary
Leishmania braziliensis Leishmaniasis (ATL) is unclear, but it has been suggested that molecules secreted/released by parasites could
Secretome modulate the clinical outcome. Here, we analyzed the infection rate and cytokine profile of macrophages pre­
Localized cutaneous leishmaniasis
treated with the secretome of two L. braziliensis strains associated with polar clinical forms of ATL: one associated
Disseminated cutaneous leishmaniasis
Quantitative proteomics
with localized self-healing cutaneous leishmaniasis (LCL) and other associated with the disseminated form (DL).
Mass spectrometry Besides, we use an iTRAQ-based quantitative proteomics approach to compare the abundance of proteins
secreted by those strains. In vitro infection demonstrated that pretreatment with secretome resulted in higher
number of infected macrophages, as well as higher number of amastigotes per cell. Additionally, macrophages
pretreated with LCL secretome exhibited a proinflammatory profile, whereas those pretreated with the DL one
did not. These findings suggest that secretomes made macrophages more susceptible to infection and that
molecules secreted by each strain modulate, differentially, the macrophages’ cytokine profile. Indeed, prote­
omics analysis showed that the DL secretome is rich in molecules involved in macrophage deactivation, while is
poor in proteins that activate proinflammatory pathways. Together, our results reveal new molecules that may
contribute to the infection, persistence and dissemination of the parasite.
Significance: Leishmania braziliensis is associated to localized self-healing cutaneous lesions (LCL), disseminated
leishmaniasis (DL), and mucocutaneous lesions (MCL). To understand the role of the parasite in those distinct
clinical manifestations we evaluated infection rates and cytokine profiles of macrophages pre-treated with
secretomes of two L. braziliensis strains associated with DL and LCL, and quantitatively compared these secre­
tomes. The infection index of macrophages pretreated with the DL secretome was significantly higher than that
exhibited by non-treated cells. Interestingly, whereas the LCL secretome stimulated a proinflammatory setting,

* Corresponding author at: Laboratório de Pesquisa em Leishmanioses, Instituto Oswaldo Cruz, FIOCRUZ. Av. Brasil 4365, Manguinhos, 21040-360, Rio de Janeiro,
RJ, Brazil.
E-mail address: patricia.cuervo@fiocruz.br (P. Cuervo).
1
Current Address: Laboratório de Neurofisiologia, Departamento de Ciências Fisiológicas, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico,
Universidade do Estado do Rio de Janeiro.
2
These authors contributed equally to this work.
3
Current Address: Coordenação de Atividades com Modelos Biológicos Experimentais - Decania do Centro de Ciências da Saúde - UFRJ
4
Current Address: Center for Genetic Engineering & Biotechnology, La Habana, Cuba

https://doi.org/10.1016/j.jprot.2020.104077
Received 31 August 2020; Received in revised form 1 December 2020; Accepted 6 December 2020
Available online 10 December 2020
1874-3919/© 2020 Elsevier B.V. All rights reserved.
A. Rodríguez-Vega et al.
favoring an effector cell response that would explain the proper resolution of the disease caused by this strain,
the DL strain was not able to elicit such response or has mechanisms to prevent this activation. Indeed, DL
secretome is rich in peptidases that may deactivate cell pathways crucial for parasite elimination, while is poor in
proteins that could activate proinflammatory pathways, favoring parasite infection and persistence.
1. Introduction
The American tegumentary leishmaniasis (ATL) comprises a group of
diseases caused by multiple Leishmania species and with a broad spec­
trum of clinical manifestations and therapy response [1,2]. Near one
million cases of ATL were reported in the Americas during the last two
decades, with an average of ~55,000 cases every year [3,4].
Leishmania (Viannia) braziliensis is one of the main species respon­
sible for ATL in the continent. This species is associated with different
clinical forms ranging from localized self-healing cutaneous lesions
(localized cutaneous leishmaniasis, LCL) to multiple cutaneous lesions
and/or oropharyngeal mucosa compromise. The persistence of the
parasite after the appearance of the single initial lesion and its capability
to metastasize, are remarks of the infection with this species. Lymphatic
or haemotogenous metastatic dissemination of parasites may result in
lesions in the oropharyngeal mucosa and cartilage (mucocutaneous
leishmaniasis, MCL), which occurs in 1–10% of infections, or may result
in the appearance of multiple skin lesions (disseminated leishmaniasis,
DL), frequently refractory to treatment [5–9]. Remarkable, cutaneous
and mucocutaneous lesions can lead to cosmetic disfiguration with
consequent social stigmatization and psychological sequels [10,11]. In
the severe forms of MCL, it can lead to the destruction of the nasal
septum and palate, resulting in a highly, life-threatening, disfiguring
condition. Disseminated leishmaniasis is characterized by ten or more
cutaneous lesions located in two or more parts of the body, few parasites
in the lesions, and a high frequency of mucosal involvement [12–18].
The clinical pleiomorphism observed in infections with L.braziliensis
results from the complex interaction of particular features of the para­
sites and host’ immune response. While proinflammatory cytokines such
as TNF-α and IFN-γ are associated with lesion resolution in the case of
localized CL, high levels of those cytokines lead to the destruction of
mucosal tissue in the case of MCL and systemic decreased levels of the
same cytokines seem to be associated with DL, indicating a weak
response mediated by T cells that leads to a failure in the control of
parasites but favors little tissue destruction [15,17,19]. Nevertheless,
the mechanisms involved in the progression of the skin lesion to MCL or
DL forms are still not well understood. How does L. braziliensis metas­
tasize? How is it explained that only in some infections does the parasite
disseminate? The parasite’s role in the dissemination process is not yet
clear, but it is suggested that the intrinsic characteristics of some strains
may modulate the clinical outcome. Studies in a L. braziliensis-rhesus
model showed that infection with a strain isolated from an LCL patient
resulted in self-resolution [20], whereas infection with a strain from a
DL patient caused multiple disseminated lesions in 100% of the monkeys
[21]. These observations suggest that the parasites that disseminated
can be distinguished from those that do not and that such strains,
associated with very polar clinical manifestations [20,21], represent a
good model for studying the parasite’ factors involved in dissemination
and clinical outcome.
Secreted molecules, in particular proteins, are key mediators of
communication between Leishmania and host cells and participate in
several physiological processes, such as cell signaling, differentiation,
invasion, dissemination, among others [22–24]. Indeed, during the
description of the L. braziliensis secretome, our group identified several
virulence factors and molecules involved in immunomodulation, rein­
forcing the idea that inherent features of parasites could have a role in
the clinical outcome [25]. Accordingly, studies of different Leishmania
spp. showed that proteins secreted in the first moments of the infection
have an essential role in the recruitment of neutrophils, macrophages,
2
Journal of Proteomics 232 (2021) 104077
and other professional phagocytic cells to the infection site. Once the
parasites come into contact with the phagocytic cells and/or are inter­
nalized, they secrete several molecules that can modulate the activation
of the macrophage, subverting the beginning of an appropriate response
to the infection [26,27].
Based on those evidence, we hypothesize that proteins secreted by
L. braziliensis specifically during the initial process of macrophages
infection may modulate the activation profile of the cells, contributing
to the disparity in the clinical outcome of infections with this species. In
order to evaluate this, here we studied the secretomes of two different L.
braziliensis strains: one isolated from a patient with LCL and one isolated
from a patient with DL. We evaluated the effect of the secretomes of each
strain on the infection rate and cytokine profile of peritoneal macro­
phages in vitro, as well as using an iTRAQ-based quantitative proteomics
approach we identified and compared the relative abundance of proteins
secreted by each strain. Our data provide evidence on the differential
infection rate and inflammatory profile of macrophages pre-stimulated
with the secretion of each strain. In addition, we describe the proteins
in those secretomes, their relative abundance, and the main quantitative
differences between those strains.
2. Materials and methods
2.1. Ethics statement
This study was carried out in accordance with the recommendations
of the Guide for the Care and Use of Laboratory Animals of the National
Institutes of Health (NIH Publications No. 8023, revised 1978). All an­
imal procedures were approved by the Instituto Oswaldo Cruz Animal
Care and Use Committee (License # L-005/2017).
2.2. Parasite culture
The L.braziliensis strains used in this study were provided by the
Collection of Leishmania of the Instituto Oswaldo Cruz (Coleção de
Leishmania do Instituto Oswaldo Cruz, CLIOC, http://clioc.fiocruz.br/).
The strain MHOM/BR/2000/LTCP13396 associated with DL and the
strain MHOM/BR/2000/LTCP13490 associated with LCL were isolated
in the same geographical region, belong to the same zymodema, had
their in vitro passages controlled and their infectious capacity main­
tained through inoculation in golden hamsters. As mentioned above,
such strains reproduced the clinical phenotype in the rhesus model
[20,21]. Through the text, tables, and figures, those strains will be
mentioned as DL and LCL. Promastigotes were cultivated at 25 ◦ C in
biphasic NNN- Schneider’s medium (Vitrocell) supplemented with 20%
fetal bovine serum (FBS) (Vitrocell, heat-inactivated at 56 ◦ C for 50 min)
at 25 ◦ C. According to the Brazilian Law of Biodiversity, this study was
registered at SisGen (AA2236F).
2.3. Secretion assays
Secretion assays were performed as previously described [25], with
minor modifications. Briefly, 5 × 108 promastigotes were centrifugated
and washed twice with PBS (0.01 M phosphate-buffered 0.145 M NaCl,
pH 7.2). Parasites were resuspended in 5 mL of RPMI 1640 medium
(Gibco) supplemented with 25 mM HEPES and incubated for 3 h at
25 ◦ C. Before used, RPMI supplemented medium was tested for endo­
toxin using the commercial kit endosafe (Charles River Labs). Subse­
quently, supernatants were collected by centrifugation at 1800 xg for 10
A. Rodríguez-Vega et al.
min for parasites removal followed by filtration through a 0.2 μm filter
unit (Millipore, Bedford, MA) for debris removal. The conditioned me­
dium so obtained contains the secretome that was used in further ex­
periments. The assays were carried out in biological quadruplicates.
During the secretion assay, parasites’ viability was evaluated every
hour by flow cytometry using propidium iodide (PI) labeling. For posi­
tive cell death control, parasites were permeabilized with 1% BSA and
0.1% saponin for 15 min at 25 ◦ C. Samples were incubated with 2 μL of
PI (1 mg/mL) at 25 ◦ C for 20 min protected from light. Acquisition
(10,000 events) was performed on a FACSCanto™ flow cytometer
(Becton Dickinson, USA). Off-line analysis was made using the Sum­
mit™ software.
2.4. Peritoneal macrophages infection and treatment with secretomes
To evaluate whether the secretome of each L.braziliensis strain could
modulate the percentage of infection of macrophages in vitro, murine
peritoneal macrophages were pre-stimulated with secretomes obtained
as described above, infected, and further analyzed by microscopy.
Macrophages were obtained by peritoneal washes from Swiss mice,
resuspended in RPMI 1640 supplemented with 10% SFB, 4 mM L-
glutamine, 1000 U/mL penicillin and 50 μg/mL streptomycin, and
grown on plates containing sterile glass coverslips for 24 h at 37 ◦ C and
5% CO2. Further, cells were stimulated for 24 h with 0.6 μg of the
endotoxin-free secretome from DL or LCL strain, which corresponds to
the quantity of protein secreted by 106 parasites, resembling a MOI of
10:1 (parasite:macrophage). Control macrophages were incubated with
supplemented RPMI for 24 h. After stimulation, the conditioned medium
was removed and macrophages (control and treated) were infected with
DL or LCL strain at a MOI of 10:1 and incubated for 3 h. Infected mac­
rophages were further washed to remove non-internalized promasti­
gotes and incubated for 24, 48, and 72 h. Cell-free supernatants were
collected at the indicated times and stored at − 80 ◦ C for further cytokine
quantitation. For calculation of the infection rate, coverslips were
stained with panoptic staining, and cells were examined by light mi­
croscopy (Nikon Eclipse E400-Tokyo, Japan) (60× objective). The
number of infected cells, as well as the number of amastigotes per cell,
were assessed. Assays were performed in triplicate.
2.5. Cytokine quantitation
Supernatants collected from peritoneal macrophages at indicated
times were analyzed by flow cytometry using a mouse CBA inflamma­
tion kit (Cytometric Beads Array, Cat No. 552364, Becton Dickinson,
USA) for quantification of IL-6, IL-10, MCP-1/CCL2, IFN-γ, TNF-α, and
IL-12p70. Samples were prepared according to the manufacturer’s in­
structions and acquired on a FACSCanto™ flow cytometer (Becton
Dickinson, USA). Data were analyzed using FCAP Array Software v3.0 ™
software.
2.6. ITRAQ-based protein quantification
Secretomes were concentrated to a final volume of 100 μL using
centrifugation filters 3 K (Millipore), and proteins were precipitated
with trichloroacetic acid (15% v/v) for 2 h at 4 ◦ C and washed twice with
cold acetone. Proteins were resuspended in triethylammonium bicar­
bonate buffer (TEAB) 50 mM pH 8.5, dosed using a Qubit® fluorometer
(Invitrogen) and processed as described previously [28]. Briefly, pro­
teins were treated with 0.1% of Rapigest (Waters), reduced by the
addition of 20 mM tris(2-carboxyethyl)-phosphine (TCEP) at 60 ◦ C for
30 min, alkylated with iodoacetamide (IAA) 20 mM at 25 ◦ C for 30 min.
Afterward, samples were digested sequentially with lysyl endopeptidase
mass spectrometry grade (Wako) and sequencing grade trypsin (Prom­
ega). Peptides were desalted in a spin column C18 (Harvard Apparatus,
USA), dried by vacuum centrifugation and resuspended in TEAB 50 mM.
Four biological replicates of secretomes of each strain were labeled
3
Journal of Proteomics 232 (2021) 104077
with iTRAQ 4-plex (AB Sciex, Framingham, MA, USA) according to the
instruction of the manufacturer. Labeling proceeded as follows: two
biological replicates of DL secretomes were labeled with iTRAQ114 and
two were labeled with iTRAQ116, whereas two biological replicates of
LCL secretomes were labeled with iTRAQ115 and two with iTRAQ117.
Labeled peptides were mixed, pooling one sample of each label, total­
izing two final mixes. Both pools were fractionated by strong cation
exchange using Macro Spin Columns (Harvard Apparatus, USA), and the
two resulting fractions were dried in a vacuum centrifuge system and
desalted using Spin Column C18 (Harvard Apparatus, USA).
2.7. LC-MS/MS analysis
Samples were analyzed by nano-liquid chromatography (Easy-nLC
1000, Thermo Fischer Scientific) coupled to tandem mass spectrometry
(nLC-MS/MS) in the mass spectrometry facility of Carlos Chagas Insti­
tute - Fiocruz Paraná (PDTIS-RPT02H). Samples were injected in
duplicate. Peptides were loaded onto a reverse phase column (30 cm
long, 75 μm inner diameter) packed in-house using a flow of 500 nL/min
ReprosilPur C18 Acqua (beads 1.9 μm in diameter, Dr. Maisch). Peptide
elution followed a flowrate of 250 nL/min, and a gradient of 5–40% of
phase B (5% DMSO, 0.1% formic acid in acetonitrile) for 180 min. Phase
A was 5% DMSO in 0.1% formic acid. Data were acquired in data-
dependent acquisition mode, automatically switching between full
scan MS (m/z 300–2000) at a resolution of 60,000 (m/z 100) and MS/
MS acquisition, with a dynamic exclusion of 90 s in the LTQ Orbitrap XL-
ETD mass spectrometer (Thermo Fisher Scientific). For each MS spec­
trum, up to the five more intense ions with +2 and + 3 charges were
isolated and fragmented through high energy collision dissociation
(HCD) with a normalized collision energy of 45 and a 30 ms activation
time. Xcalibur 2.0 software (Thermo Fischer Scientific) was used to
control the scan functions and solvent gradients in the nLC.
2.8. Protein identification and quantification
Proteins were identified using the software PatternLab for Prote­
omics (V.3.2.0.3) (http://patternlabforproteomics.org/) and a custom­
ized database containing protein sequences available for Leishmania spp.
at UniProt in January 2017 (http://www.uniprot.org/, [29]). Peptide
and protein false identification rate (FDR) was set at 1% based on the
target-decoy reverse database, including also common contaminants
available in the PatternLab’s Search Database Generator [30]. Search
parameters used for identification were: tryptic and semi-tryptic pep­
tides; three missed cleavages; fixed modification of cysteine carbami­
domethylation; fixed iTRAQ (+144.1 Da) at the N-terminal; variable
iTRAQ modification (+144.1 Da) in lysine (K) side chain; and 40 ppm
mass tolerance of the precursor. Peptide-spectrum matches (PSM) were
validated with the Search Engine Processor (SEPro), in PatternLab’s
environment [30]. Identifications were grouped by charge state (2+ and
3+) and then by a fully tryptic or semi-tryptic state. Six amino acid
residues were set as a minimum for sequence length. A bayesian
discriminator for each result was produced based on the ProLuCID
XCorr, DeltaCN, and ZScore values. In addition, results with two or more
evidence of the protein in the sample and sequences with less than five
ppm of variation at the protein level were accepted. Identified proteins
in the database corresponding to L. braziliensis species, as well as some
other Leishmania species, were accepted and cured manually.
Relative quantification of proteins was made as described previously
[28], using the ‘Isobaric Analyzer’ module of PatternLab [31,32]. A two-
class analysis was made using the quantified peptides for comparing DL
strain (iTRAQ114 and iTRAQ116) to LCL strain (iTRAQ115 and
iTRAQ117 labels). A cutoff of at least two unique peptides per protein,
0.30 of “peptide Log Fold Change Cutoff” and 0.05 of “Peptide p-value
Cutoff” for the t-paired test or p binomial value was set. p-value was
calculated according to the Benjamini-Hochberg procedure [32]. Pro­
teins with a fold change of abundance ≥1.5 and p ≤ 0.05 were accepted.
A. Rodríguez-Vega et al.
Identified proteins were classified according to cellular component,
molecular function, and biological process through Gene Ontology
database (http://geneontology.org/) using the Gene Ontology Explorer
tool (GOEx) from the PatternLab [31]. Interactome network analysis
was built using the web-accessible program IIS- Integrated Interactome
system (http://www.lge.ibi.unicamp.br/lnbio/IIS/) [33]. The enrich­
ment level of proteins with any annotation of the biological process was
calculated using the orthologous coding genes in humans. Data were
visualized using Cytoscape version 2.8.3 (http://www.cytoscape.org/).
2.9. Bioinformatics analysis of secreted proteins
Different servers and databases were used to analyze functional do­
mains, secretion pathways, and exosomal origin of the identified pro­
teins. Prediction of functional domains for uncharacterized proteins was
made using Pfam V27.0 (http://pfam.xfam.org/) [34]. SignalP 4.1 (http
://www.cbs.dtu.dk/services/SignalP-4.1/) was used for the prediction
of N-terminal signal peptide [35]. Potential non-classical secretion
pathways and potential subcellular location were predicted with
SecretomeP 2.0 (http://www.cbs.dtu.dk/services/SecretomeP/) [36]
and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) [37],
respectively. The TMHMM v.2.0 server (http://www.cbs.dtu.dk/servic
es/TMHMM/) [38] was used for the prediction of transmembrane do­
mains. Finally, our dataset was compared against the ExoCarta database
(http://www.exocarta.org/) [39] to determine the potential exosomal
origin of the identified proteins.
2.10. Gene expression analysis
To assess whether the differential protein abundance in the secre­
tome could correlate with the relative abundance of their transcripts, a
gene expression analysis was performed for six genes of both strains
using quantitative real-time PCR (qPCR) following protocols previously
described [40]. Total RNA was extracted from promastigotes using
Trizol; RNA was quantified, and the cDNA was synthesized using the
SuperScript III reverse transcription system (Invitrogen). qPCR was
performed using a StepOne equipment and SYBR® Green PCR Master
Mix (Applied Biosystems). Oligonucleotide sequences of the target and
reference genes were designed from available public sequences using
Primer Blast software (Supplementary Table 1). PCR consisted of 40
cycles of 10 s at 95 ◦ C, 15 s at 56 ◦ C and 10 s at 72 ◦ C, followed by
melting curve analyzes in order to verify the reaction specificity. Stan­
dard curves for all genes were generated using serial cDNA dilutions
obtained from all samples. Gene expression was quantified using the
comparative Ct method (ΔΔCt). Data are shown as normalized re­
lationships between target gene expression and geometric mean refer­
ence genes [41]. The experiments followed the Minimum Information
for Publication of Quantitative Real-Time PCR Experiments (MIQE)
guidelines [41,42].
2.11. Statistical analysis
Statistical analysis was performed using GraphPad Prism 5.0 soft­
ware. To analyze the differences between the two conditions studied,
Student’s t-test was applied. Data are presented as means ± standard
deviation.
3. Results
3.1. The secretome of L. braziliensis increases the infection rate of
peritoneal macrophages in vitro
First, to ensure that during the 3 h of secretion assays the proteins
found in the conditioned medium are derived from active secretion
rather than released by cell death, parasite viability was measured every
hour by flow cytometry through PI labeling. Both DL and LCL strains
4
Journal of Proteomics 232 (2021) 104077
displayed viability above 97% after 1, 2, and 3 h of secretion (Supple­
mentary Fig. 1).
To assess whether the secretome from DL or LCL strains could
differentially influence the infection rate, peritoneal macrophages were
treated with the secretome of each strain and subsequently were infec­
ted. The treatment with parasite secretome, DL or LCL, before challenge
with DL or LCL parasites significantly increased the number of infected
cells at all analyzed times for both strains (Fig. 1A). Also, the number of
parasites per cell at 24 and 72 h post-infection with DL strain, and at 48 h
post-infection with LCL strain, was significantly increased when mac­
rophages were pretreated with secretion (Fig. 1B). Remarkably, the
infection index of macrophages pretreated with the DL secretome was
significantly higher than that exhibited by non-treated macrophages in
all the times evaluated (Fig. 1C). For macrophages pretreated with LCL
secretome, the infection index was higher than for non-treated ones only
at 48 h.
3.2. LCL secretome and infection stimulated a proinflammatory cytokine
profile in peritoneal macrophages in vitro whereas DL secretome did not
As we observed that the infection index of peritoneal macrophages
was significantly increased when the cells were pretreated with the
secretome of DL strain, we decided to evaluate the cytokine profile of
those macrophages and compare it to the profile of macrophages stim­
ulated with LCL secretome. We observed significant differences in the
levels of cytokines produced by pretreated-infected macrophages
compared to untreated-infected ones, as well as significant differences
between the cytokine profiles induced by each secretome (DL or LCL).
Macrophages pre-stimulated with LCL secretome and further infec­
ted with that strain showed significantly higher levels of TNF-α and IL-6
(p < 0.05) compared with the levels measured for macrophages pre­
treated and infected with DL strain, at all analyzed times (Fig. 2). TNF-α
and IL-6 levels were 4 to 8-fold higher in the LCL pretreated-infected
cells than in the DL pretreated-infected macrophages (Fig. 2). Interest­
ingly, higher levels of those cytokines were also observed when unsti­
mulated macrophages were infected with LCL strain; however, the TNF-
α and IL-6 concentrations were 2-fold lower than those observed in pre-
stimulated macrophages (Figs. 2 and 3). In addition, at 72 h post-
infection, the concentration of CCL2 was significantly higher (3-fold)
in macrophages pre-treated and infected with LCL strain than in those
stimulated with DL secretome and infected (Fig. 2, p < 0.001). The
concentration of CCL2 was also significantly higher in untreated mac­
rophages infected with LCL than in cells infected with DL strain (Fig. 3).
Remarkably, although IL-12 and IL-10 were detected at 24 h and 48 h
in macrophages pre-stimulated with DL secretome and infected, the
levels of those cytokines were abrogated at 72 h post-infection. More­
over, IL-12 and IFN-γ levels were almost undetectable in cells pre-
treated and infected with LCL strain (Fig. 2) at all times evaluated
here, whereas IL-10 was significantly higher in those cells, at 72 h post-
infection, when compared to macrophages pre-treated and infected with
DL strain. In contrast, unstimulated macrophages infected with LCL
strain displayed detectable levels of IL-12, IL-10, and IFN-γ at all
analyzed times. In contrast, those cytokines were barely detected in
untreated macrophages infected with DL strain (Fig. 3).
3.2.1. Secretome contains proteins secreted mainly by alternative pathways
involving exosomes
As the secretomes of the LCL and DL strains differentially altered the
infection rate and cytokine profile of macrophages pre-treated with
them, we hypothesized that the proteins secreted by each strain can
differentially modulate the host’s immune response, leading to polarized
clinical manifestations. Therefore, we applied iTRAQ labeling to identify
and quantify the differences in protein abundance between LCL and DL
secretomes. A total of 1176 peptides identified were assigned to 252
proteins at 1% FDR (Supplementary Table 2). From these proteins, 80%
(201/252) were identified with two peptides at least, while 20% (51/
A. Rodríguez-Vega et al. Journal of Proteomics 232 (2021) 104077

Fig. 1. Effect of pre-treatment with L. (V.) braziliensis secretome on in vitro macrophage infection. Murine peritoneal macrophages were pre-treated with secretion of
each L. (V.) braziliensis strain and infected with promastigotes of the corresponding strain. Infection was followed at 24, 48 and 72 h post infection. (A) Percentage of
infected cells; (B) number of parasites per macrophage; (C) infection index. Statistical differences between control and pretreated cells were analyzed by Student’s t-
test (*p < 0.05, **p < 0.005 and ***p < 0.001) (n = 3). Graphs represent mean ± standard deviation.

252) were identified with a single peptide (Supplementary Table 2).
Proteins identified with only one peptide were accepted since there was
more than one evidence of their presence in the sample, such as two or
more spectra and two or more charge states.
Bona fide secreted proteins were identified in the L. braziliensis
secretome, including GP63, secreted acid phosphatase, disulfide isom­
erase protein, and activated protein kinase c receptor, among others
(Supplementary Table 2). Bioinformatics analyses showed that ~80%
identified proteins could be secreted by either classical or alternatives
routes, and for several proteins, one or more pathways were predicted
(Supplementary Table 2 and Fig. 4). In addition, those predictions
showed that the predominant pathway of secretion involves exosomes
(Supplementary Table 2 and Fig. 4). TMHMM analysis showed that ~9%
of identified proteins contain at least one transmembrane domain
(Supplementary Table 2). However, ~6% of those proteins also can be
secreted by classical or alternative pathways according to the pre­
dictions obtained by SignalP, SecretomeP and/or exocarta, suggesting
that they could be truly exported to the extracellular milieu. The other
~3% proteins could be part of apoptotic vesicles or shedding products,
known to be part of the parasite secretion and important for the infective
process [24]. Finally, for ~20% proteins, it was not possible to predict
any signal of secretion with the tools used here, but several of them have
been found enriched in the conditioned medium of several Leishmania
spp. [24,25,43].
To obtain information about the potential function of the unchar­
acterized proteins, we analyzed functional domains using the Pfam 27.0
server. We found at least one functional domain for some regions of the
proteins in ~45% of the cases (25/56). Among these, we highlight heat
shock proteins, thioredoxin, and phosphatase domains, among others
(Supplementary Table 3).

5
A. Rodríguez-Vega et al. Journal of Proteomics 232 (2021) 104077

Fig. 2. Cytokines secreted by murine macrophages pre-treated with secretome of the DL or LCL strain of L. (V.) braziliensis and infected. Cytokines were quantified by
flow cytometry in supernatants of macrophages pre-treated with the secretome of each strain and infected with the homologous strain. Statistically significant
differences between control and treatment were calculated using the Student’s t-test, using the GraphPad Prism 5.0 program. (*) p < 0.04 and (**) p < 0.008; (***) p
< 0.0009. The bars represent mean ± standard deviation.

3.2.2. Proteins secreted by L. braziliensis are mainly involved in catalytic
activities
In order to obtain more information about the function of identified
proteins, we retrieved their annotations regarding the main gene
ontology categories: biological process, molecular function, and cellular
component (Supplementary Fig. 2). We observed that 71% (180/252)
proteins have an annotation in at least one of the three categories, while
~29% (72/252) of the proteins did not have an associated classification.
According to the molecular function annotations, the subcategories
associated with catalytic activity (44%) or binding (40%) were more
represented in our dataset. Ninety-four proteins were classified into six
different categories: (i) stress response and protein folding, (ii) carbohy­
drate metabolism, (iii) amino acid metabolism, (iv) nucleotide metabolism,
(v) protein degradation and (vi) protein translation and synthesis.
Furthermore, the ontological classification allowed us to focus on cate­
gories of interest for the study of the first interactions of the parasite
with host cells. In response to stress and protein folding, for example, 18
proteins were found, including heat shock proteins and proteins of the
redox metabolism such as SOD, trypanothione reductase, and trypar­
edoxin peroxidase, among others (Table 1), and in the case of proteins
involved in protein degradation, 17 were identified, including peptidases
recognized for their role in virulence, such as GP63 and calpain-like
cysteine peptidases (Table 1).
3.3. LCL and DL associated strains show significant differences in protein
abundance in their secretomes
To gain insights into the quantitative differences of protein abun­
dance between the two strains of L. braziliensis studied here, we used a
combination of stable isotope labeling with tandem mass spectrometry
to compare the relative abundance of protein in the secretome of LCL
and DL strains. Differences in the abundance of secreted proteins be­
tween the strains were calculated in the Quant-Isobaric analyzer module
of PatternLab by comparing the iTRAQ normalized reporter ion in­
tensities. A total of 26 proteins showed significant differences in abun­
dance between the strains (Table 2). The relative abundance of 13

6
A. Rodríguez-Vega et al. Journal of Proteomics 232 (2021) 104077

Fig. 3. Cytokines secreted by murine macrophages infected with DL or LCL strain of L. (V.) braziliensis. Cytokines were quantified by flow cytometry in macrophages
infected with the DL or LCL strain. Statistically significant differences between control and treatment were calculated using the Student’s t-test, using the GraphPad
Prism 5.0 program. (*) p < 0.03; (**) p < 0.006 and (***) p < 0.0009. The bars represent mean ± standard deviation.

proteins was increased in the DL strain compared to the LCL strain,
whereas the relative abundance of 13 proteins was decreased (Table 2).
Interestingly, among the proteins with increased abundance in the
secretome of the DL strain, there are several proteins involved in the
response to stress and redox homeostasis, such as the thioredoxin-like_fold
domain containing protein and a trypanothione reductase. We also
observed that proteins involved in protein degradation and cell adhesion
such as the dipeptidyl-peptidase 3 and the uncharacterized protein
A4H3T9, as well as in translation and protein synthesis such as the 40S
ribosomal protein S3, T-complex protein 1, eta subunit and T-complex
protein 1, theta subunit, presented higher abundance in DL secretome
than LCL one. Interestingly, an uncharacterized protein with increased
abundance in the secretome of DL strain (protein A4HAX5) has an Alba
domain (Table 2 and Supplementary Table 3), which has been involved
in parasite differentiation process as well as in binding to RNA and
regulation of virulence factors [44,45].
As the canonical function described for various of those proteins do
not allow understanding their role in the extracellular milieu, we
analyzed if those proteins could have an alternative function previously
described. Remarkably, four proteins that were found differentially
abundant between the strains showed similarity with multitasking
proteins described in other organisms (Supplementary Table 4). Dihy­
drolipoyl dehydrogenase and 60S acidic ribosomal protein P2, which
were less abundant in the secretome of the DL strain, have moonlighting
functions as protease and iron-binding protein, respectively (Table 2 and
Supplementary Table 4). In addition, the elongation factor 1-alpha, and
40S ribosomal protein S3, which were more abundant in the secretome
of the DL strain, have alternative functions as cytoskeleton structure

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A. Rodríguez-Vega et al. Journal of Proteomics 232 (2021) 104077

Table 1
Proteins identified in the secretion of L. (V.) braziliensis and classified in pro­
cesses that are relevant for parasite-host interaction.
Access number Name Number of
peptides

Stress response and protein

folding
A0A088RUF5 Glucose-regulated protein 78, 7
A4HII0 Heat shock 70-related protein 1, 12
mitochondrial
A4H9P0 Heat shock protein 10
A4HL70 Heat shock protein 83–1 18
A0A088RU16 Heat shock protein DNAJ, 5
A4HGY1 Heat-shock protein hsp70 21
A4HKX2 T-complex protein 1 subunit alpha 6
E9AIQ3 T-complex protein 1 subunit delta 5
E9AWE0 T-complex protein 1 subunit gamma 7
Fig. 4. Prediction of the secretion pathways for the proteins identified in the A4HN57 T-complex protein 1, eta subunit 4
L. (V.) braziliensis secretome. The figure shows the percentage of proteins with A4HQL2 T-complex protein 1, theta subunit 4
prediction for secretion via classical pathway (Signal P), alternative pathways A4H879 Tryparedoxin peroxidase 6
A4HKH1 Superoxide dismutase 3
(Secretome P), alternative-exosomal pathway (SecretomaP-exo), exosomal
E9JUH4 Trypanothione reductase 8
pathway (Exo), or without prediction.
A4HHC7 Tryparedoxin 4
A4HFW1 Trypanothione synthetase 1
regulator and DNA repair, respectively (Table 2 and Supplementary A4HCL7 Peroxidoxin 7
A0A088RZE8 Thiol-dependent reductase 1 5
Table 4). Interestingly, in L. donovani was described other alternative
Proteins involved in protein
function for the elongation factor 1-alpha as a peptidase [46], thus, for degradation
that protein, at least three different functions have been described. A0A088RI51 20S proteasome beta 6 subunit, 2
A4H6I8 Aminopeptidase 11
A4HMQ9 Aminopeptidase P 2
3.4. Functional interaction network of differentially abundant proteins in
A4HFH6 Calpain-like cysteine peptidase 25
the secretome of L. (V.) braziliensis strains Q4QCS4 Calpain-like cysteine peptidase, Clan 1
CA, family C2
To explore the biological processes of differentially abundant pro­ A4H716 Carboxypeptidase 11
teins in the secretome of DL and LCL strains, we also performed a A0A088RIB2 Dipeptidyl-peptidase III, 17
A4H638 GP63, leishmanolysin 15
functional interaction analysis. Our dataset of differential proteins was A4H5Q8 Oligopeptidase b 8
used to identify the orthologous proteins in Homo sapiens, and these A4HJV3 Phosphoglycan beta 1,3 6
were used to build a functional interaction analysis using the IIS- galactosyltransferase 5
interactome system web-accessible software. From the 26 differentially A4HQJ7 Prolyl oligopeptidase 3
A4HMA7 Proteasome activator protein pa26 3
abundant proteins, 15 presented orthologous in humans (Table 2), and
A4H9T4 Proteasome alpha 7 subunit 5
they were used for the interaction network (Fig. 5). The secretion of the A4HNP0 Proteasome beta 2 subunit 3
disseminated strain displayed two defined groups of proteins with A4HC34 Proteasome subunit alpha type 5
increased abundance: cellular protein metabolic processes (orthologs A4HP29 Proteasome subunit beta type 5
HSPA5, CCT8, CCT7) (Fig. 5), and gene expression (orthologs RPS3 and A4HF12 Thimet oligopeptidase 7
Proteins associated with
EEF1A1). Proteins with increased abundance (orthologs GOT1 and GSP) carbohydrate metabolism
and proteins with decreased abundance (orthologs MTAP, NME1, A4HQI8 2,3-bisphosphoglycerate-independent 6
AHCY, DLD) were grouped in the small molecule metabolic process cluster phosphoglycerate mutase
(Fig. 5). The remaining proteins were clustered in different functional A4H7T6 Enolase 9
A4HNY6 Fructose-bisphosphate aldolase 3
groups such as innate immune response, proteolysis, viral process, and
A4HMU1 Galactokinase-like protein 1
protein folding (Fig. 5). A4H643 Glycerol-3-phosphate dehydrogenase 7
Some proteins with increased abundance in the DL strain have direct [NAD(+)]
interactions between them: RPS3-YWHAE, RPS3-HSPA5 and CCT7- A4HFV1 Glycosomal phosphoenolpyruvate 2
CCT8 (Fig. 5). The first interaction corresponds to a functional associ­ carboxykinase
A7UFI6 Malate dehydrogenase 11
ation between gene expression (RPS3) and viral process (YWHAE); the A4HCQ1 NADP-dependent alcohol 2
second corresponds to the interaction between gene expression and pro­ dehydrogenase
tein metabolic processes (HSPA5); and the third one corresponds to an A4HBH9 Phosphoglycerate kinase 4
interaction between proteins involved in protein metabolic processes A0A088S299 Phosphomannomutase, 6
A4HM36 Pyruvate kinase 6
(CCT7 and CCT8). Interestingly, the proteins with increased abundance
A4H6M6 Pyruvate phosphate dikinase 2
showed a higher number of direct interactions with other proteins than A4H8J8 Transaldolase 4
those with reduced abundance. HSPA5 presented a total of 154 in­ A0A088SAQ9 Triosephosphate isomerase 6
teractions followed by EEF1A1 with 138, RPS3 with 121, and YWHAE A4HLU3 Udp-glc 4′ -epimerase 5
with 100 (Table 2). Amino acid metabolism
A4HQG9 Histidine secretory acid phosphatase 6
Most of the proteins with reduced abundance in the secretome of the A4H9Q9 Nonspecific nucleoside hydrolase 3
DL strain were not assigned to functional clusters but displayed in­ A0A088RTT3 Nucleoside 2-deoxyribosyltransferase, 3
teractions with different kinds of proteins (Fig. 5). We found, for A4HKT7 Nucleoside diphosphate kinase 8
example, that CALM1 (ortholog of calmodulin) contains the highest A4H4C5 S-methyl-5′ -thioadenosine 3
phosphorylase
number of interactions with other proteins (84 interactions), including
Nucleotide metabolism
proteins involved with regulation of gene expression and innate immune A4HPQ8 Adenosylhomocysteinase 16
response. In addition, three direct interactions were observed between (continued on next page)
increased (I) and decreased proteins(D): CALM1 (D)-YWHAE (I), DLD

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A. Rodríguez-Vega et al. Journal of Proteomics 232 (2021) 104077

Table 1 (continued ) LCL strain. Conversely, the tyrosine aminotransferase (tat) and Glucose-
Access number Name Number of regulated protein 78 (Lpmp_281290) genes showed similar mRNA levels
peptides between the strains, whereas its protein abundance was significantly
A4HMB3 Aspartate aminotransferase 10
increased in the DL strain in comparison with the LCL strain (Fig. 6).
A0A088S0Y7 Cystathione gamma lyase, 5 Finally, we compared the gene expression of the uncharacterized protein
A4H3J2 Delta-1-pyrroline-5-carboxylate 4 (Lbrm_26_2610) that exhibited similar protein abundances between the
dehydrogenase DL and LCL strains in our dataset and, accordingly, the mRNA levels
Proteins involved in protein
were similar between the strains (Fig. 6).
translation and synthesis
A4H746 40S ribosomal protein S12 7
Q4Q9A5 40S ribosomal protein S16 2 4. Discussion
A4H3R3 40S ribosomal protein S19 protein 2
A4H9W3 40S ribosomal protein S2 2
Leishmania braziliensis is often associated with large clinical plas­
A4HLE6 40S ribosomal protein S3 4
A4HM77 40S ribosomal protein S3a 3
ticity, but the role of the parasite in such pleomorphism is not clear.
Q4QG31 40S ribosomal protein S4 3 Although the clinical outcome is a multifactorial phenomenon that in­
A4HMM1 40S ribosomal protein S6 1 volves several aspects of the host such as immune status, nutritional
A4H519 40S ribosomal protein S9 3 status, and genetic background, intrinsic aspects of the parasite may also
A4HQ28 40S ribosomal protein SA 2
modulate the pathogenic process and might influence the clinical form
E9AI48 60S acidic ribosomal protein 1
O44010 60S acidic ribosomal protein P2 2 of the disease [20,21,47]. However, it is not yet clear how and why some
A4H3Y8 60S ribosomal protein L10 3 strains of L. braziliensis spread metastatically while others do not. Here
A0A088SL38 60S ribosomal protein L10a, 3 we hypothesized that different strains modulate the activation of mac­
A4HDT2 60S ribosomal protein L12 2
rophages differentially and that such modulation would be one of the
A4HHP5 60S ribosomal protein L13 1
A4HAJ9 60S ribosomal protein L13a 1
first mechanisms contributing to the disparity in the clinical outcome.
A4H745 60S ribosomal protein L18 2 The release of molecules into the extracellular environment by par­
A4HK69 60S ribosomal protein L18a 1 asites of the Leishmania genus has been identified as one of the mecha­
A4HML2 60S Ribosomal protein L36 1 nisms used by this protozoan to evade or subvert the host’s immune
A4HMK9 60S ribosomal protein L5 3
response [27]. We observed essential changes in the parasite infection of
A4H873 60S ribosomal protein L6 3
A4H500 60S ribosomal protein L7a 1 macrophages pre-incubated with the secretome of each L. braziliensis
A4HFQ8 Arginyl-tRNA synthetase 5 strain. The pre-stimulation of peritoneal macrophages with secretome
A4H8V4 Elongation factor 1-alpha 6 significantly increases the percentage of infection, numbers of amasti­
Q2HZY7 Elongation factor 2 18
gotes per cell, and infection index over time. Those results suggested that
A4H5S5 Elongation factor-1 gamma 5
A4HCN4 Endoribonuclease L-PSP (Pb5) 4
the secretomes have proparasitic features that make macrophages more
A4HE05 Eukaryotic initiation factor 5a 3 susceptible to infection and favor the parasite proliferation in the early
A0A088S3S2 Eukaryotic translation initiation factor 2 stages of infection. Our data are in good agreement with previous studies
3 subunit 8, that showed that the treatment of C57BL/6 mice with L. donovani exo­
A4HGT5 Eukaryotic translation initiation factor 1
somes before to be challenged with the parasite exacerbated infection
3 subunit E
A4HNN3 Eukaryotic translation initiation factor 1 [48]. Also, co-inoculation of mice with L. major exosomes plus parasites
3 subunit L resulted in exacerbated lesions [43].
A4HNU9 Eukaryotic translation initiation factor 1 We also observed that the pre-treatment with the secretome of DL
6 strain resulted in a higher infection index than the pre-treatment with
Q25225 Probable eukaryotic initiation factor 11
4A
LCL secretome in all evaluated times, which suggested that the
A4HAJ7 Translation elongation factor 1-beta 6 biochemical composition of those secretomes might be different be­
tween the strains. The significant differences in the levels of proin­
flammatory cytokines secreted by macrophages pretreated and infected
(D)-DPP3 (I) and MTAP (D)-GOT1 (I). These direct interactions associate with each one of the strains reinforce such idea. We found that IL-6,
innate immune response with the cluster viral process (CALM1-YWHAE), TNF-α, and CCL2 levels were higher in macrophages infected with the
small molecule metabolic process with proteolysis (DLD-DPP3) and proteins LCL strain than in those infected with the disseminated one. This result
grouped in the category of metabolic process of small molecules (MTAP- supports the hypothesis that there is a strain-specific crosstalk between
GOT) (Fig. 5). each L.braziliensis strain and macrophages and, therefore, the composi­
Finally, the 894 direct interactions presented by the 15 differentially tion of the secretomes may also be distinct. Remarkably, IL-6 and TNF-α
abundant proteins (Fig. 5), indicate that there are a large number of concentration were two-fold higher in macrophages pre-treated with the
processes modulated by the secreted proteins and that different pro­ secretome of LCL strain concerning the same strain without pre-
cesses are affected by the proteins secreted by each strain. It is worth treatment, indicating that not only the direct parasite contact, but also
noting that proteins with increased abundance in the secretome of the the L. braziliensis secretome per se, is able to elicit macrophage responses.
DL strain showed a higher number of interactions when compared to Interestingly, LCL parasites, but not their secretome, were able to elicit
proteins increased in the LCL strain. IFN-γ responses, suggesting that direct contact between LCL parasites
and macrophages are necessary to IFN-γ production and reinforcing the
3.5. Gene expression of Lbrm_18_1400, lbrm_05_0800, and lip2 idea that LCL parasites activate responses in macrophages that
correlates with protein abundance contribute for the proper control of parasites. In contrast, neither DL
secretome nor parasites were able to elicit such responses.
To gain insights about the transcript levels of some of the differen­ Our results show that LCL secretome and/or parasites are able to
tially abundant proteins, we analyzed their gene expression by qPCR. In elicit the production of molecules involved in cell activation and
agreement with the relative quantification obtained by proteomics, the recruitment of monocyte and macrophages (IL-6, CCL2 and TNF-α) at
DL strain exhibited significant lower mRNA levels of the heat shock early times of stimulation/infection, whereas DL secretome and/or
protein (Lbrm_18_1400), S-methyl-5′ -thioadenosine phosphorylase parasites do not. Such results suggest that (i) the components of the LCL
(Lbrm_05_0800), and 60S acidic ribosomal protein P2 (Lip2) with a secretome that induce such markers are absent or at low abundance in
reduction of 3.8, 2.6 and 2.2-fold, respectively, when compared to the the DL secretome, or (ii) there are components in the DL secretome that

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A. Rodríguez-Vega et al. Journal of Proteomics 232 (2021) 104077

Table 2
Differentially abundant proteins in the secretome of L. (V.) braziliensis strain associated with disseminated leishmaniasis compared to the strain associated with
localized cutaneous leishmaniasis.
Protein ID Name Number of Fold Stouffer P Abundance Secretion Orthology with Homo Number of
quantified Change value (D/I) pathway sapiens interactions
peptides
Gene Homo
name sapiens
code

A4HKT7 Nucleoside diphosphate kinase (EC 3 − 2.5 0.003 D Alternative - NME1 P15531 20
2.7.4.6) Exo
O44010 60S acidic ribosomal protein P2 2 − 2.3 0.007 D
(P2B-protein)
A4H440 Surface antigen-like protein 2 − 1.8 0.006 D Classical
A4HKX6 Dihydrolipoyl dehydrogenase (EC 2 − 1.8 0.017 D DLD P09622 20
1.8.1.4)
A4H5R9 Calmodulin 5 − 1.8 0.000 D Exo CALM1 P62158 84
A4H9P0 Putative heat shock protein 2 − 1.8 0.016 D HSPA4L O95757 17
A4H4C5 S-methyl-5′ -thioadenosine 2 − 1.6 0.005 D Alternative - MTAP Q13126 16
phosphorylase (EC 2.4.2.28) Exo
(MTAPase)
A4H746 40S ribosomal protein S12 2 − 1.6 0.026 D Alternative -
Exo
A4HPQ8 Adenosylhomocysteinase (EC 3 − 1.6 0.003 D Exo AHCY P23526 34
3.3.1.1)
A4H638 Leishmanolysin (EC 3.4.24.36) 5 − 1.6 0.001 D Alternative
A4H643 Glycerol-3-phosphate 3 − 1.6 0.004 D Alternative -
dehydrogenase [NAD(+)] (EC Exo
1.1.1.8)
A4H537 Splicing factor ptsr1-like protein 2 − 1.5 0.008 D Alternative
A4HFU7 Uncharacterized protein 4 − 1.5 0.001 D Alternative
A4HMB3 Putative aspartate aminotransferase 2 1.9 0.008 I GOT1 P17174 11
(EC 2.6.1.1)
A4HLE6 Putative 40S ribosomal protein S3 2 1.9 0.008 I Exo RPS3 P23396 121
A4HN57 T-complex protein 1 subunit eta 2 1.9 0.009 I Exo CCT7 Q99832 75
(TCP-1-eta)
A0A088RUF5 Glucose-regulated protein 78, 2 1.9 0.013 I Classical- HSPA5 P11021 154
putative Exo
A4HQL2 Putative T-complex protein 1, theta 2 1.8 0.008 I CCT8 P50990 78
subunit
A4H6F2 Putative 14–3-3 protein 3 1.8 0.002 I Exo YWHAE P62258 100
A4H3T9 Uncharacterized protein 6 1.7 0.000 I Classical
A4HPA3 TAT protein (EC 2.6.1.5) 8 1.7 0.000 I
A4H7H9 Thioredoxin-like_fold domain- 2 1.6 0.007 I
containing protein
E9JUH4 Trypanothione reductase 3 1.6 0.004 I Exo GSR P00390 12
(Fragment)
A4H8V4 Elongation factor 1-alpha 2 1.5 0.008 I Alternative - EEF1A1 P68104 138
Exo
A0A088RIB2 Dipeptidyl peptidase 3 (EC 2 1.5 0.008 I Exo DPP3 Q9NY33 14
3.4.14.4) (Dipeptidyl
aminopeptidase III)
A4HAX5 Contig, possible fusion of 3 1.5 0.011 I Alternative
chromosomes 20 and 34

abrogate the proper induction of these cytokines and chemokine.
The induction of higher levels of those molecules by LCL secretome/
parasites suggests that early activation and recruitment of monocytes by
this strain could promote efficient parasite elimination. In contrast, DL
secretome/parasites failed to elicit such responses. Consistently, it has
been shown that IL-6 is highly expressed in localized cutaneous lesions
[49], is upregulated in macrophages exposed to L.mexicana promasti­
gotes [50], and individuals with low macrophage IL-6 levels have
increased risk of developing mucocutaneus form, which implies para­
site’s dissemination [51]. CCL2 is central for regulating the recruitment
and activation of macrophages and monocytes, and other cells required
for early control of Leishmania infection [52–54]. Self-healing LCL is
associated with high levels of CCL2 in the skin lesions, where probably
chemoattracts monocyte and macrophage and stimulates microbicidal
mechanisms [55]. In addition, in mice infected with L. donovani, CCL2
favors the Th1 response by induction of IL-12 secretion and inhibition of
IL-10 production in infected macrophages [56]. Recently, it was
observed that the expression of CCL2 is significantly higher in pre-
treatment lesion samples from patients who responded well to
treatment with pentavalent antimonial than in samples from those who
failed [57]. TNF-α is a proinflammatory cytokine associated with a
strong Th1 response and produced mainly by activated macrophages.
This molecule is also essential to control parasite proliferation and
dissemination by the recruitment of cells to the site of infection and
subsequent formation of the granuloma [20,58,59]. Dendritic cells
exposed to L. braziliensis secreted molecules upregulate activation
markers, produce IL-12 and TNF-α and are more efficient at presenting
antigen [60]. However, in L. braziliensis infections, excessively high
levels of TNF-α, together with a decreased ability of IL-10 to down­
regulate this cytokine, can lead to severe tissue damage such as that
observed in patients with MCL [61,62].
Interestingly, IL-10, which is recognized by its immunosuppressive
role, was produced by macrophages pre-treated and/or infected with the
LCL strain, in all times evaluated. Downregulation of proinflammatory
molecules mediated by IL-10 could favor a wound healing response
following parasite killing in infections with LCL strain, dampening the
immunopathogenic activity of cell recruitment and TNF-α production.
Consistently, it has been shown that IL-10 is important for

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A. Rodríguez-Vega et al. Journal of Proteomics 232 (2021) 104077

Fig. 5. Functional interaction network among differentially abundant proteins identified in the secretome of L. (V.) braziliensis strains. Proteins with differential
abundance in the secretome were clustered according to the biological process enriched with p < 0.05. Red circles: proteins with decreased abundance in the DL
strain in comparison with the LCL strain. Green circles: proteins with increased abundance in the DL strain in comparison with the LCL strain. The network was built
using the IIS (Integrated interactome system) platform and viewed in Cytoscape software version 2.8.3. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

Fig. 6. Gene expression analysis of selected genes with differential protein abundance in the secretome of two L. (V.) braziliensis strains. The mRNA levels of
lbrm_18_1400, lbrm_05_0800, lip2, tat, lpmp_281290, and lbrm_26_2610, were measured by qPCR in promastigotes of DL or LCL L. (V.) braziliensis strains. The values are
expressed as normalized relationships between the expression of the target gene and a geometric mean of two constitutive genes. Statistically analysis between the
strains were calculated using the Student t-test, using the GraphPad Prism 5.0 program. (*) p < 0.05 and (***) p < 0.0007. The graphs represent mean ± standard
deviation of biological triplicate.

11
A. Rodríguez-Vega et al.
counterbalance the exaggerated inflammatory response associated with
pathology in infections with L. braziliensis [63].
Together, the induction of higher and sustained levels of IL-6, TNF-α,
and CCL2 by LCL strain, associated with balanced levels of IL-10, can
favor an effector cell response and a proper regulation of Th1 response
that result in the effective resolution of the disease caused by this strain.
In contrast, the DL strain is not able to elicit such response or has
mechanisms to prevent this activation, supporting efficient infection,
proliferation, and persistence of the parasite, which are indispensable
requirements for metastatic dissemination. Hence, despite macrophages
are programmed to destroy intracellular pathogens, their activation can
be modulated by the infectious agent resulting in successful colonization
of the intracellular environment that would otherwise be hostile.
Results from several studies have shown that Leishmania spp. use
different mechanisms to evade elimination by macrophages
[22,23,26,27,46,64]. Particularly, the interference of host signaling
pathways prevents effective macrophage activation and favors para­
sites’ survival and proliferation [22,46,65,66]. In order to obtain in­
formation about the molecules that could be associated to such
modulation, we used a proteomics approach to identify and compara­
tively quantify proteins secreted by LCL and DL strains.
Our dataset shows 252 proteins that were reproducibly identified in
the secretome of L. braziliensis. Various of those proteins have been
systematically reported in the secretions of Leishmania spp. including
HSP70, acid phosphatase, elongation factor 1β, tryparedoxin peroxi­
dase, and Leishmania homologue of activated C kinase (LACK), which are
involved in protein folding, regulation of the cell cycle and resistance to
oxidative stress [23–25]. We also observed that ~75% proteins secreted
by L. braziliensis are exported by alternative pathways, mainly involving
exosomes, which is in good agreement with previous reports that
showed that exosomes are a main route for protein secretion in Leish­
mania spp. [25,43,50,64,67,68]. Remarkably, several proteins identified
in the secretome have potential transmembrane domains, suggesting
membrane localization, but various of them were also predicted to be
secreted by exosomes and are recurrently reported in the extracellular
milieu. Many of these proteins could be released by membrane shedding,
which is an important mechanism for communication in trypanosoma­
tids [69,70]. Consistently, L. donovani and L. mexicana secretomes also
present proteins that are potentially targeted to membranes [24,71] or
intracellular compartments such as the mitochondrion, reinforcing the
idea that Leishmania proteins can perform more than one function in the
intra- and extra-cellular space, similarly to the observed for other or­
ganisms [72,73]. Our dataset still presented various proteins for which
no prediction of conventional or alternative secretion was found, indi­
cating that there are several proteins with unknown routes of secretion/
releasing or that there is a limitation of the prediction tools for working
with these protozoa.
We observed that the abundance of several proteins was significantly
reduced in the secretome of the DL strain compared to the LCL. Among
those proteins, the 60S acidic ribosomal protein P2, which is involved in
protein synthesis, has a non-canonical function as iron-binding protein,
and is associated with intracellular iron uptake [72,74]. As iron is
essential for the Leishmania replication [75], the increased expression of
molecules involved in iron uptake should favor its proliferation inside
the cell. However, infected macrophages rapidly upregulate the
expression of iron transporters, sequestering the available metal, and
controlling parasite proliferation [75]. In this context, the potential
decrease in iron uptake by DL strain could be a “silent” way of invading
host cells, as it would not alert macrophages for overexpressing iron
transporters [75]. In addition, it was observed that limited iron condi­
tions induce Leishmania differentiation [76], which leads us to consider
that the disseminated strain may also have its differentiation favored
inside the host cells.
A heat shock protein (HSP-A4H9P0) was also less abundant in the
secretome of DL strain than LCL strain. It is currently recognized that
HSPs have functions other than chaperones in the extracellular
12
Journal of Proteomics 232 (2021) 104077
environment [77–79]. In the case of cells of the immune system,
extracellular HSPs act as a signaling molecule for stress conditions,
alerting other cells, thus preventing the extension of damage and pro­
moting resolution [78,80]. In human cell lines, extracellular HSP70
exerts immunomodulatory functions, activating macrophages and
inducing the production of TNF-α [80]. Given the immunomodulatory
functions of HSPs, it is possible to suggest that the low abundance of
HSP-A4H9P0 in the DL secretome, which was also decreased at the level
of transcripts, could prevent the activation of proinflammatory path­
ways, inhibiting the production of TNF-α and other cytokines. This
proposition is supported by the almost undetectable levels of proin­
flammatory cytokines in macrophages pre-treated with DL secretome
and/or infected with this strain, compared to the LCL strain. In fact, both
the LCL secretome as well as the LCL parasite, were able to stimulate
high levels of TNF-α, IL-6, and CCL2 in murine macrophages, indicating
an appropriate activation of the cells to control the infection.
A metallopeptidase GP63 was also decreased in the DL secretome
when compared to the LCL secretome. This result is intriguing since it
has been shown that a GP63 isoform secreted by L.major can be inter­
nalized by macrophages, activating different tyrosine phosphatases
(PTPs), such as Src homology 2 domain containing tyrosine
phosphatase-1 (SHP-1), protein tyrosine phosphatase 1B (PTP1B) and
protein tyrosine phosphatase T cell phosphatase (TCPTP), which induce
inactivation of the macrophage [22] and, consequently, favors the
establishment of the parasite. Thus, considering that GP63 belongs to an
extensive gene family composed of more than 30 members with
different potential functions in L. braziliensis [81], it is possible to sug­
gest that the GP63 isoform identified here might play a different role
than that described in L. major [22]. In line with this proposition, we
showed in previous studies that strains associated to different forms of
ATL exhibit different profiles of secreted metallopeptidases [82,83].
Furthermore, the low abundance of this GP63 in the DL secretome
suggests the existence of alternative mechanisms of macrophage deac­
tivation in this strain, such as those mediated by the elongation factor 1-
alpha 1 (EEF1A) [46].
In fact, we detected an increased abundance of EEF1A in the secre­
tome of DL strain compared to the LCL one. EEF1A catalyzes the binding
of aminoacyl-tRNAs to the ribosome site A during the growth of the
peptide chain but also plays a non-canonical function of peptidase.
Peptidases can cleave and activate macrophage phosphatases that
inhibit the signaling cascade responsible for activating NO production,
favoring parasite establishment. Such a non-canonical function of
EEF1A as peptidase was observed in the secretion of L. donovani [46,66].
EEF1A activates SHP-1, a tyrosine phosphatase that negatively modu­
lates the JACK/STAT (Janus kinase/signal transducer and activator of
transcription) and MAPK (mitogen-activated protein kinase) pathways
resulting in the deactivation of the macrophage and decreased NO
production [46,66,84]. Furthermore, in our functional analysis network,
EEF1A presented high complexity concerning the number of direct in­
teractions, suggesting that the increase in its abundance would modulate
many processes. Additionally, we observed that the dipeptidyl-peptidase
III (DPP3) was also more abundant in the DL secretome. According to the
interaction network (A0A088RIB2), this peptidase affects critical pro­
cesses, including activation of the MAPK pathway (UBC), cell death
(NDRG1), and regulation of the apoptotic process (TNFAIP8). Therefore,
the secretome of DL strain is enriched in peptidases that might deacti­
vate macrophage’ signaling pathways crucial for parasite elimination.
The glucose-regulated protein 78 (GRP78) was also more abundant
in the DL secretome than the LCL strain. This protein has also been found
in the secretome of L. donovani [24] and is actively secreted by cancer
cells, and supports metastatic dissemination [85–87]. It has been sug­
gested that this protein can modulate parasite’s differentiation process
[88], which is a fundamental requirement for Leishmania’s survival.
Thus, our results suggest that DL strain could have higher efficiency in
differentiation and proliferation, mediated by GRP78, which favors
rapid adaptation to the phagolysosomal environment. As seen in cancer
A. Rodríguez-Vega et al.
cells, the secretion of this protein to the extracellular milieu could also
promote the metastatic dissemination of parasites; however, this aspect
needs to be studied.
Greater efficiency in the proliferation of DL strain could also be
mediated by the increase in aspartate aminotransferase, GOT1, which
regenerates methionine from methylthioadenosine [89]. The synthesis
of polyamines is fundamental for rapid cell proliferation and is a process
highly exploited by parasites and cancer cells [89,90]. The increase of
this protein in the secretion of the disseminated strain could favor the
rapid multiplication of the parasite in the first stages of infection in the
host cell, ensuring the establishment and dissemination of the infection
[89,90].
The DL strain might also have a greater resistance to oxidative stress,
mediated by the increased abundance of trypanothione reductase, and
thioredoxin-like fold domain containing protein. Consistently, disrup­
tion of the trypanothione reductase gene decreases the Leishmania
infectivity and its ability to survive within the macrophage [91,92].
Besides, the resistance to oxidative stress has been shown to be associ­
ated with metastasis in mucocutaneous leishmaniasis caused by L.
panamensis and L.guyanensis strains [93]. Remarkably, the enriched
group of proteins involved in response to oxidative stress was abundant
only in the DL strain, suggesting that this process may be crucial for
dissemination. The mass spectrometry proteomics data have been
deposited to the ProteomeXchange Consortium via the PRIDE [94]
partner repository with the dataset identifier #440425.
5. Conclusions
Using an iTRAQ-based quantitative proteomics approach we char­
acterized and compared the abundance of proteins secreted by L. bra­
ziliensis strains associated with very different clinical manifestations, DL
and LCL. Our dataset provides new information on molecules potentially
involved in the process of infection and pathogenicity of those strains.
Our data indicate that higher efficiency in detoxification and differen­
tiation can favor the infection and proliferation of the DL strain. In line
with that evidence, macrophages pre-treated with the secretome of the
DL strain have a higher infection index than those pre-treated with the
LCL secretome. Besides, a better efficiency in the “silencing” of the
macrophage, mediated by peptidases, would further favor the estab­
lishment of infection and persistence of the DL strain. This “silencing”
can also be corroborated by the reduced inflammatory response elicited
in macrophages pre-treated with the secretome of the DL strain, and by
the DL parasites per se, in comparison with the levels observed for the
macrophages pre-treated and infected with the LCL strain. Thus, the DL
strain has mechanisms to prevent macrophage activation, favoring
parasite proliferation, and persistence, which are indispensable re­
quirements for metastatic dissemination. We also identified in the
secretome of each strain several molecules that potentially mediated this
activation/deactivation and pointed out some molecules that could
potentially be associated with the metastatic process. Additional studies
of the secretome of other L. braziliensis strains need to be done to validate
the association of these molecules with the metastatic phenotype.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jprot.2020.104077.
Author contributions
Designed the research: P⋅C., J.B.J., G.P.; conducted the research: A.R.
V., M.L.B., L.R.B., C.M.R., A.C.B., P.A.; provided essential reagents/
materials/analysis tools: P.C., R.M.B., P.C.C.; analyzed the data: P.C., A.
R.V., J.B.J.; wrote the manuscript: P.C., M.L.B. A.R.V. All authors have
read and approved the final manuscript.
Funding
This work was supported by the Conselho Nacional de
13
Journal of Proteomics 232 (2021) 104077
Desenvolvimento Científico e Tecnológico - CNPq (P⋅C. Universal grant
No. 423300/2018–0); FIOCRUZ (P.C., A.R.V., M.L.B, N.P., G.P. PROEP
grant No. 476727/2010–3); Fundação de Amparo à Pesquisa do Estado
de Rio de Janeiro - FAPERJ (P.C. JCNE E-26/201.545/2014 and JCNE E-
26/203.253/2017; A.R.V. TCT No. E-26/202.539/2016), and Coor­
denação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES, −
Finance Code 001 (P.C Process No. 88887.374332/2019–00). G.P. was a
CAPES fellow of the Visitant Professor Program (Process No. 0344141).
P.C, and J.B.J. are CNPq Researchers fellows (P.C. Process No. 306393/
2014–0 and 305796/2017–8; J.B.J Process No. 309732/2018–2).
All authors have read and approved the final manuscript.
Data availability
The mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium via the PRIDE
Acknowledgements
We thank all the staff of the proteomic facilities of Fiocruz-Paraná
(Plataforma Proteômica RPT02H – FIOCRUZ) for technical help with
mass spectrometric measurements, and Dr. Rosane Temporal and all the
staff of CLIOC for quality management advising.
Conflict of interest
The authors declare no conflict of interests.
References
[1] S. Burza, S.L. Croft, M. Boelaert, Leishmaniasis, Lancet 392 (2018) 951–970.
[2] H.W. Murray, J.D. Berman, C.R. Davies, N.G. Saravia, Advances in leishmaniasis,
Lancet 366 (2005) 1561–1577.
[3] J. Alvar, I.D. Velez, C. Bern, M. Herrero, P. Desjeux, J. Cano, J. Jannin, M. den
Boer, Team WHOLC, Leishmaniasis worldwide and global estimates of its
incidence, PloS one 7 (2012), e35671.
[4] PAHO, Pan American Health Organization. Leishmaniasis: Epidemiological Report
in the Americas, PAHO, Washington, D.C, 2019.
[5] de Oliveira Camera P, Junger J, do Espirito Santo Silva Pires F, Mattos M, Oliveira-
Neto MP, Fernandes O, Pirmez C. Haematogenous dissemination of Leishmania
(Viannia) braziliensis in human American tegumentary leishmaniasis. Trans. R. Soc.
Trop. Med. Hyg. 2006;100:1112–7.
[6] L. Jirmanus, M.J. Glesby, L.H. Guimaraes, E. Lago, M.E. Rosa, P.R. Machado, E.
M. Carvalho, Epidemiological and clinical changes in American tegumentary
leishmaniasis in an area of Leishmania (Viannia) braziliensis transmission over a 20-
year period. Am. J. Trop. Med. Hyg. 86 (2012) 426–433.
[7] P.D. Marsden, Mucosal leishmaniasis (“espundia” Escomel, 1911), Trans. R. Soc.
Trop. Med. Hyg. 80 (1986) 859–876.
[8] J.E. Martinez, Arias L. Alba, M.A. Escobar, N.G. Saravia, Haemoculture of
Leishmania (Viannia) braziliensis from two cases of mucosal leishmaniasis: re-
examination of haematogenous dissemination, Trans. R. Soc. Trop. Med. Hyg. 86
(1992) 392–394.
[9] E. Thomaidou, L. Horev, D. Jotkowitz, M. Zamir, A. Ingber, C.D. Enk, V. Molho-
Pessach, Lymphatic dissemination in cutaneous Leishmaniasis following local
treatment, Am. J. Trop. Med. Hyg. 93 (2015) 770–773.
[10] I. Bennis, S. Thys, H. Filali, V. De Brouwere, H. Sahibi, M. Boelaert, Psychosocial
impact of scars due to cutaneous leishmaniasis on high school students in
Errachidia province, Morocco. Infect. Dis. Poverty. 6 (2017) 46.
[11] M. Yanik, M.S. Gurel, Z. Simsek, M. Kati, The psychological impact of cutaneous
leishmaniasis, Clin. Exp. Dermatol. 29 (2004) 464–467.
[12] C. Cincura, C.M.F. de Lima, P.R.L. Machado, J. Oliveira-Filho, M.J. Glesby, M.
M. Lessa, E.M. Carvalho, Mucosal leishmaniasis: a retrospective study of 327 cases
from an endemic area of Leishmania (Viannia) braziliensis, Am. J. Trop. Med. Hyg.
97 (2017) 761–766.
[13] A.L. Garcia, R. Parrado, E. Rojas, R. Delgado, J.C. Dujardin, R. Reithinger,
Leishmaniases in Bolivia: comprehensive review and current status, Am. J. Trop.
Med. Hyg. 80 (2009) 704–711.
[14] Y. Hashiguchi, E.L. Gomez, H. Kato, L.R. Martini, L.N. Velez, H. Uezato, Diffuse and
disseminated cutaneous leishmaniasis: clinical cases experienced in Ecuador and a
brief review, Trop. Med. Health. 44 (2016) 2.
[15] P.R. Machado, M.E. Rosa, D. Costa, M. Mignac, J.S. Silva, A. Schriefer, M.
M. Teixeira, O. Bacellar, E.M. Carvalho, Reappraisal of the immunopathogenesis of
disseminated leishmaniasis: in situ and systemic immune response, Trans. R. Soc.
Trop. Med. Hyg. 105 (2011) 438–444.
[16] M. Solomon, N. Sahar, F. Pavlotzky, A. Barzilai, C.L. Jaffe, A. Nasereddin,
E. Schwartz, Mucosal Leishmaniasis in travelers with Leishmania braziliensis
complex returning to Israel, Emerg. Infect. Dis. 25 (2019) 642–648.
A. Rodríguez-Vega et al.
[17] M.L. Turetz, P.R. Machado, A.I. Ko, F. Alves, A. Bittencourt, R.P. Almeida,
N. Mobashery, W.D. Johnson Jr., E.M. Carvalho, Disseminated leishmaniasis: a
new and emerging form of leishmaniasis observed in northeastern Brazil, J. Infect.
Dis. 186 (2002) 1829–1834.
[18] I.D. Velez, A. Jimenez, D. Vasquez, S.M. Robledo, Disseminated cutaneous
Leishmaniasis in Colombia: report of 27 cases, Case reports in dermatology 7
(2015) 275–286.
[19] K.J. Gollob, A.G. Viana, W.O. Dutra, Immunoregulation in human American
leishmaniasis: balancing pathology and protection, Parasite Immunol. 36 (2014)
367–376.
[20] C. Souza-Lemos, S.N. De-Campos, A. Teva, S. Corte-Real, E.C. Fonseca, R. Porrozzi,
G. Grimaldi Jr., Dynamics of immune granuloma formation in a Leishmania
braziliensis-induced self-limiting cutaneous infection in the primate Macaca
mulatta, J. Pathol. 216 (2008) 375–386.
[21] C. Souza-Lemos, S.N. De-Campos, A. Teva, R. Porrozzi, G. Grimaldi Jr., In situ
characterization of the granulomatous immune response with time in nonhealing
lesional skin of Leishmania braziliensis-infected rhesus macaques (Macaca mulatta),
Vet. Immunol. Immunopathol. 142 (2011) 147–155.
[22] M.A. Gomez, I. Contreras, M. Halle, M.L. Tremblay, R.W. McMaster, M. Olivier,
Leishmania GP63 alters host signaling through cleavage-activated protein tyrosine
phosphatases, Sci. Signal. 2 (2009) ra58.
[23] B. Perez-Cabezas, N. Santarem, P. Cecilio, C. Silva, R. Silvestre, J. AMC, A. Cordeiro
da Silva, More than just exosomes: distinct Leishmania infantum extracellular
products potentiate the establishment of infection, J. Extracell. Vesicles. 8 (2019)
1541708.
[24] J.M. Silverman, S.K. Chan, D.P. Robinson, D.M. Dwyer, D. Nandan, L.J. Foster, N.
E. Reiner, Proteomic analysis of the secretome of Leishmania donovani, Genome
Biol. 9 (2008) R35.
[25] P. Cuervo, J.B. De Jesus, L. Saboia-Vahia, L. Mendonca-Lima, G.B. Domont,
E. Cupolillo, Proteomic characterization of the released/secreted proteins of
Leishmania (Viannia) braziliensis promastigotes, J. Proteomics. 73 (2009) 79–92.
[26] N. Moradin, A. Descoteaux, Leishmania promastigotes: building a safe niche within
macrophages, Front. Cell. Infect. Microbiol. 2 (2012) 121.
[27] U. Lambertz, J.M. Silverman, D. Nandan, W.R. McMaster, J. Clos, L.J. Foster, N.
E. Reiner, Secreted virulence factors and immune evasion in visceral leishmaniasis,
J. Leukoc. Biol. 91 (2012) 887–899.
[28] M. Losada-Barragan, A. Umana-Perez, A. Rodriguez-Vega, S. Cuervo-Escobar,
R. Azevedo, F.N. Morgado, Carvalho V. de Frias, P. Aquino, P.C. Carvalho,
R. Porrozzi, M. Sanchez-Gomez, G. Padron, P. Cuervo, Proteomic profiling of
splenic interstitial fluid of malnourished mice infected with Leishmania infantum
reveals defects on cell proliferation and pro-inflammatory response, J. Proteomics.
208 (2019) 103492.
[29] C. UniProt, UniProt: a worldwide hub of protein knowledge, Nucleic Acids Res. 47
(2019) D506–D515.
[30] P.C. Carvalho, J.S. Fischer, T. Xu, D. Cociorva, T.S. Balbuena, R.H. Valente,
J. Perales, J.R. Yates 3rd, V.C. Barbosa, Search engine processor: filtering and
organizing peptide spectrum matches, Proteomics 12 (2012) 944–949.
[31] P.C. Carvalho, J.S. Fischer, E.I. Chen, G.B. Domont, M.G. Carvalho, W.M. Degrave,
J.R. Yates 3rd, V.C. Barbosa, GO explorer: a gene-ontology tool to aid in the
interpretation of shotgun proteomics data, Proteome Sci. 7 (2009) 6.
[32] P.C. Carvalho, D.B. Lima, F.V. Leprevost, M.D. Santos, J.S. Fischer, P.F. Aquino, J.
J. Moresco, J.R. Yates 3rd, V.C. Barbosa, Integrated analysis of shotgun proteomic
data with PatternLab for proteomics 4.0, Nat. Protoc. 11 (2016) 102–117.
[33] M.F. Carazzolle, L.M. de Carvalho, H.H. Slepicka, R.O. Vidal, G.A. Pereira,
J. Kobarg, G.V. Meirelles, IIS–integrated Interactome system: a web-based platform
for the annotation, analysis and visualization of protein-metabolite-gene-drug
interactions by integrating a variety of data sources and tools, PLoS One 9 (2014),
e100385.
[34] R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J.E. Pollington, O.L. Gavin,
P. Gunasekaran, G. Ceric, K. Forslund, L. Holm, E.L. Sonnhammer, S.R. Eddy,
A. Bateman, The Pfam protein families database, Nucleic Acids Res. 38 (2010)
D211–D222.
[35] T.N. Petersen, S. Brunak, G. von Heijne, H. Nielsen, SignalP 4.0: discriminating
signal peptides from transmembrane regions, Nat. Methods 8 (2011) 785–786.
[36] J.D. Bendtsen, L.J. Jensen, N. Blom, G. Von Heijne, S. Brunak, Feature-based
prediction of non-classical and leaderless protein secretion, Protein Eng. Des. Sel.
17 (2004) 349–356.
[37] J.J. Almagro Armenteros, M. Salvatore, O. Emanuelsson, O. Winther, G. von
Heijne, A. Elofsson, H. Nielsen, Detecting sequence signals in targeting peptides
using deep learning, Life Sci. Alliance. 2 (2019).
[38] A. Krogh, B. Larsson, G. von Heijne, E.L. Sonnhammer, Predicting transmembrane
protein topology with a hidden Markov model: application to complete genomes,
J. Mol. Biol. 305 (2001) 567–580.
[39] S. Keerthikumar, D. Chisanga, D. Ariyaratne, H. Al Saffar, S. Anand, K. Zhao,
M. Samuel, M. Pathan, M. Jois, N. Chilamkurti, L. Gangoda, S. Mathivanan,
ExoCarta: a web-based compendium of Exosomal cargo, J. Mol. Biol. 428 (2016)
688–692.
[40] M. Losada-Barragan, A. Umana-Perez, S. Cuervo-Escobar, L.R. Berbert, R. Porrozzi,
F.N. Morgado, D.A. Mendes-da-Cruz, W. Savino, M. Sanchez-Gomez, P. Cuervo,
Protein malnutrition promotes dysregulation of molecules involved in T cell
migration in the thymus of mice infected with Leishmania infantum, Sci. Rep. 7
(2017) 45991.
[41] S.A. Bustin, J.F. Beaulieu, J. Huggett, R. Jaggi, F.S. Kibenge, P.A. Olsvik, L.
C. Penning, S. Toegel, MIQE precis: practical implementation of minimum standard
guidelines for fluorescence-based quantitative real-time PCR experiments, BMC
Mol. Biol. 11 (2010) 74.
14
Journal of Proteomics 232 (2021) 104077
[42] E. Willems, L. Leyns, J. Vandesompele, Standardization of real-time PCR gene
expression data from independent biological replicates, Anal. Biochem. 379 (2008)
127–129.
[43] V.D. Atayde, H. Aslan, S. Townsend, K. Hassani, S. Kamhawi, M. Olivier, Exosome
secretion by the parasitic protozoan Leishmania within the sand Fly Midgut, Cell
Rep. 13 (2015) 957–967.
[44] A. Dupe, C. Dumas, B. Papadopoulou, An Alba-domain protein contributes to the
stage-regulated stability of amastin transcripts in Leishmania, Mol. Microbiol. 91
(2014) 548–561.
[45] A. Dupe, C. Dumas, B. Papadopoulou, Differential subcellular localization of
Leishmania Alba-domain proteins throughout the parasite development, PLoS One
10 (2015), e0137243.
[46] D. Nandan, T. Yi, M. Lopez, C. Lai, N.E. Reiner, Leishmania EF-1alpha activates the
Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to
macrophage deactivation, J. Biol. Chem. 277 (2002) 50190–50197.
[47] J.E. Martinez, B.L. Travi, A.Z. Valencia, N.G. Saravia, Metastatic capability of
Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis in golden
hamsters, J. Parasitol. 77 (1991) 762–768.
[48] J.M. Silverman, J. Clos, E. Horakova, A.Y. Wang, M. Wiesgigl, I. Kelly, M.A. Lynn,
W.R. McMaster, L.J. Foster, M.K. Levings, N.E. Reiner, Leishmania exosomes
modulate innate and adaptive immune responses through effects on monocytes and
dendritic cells, J. Immunol. 185 (2010) 5011–5022.
[49] G. Caceres-Dittmar, F.J. Tapia, M.A. Sanchez, M. Yamamura, K. Uyemura, R.
L. Modlin, B.R. Bloom, J. Convit, Determination of the cytokine profile in American
cutaneous leishmaniasis using the polymerase chain reaction, Clin. Exp. Immunol.
91 (1993) 500–505.
[50] K. Hassani, M. Olivier, Immunomodulatory impact of Leishmania-induced
macrophage exosomes: a comparative proteomic and functional analysis, PLoS
Negl. Trop. Dis. 7 (2013), e2185.
[51] L. Castellucci, E. Menezes, J. Oliveira, A. Magalhaes, L.H. Guimaraes, M. Lessa,
S. Ribeiro, J. Reale, E.F. Noronha, M.E. Wilson, P. Duggal, T.H. Beaty, S. Jeronimo,
S.E. Jamieson, A. Bales, J.M. Blackwell, A.R. de Jesus, E.M. Carvalho, IL6–174 G/C
promoter polymorphism influences susceptibility to mucosal but not localized
cutaneous leishmaniasis in Brazil, J. Infect. Dis. 194 (2006) 519–527.
[52] P. Allavena, G. Bianchi, D. Zhou, J. van Damme, P. Jilek, S. Sozzani, A. Mantovani,
Induction of natural killer cell migration by monocyte chemotactic protein-1, − 2
and − 3, Eur. J. Immunol. 24 (1994) 3233–3236.
[53] S. Oghumu, C.M. Lezama-Davila, A.P. Isaac-Marquez, A.R. Satoskar, Role of
chemokines in regulation of immunity against leishmaniasis, Exp. Parasitol. 126
(2010) 389–396.
[54] U. Ritter, H. Moll, Monocyte chemotactic protein-1 stimulates the killing of
Leishmania major by human monocytes, acts synergistically with IFN-gamma and is
antagonized by IL-4, Eur. J. Immunol. 30 (2000) 3111–3120.
[55] U. Ritter, H. Moll, T. Laskay, E. Brocker, O. Velazco, I. Becker, R. Gillitzer,
Differential expression of chemokines in patients with localized and diffuse
cutaneous American leishmaniasis, J. Infect. Dis. 173 (1996) 699–709.
[56] R. Dey, N. Majumder, S. Bhattacharyya Majumdar, S. Bhattacharjee, S. Banerjee,
S. Roy, S. Majumdar, Induction of host protective Th1 immune response by
chemokines in Leishmania donovani-infected BALB/c mice, Scand. J. Immunol. 66
(2007) 671–683.
[57] A. Navas, O. Fernandez, C. Gallego-Marin, M.D.M. Castro, M. Rosales-Chilama,
J. Murillo, A. Cossio, D. McMahon-Pratt, N.G. Saravia, M.A. Gomez, Profiles of
local and systemic inflammation in the outcome of treatment of human cutaneous
Leishmaniasis caused by Leishmania (Viannia), Infect. Immun. 88 (2020) e00764.
[58] H.W. Murray, A. Jungbluth, E. Ritter, C. Montelibano, M.W. Marino, Visceral
leishmaniasis in mice devoid of tumor necrosis factor and response to treatment,
Infect. Immun. 68 (2000) 6289–6293.
[59] P. Wilhelm, U. Ritter, S. Labbow, N. Donhauser, M. Rollinghoff, C. Bogdan,
H. Korner, Rapidly fatal leishmaniasis in resistant C57BL/6 mice lacking TNF,
J. Immunol. 166 (2001) 4012–4019.
[60] L.P. Carvalho, E.J. Pearce, P. Scott, Functional dichotomy of dendritic cells
following interaction with Leishmania braziliensis: infected cells produce high levels
of TNF-alpha, whereas bystander dendritic cells are activated to promote T cell
responses, J. Immunol. 181 (2008) 6473–6480.
[61] O. Bacellar, H. Lessa, A. Schriefer, P. Machado, A. Ribeiro de Jesus, W.O. Dutra, K.
J. Gollob, E.M. Carvalho, Up-regulation of Th1-type responses in mucosal
leishmaniasis patients, Infect. Immun. 70 (2002) 6734–6740.
[62] A.M. Da-Cruz, M.P. de Oliveira, P.M. De Luca, S.C. Mendonca, S.G. Coutinho,
Tumor necrosis factor-alpha in human american tegumentary leishmaniasis, Mem.
Inst. Oswaldo Cruz. 91 (1996) 225–229.
[63] D.L. Costa, T.M. Cardoso, A. Queiroz, C.M. Milanezi, O. Bacellar, E.M. Carvalho, J.
S. Silva, Tr-1-like CD4+CD25-CD127-/lowFOXP3- cells are the main source of
interleukin 10 in patients with cutaneous leishmaniasis due to Leishmania
braziliensis, J. Infect. Dis. 211 (2015) 708–718.
[64] J.M. Silverman, N.E. Reiner, Leishmania exosomes deliver preemptive strikes to
create an environment permissive for early infection, Front. Cell. Infect. Microbiol.
1 (2011) 26.
[65] J. Blanchette, N. Racette, R. Faure, K.A. Siminovitch, M. Olivier, Leishmania-
induced increases in activation of macrophage SHP-1 tyrosine phosphatase are
associated with impaired IFN-gamma-triggered JAK2 activation, Eur. J. Immunol.
29 (1999) 3737–3744.
[66] D. Nandan, R. Lo, N.E. Reiner, Activation of phosphotyrosine phosphatase activity
attenuates mitogen-activated protein kinase signaling and inhibits c-FOS and nitric
oxide synthase expression in macrophages infected with Leishmania donovani,
Infect. Immun. 67 (1999) 4055–4063.
A. Rodríguez-Vega et al.
[67] J.M. Silverman, J. Clos, C.C. De’oliveira, O. Shirvani, Y. Fang, C. Wang, L.J. Foster,
N.E. Reiner, An exosome-based secretion pathway is responsible for protein export
from Leishmania and communication with macrophages, J. Cell Sci. 123 (2010)
842–852.
[68] N. Santarem, G. Racine, R. Silvestre, A. Cordeiro-da-Silva, M. Ouellette,
Exoproteome dynamics in Leishmania infantum, J. Proteomics. 84 (2013) 106–118.
[69] G. Arango Duque, A. Jardim, E. Gagnon, M. Fukuda, A. Descoteaux, The host cell
secretory pathway mediates the export of Leishmania virulence factors out of the
parasitophorous vacuole, PLoS Pathog. 15 (2019), e1007982.
[70] M.F. Goncalves, E.S. Umezawa, A.M. Katzin, W. de Souza, M.J. Alves, B. Zingales,
W. Colli, Trypanosoma cruzi: shedding of surface antigens as membrane vesicles,
Exp. Parasitol. 72 (1991) 43–53.
[71] K. Hassani, E. Antoniak, A. Jardim, M. Olivier, Temperature-induced protein
secretion by Leishmania mexicana modulates macrophage signalling and function,
PLoS One 6 (2011), e18724.
[72] L. Franco-Serrano, S. Hernandez, A. Calvo, M.A. Severi, G. Ferragut, J. Perez-Pons,
J. Pinol, O. Pich, A. Mozo-Villarias, I. Amela, E. Querol, J. Cedano,
MultitaskProtDB-II: an update of a database of multitasking/moonlighting
proteins, Nucleic Acids Res. 46 (2018) D645–D648.
[73] F. Meissner, R.A. Scheltema, H.J. Mollenkopf, M. Mann, Direct proteomic
quantification of the secretome of activated immune cells, Science 340 (2013)
475–478.
[74] T. Furukawa, T. Uchiumi, R. Tokunaga, S. Taketani, Ribosomal protein P2, a novel
iron-binding protein, Arch. Biochem. Biophys. 298 (1992) 182–186.
[75] C. Huynh, N.W. Andrews, Iron acquisition within host cells and the pathogenicity
of Leishmania, Cell. Microbiol. 10 (2008) 293–300.
[76] B. Mittra, N.W. Andrews, IRONy OF FATE: role of iron-mediated ROS in Leishmania
differentiation, Trends Parasitol. 29 (2013) 489–496.
[77] S.K. Calderwood, J. Gong, A. Murshid, Extracellular HSPs: the complicated roles of
extracellular HSPs in immunity, Front. Immunol. 7 (2016) 159.
[78] A. De Maio, D. Vazquez, Extracellular heat shock proteins: a new location, a new
function, Shock 40 (2013) 239–246.
[79] E.A. Taha, K. Ono, T. Eguchi, Roles of extracellular HSPs as biomarkers in immune
surveillance and immune evasion, Int. J. Mol. Sci. 20 (2019).
[80] V.L. Vega, M. Rodriguez-Silva, T. Frey, M. Gehrmann, J.C. Diaz, C. Steinem,
G. Multhoff, N. Arispe, A. De Maio, Hsp70 translocates into the plasma membrane
after stress and is released into the extracellular environment in a membrane-
associated form that activates macrophages, J. Immunol. 180 (2008) 4299–4307.
[81] C. Yao, Major surface protease of trypanosomatids: one size fits all? Infect. Immun.
78 (2010) 22–31.
[82] P. Cuervo, L. Saboia-Vahia, F. Costa Silva-Filho, O. Fernandes, E. Cupolillo, J.B. De
Jesus, A zymographic study of metalloprotease activities in extracts and
15
Journal of Proteomics 232 (2021) 104077
extracellular secretions of Leishmania (Viannia) braziliensis strains, Parasitology
132 (2006) 177–185.
[83] P. Cuervo, A.L. Santos, C.R. Alves, G.C. Menezes, B.A. Silva, C. Britto,
O. Fernandes, E. Cupolillo, J. Batista De Jesus, Cellular localization and expression
of gp63 homologous metalloproteases in Leishmania (Viannia) braziliensis strains,
Acta Trop. 106 (2008) 143–148.
[84] G. Forget, D.J. Gregory, L.A. Whitcombe, M. Olivier, Role of host protein tyrosine
phosphatase SHP-1 in Leishmania donovani-induced inhibition of nitric oxide
production, Infect. Immun. 74 (2006) 6272–6279.
[85] J. Kern, G. Untergasser, C. Zenzmaier, B. Sarg, G. Gastl, E. Gunsilius, M. Steurer,
GRP-78 secreted by tumor cells blocks the antiangiogenic activity of bortezomib,
Blood 114 (2009) 3960–3967.
[86] R. Langer, M. Feith, J.R. Siewert, H.J. Wester, H. Hoefler, Expression and clinical
significance of glucose regulated proteins GRP78 (BiP) and GRP94 (GP96) in
human adenocarcinomas of the esophagus, BMC Cancer 8 (2008) 70.
[87] G. Zhao, J. Kang, K. Jiao, G. Xu, L. Yang, S. Tang, H. Zhang, Y. Wang, Y. Nie, K. Wu,
D. Fan, H. Zhang, D. Zhang, High expression of GRP78 promotes invasion and
metastases in patients with Esophageal squamous cell carcinoma, Dig. Dis. Sci. 60
(2015) 2690–2699.
[88] N. Biyani, R. Madhubala, Quantitative proteomic profiling of the promastigotes
and the intracellular amastigotes of Leishmania donovani isolates identifies novel
proteins having a role in Leishmania differentiation and intracellular survival,
Biochim. Biophys. Acta 1824 (2012) 1342–1350.
[89] L.C. Berger, J. Wilson, P. Wood, B.J. Berger, Methionine regeneration and aspartate
aminotransferase in parasitic protozoa, J. Bacteriol. 183 (2001) 4421–4434.
[90] L.J. Marton, A.E. Pegg, Polyamines as targets for therapeutic intervention, Annu.
Rev. Pharmacol. Toxicol. 35 (1995) 55–91.
[91] C. Dumas, M. Ouellette, J. Tovar, M.L. Cunningham, A.H. Fairlamb, S. Tamar,
M. Olivier, B. Papadopoulou, Disruption of the trypanothione reductase gene of
Leishmania decreases its ability to survive oxidative stress in macrophages, EMBO
J. 16 (1997) 2590–2598.
[92] S. Krieger, W. Schwarz, M.R. Ariyanayagam, A.H. Fairlamb, R.L. Krauth-Siegel,
C. Clayton, Trypanosomes lacking trypanothione reductase are avirulent and show
increased sensitivity to oxidative stress, Mol. Microbiol. 35 (2000) 542–552.
[93] N. Acestor, S. Masina, A. Ives, J. Walker, N.G. Saravia, N. Fasel, Resistance to
oxidative stress is associated with metastasis in mucocutaneous leishmaniasis,
J. Infect. Dis. 194 (2006) 1160–1167.
[94] Y. Perez-Riverol, A. Csordas, J. Bai, M. Bernal-Llinares, S. Hewapathirana, D.
J. Kundu, A. Inuganti, J. Griss, G. Mayer, M. Eisenacher, E. Perez, J. Uszkoreit,
J. Pfeuffer, T. Sachsenberg, S. Yilmaz, S. Tiwary, J. Cox, E. Audain, M. Walzer, A.
F. Jarnuczak, T. Ternent, A. Brazma, J.A. Vizcaino, The PRIDE database and related
tools and resources in 2019: improving support for quantification data, Nucleic
Acids Res. 47 (2019) D442–D450.