VIRULENCE

2022, VOL. 13, NO. 1, 903–935

https://doi.org/10.1080/21505594.2022.2074130

SIGNATURE REVIEWS

The pathogenicity and virulence of Leishmania - interplay of virulence factors

with host defenses
Anand Kumar Gupta, Sonali Das, Mohd Kamran, Sarfaraz Ahmad Ejazi, and Nahid Ali
Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India

ABSTRACT ARTICLE HISTORY

Leishmaniasis is a group of disease caused by the intracellular protozoan parasite of the genus Received 14 December 2021
Leishmania. Infection by different species of Leishmania results in various host immune responses, Revised 20 April 2022
which usually lead to parasite clearance and may also contribute to pathogenesis and, hence, Accepted 21 April 2022
increasing the complexity of the disease. Interestingly, the parasite tends to reside within the
KEYWORDS
unfriendly environment of the macrophages and has evolved various survival strategies to evade Leishmania; macrophage;
or modulate host immune defense. This can be attributed to the array of virulence factors of the virulence factor; signaling
vicious parasite, which target important host functioning and machineries. This review encom­ pathways; ncRNAs;
passes a holistic overview of leishmanial virulence factors, their role in assisting parasite-mediated diagnosis; therapeutics;
evasion of host defense weaponries, and modulating epigenetic landscapes of host immune immunomodulators
regulatory genes. Furthermore, the review also discusses the diagnostic potential of various
leishmanial virulence factors and the advent of immunomodulators as futuristic antileishmanial
drug therapy.

Introduction
Charles Donovan provided the clinical description of
Leishmaniasis is a multifarious vector-borne neglected VL (termed as “Kala-azar”) [4].
tropical disease (NTD) varying in the form of disease Currently, the disease is prevalent in tropical and
onset and includes cutaneous (CL), muco-cutaneous sub-tropical countries and southern Europe and covers
(MCL), visceral (VL), and post kala-azar dermal leish­ a geographic range of approximately 90 countries.
maniasis (PKDL) caused by at least 20 species of the According to WHO reports, apart from Australia and
obligate intracellular parasite, Leishmania [1,2]. Of Antarctica, the disease can be found in people of every
these, VL is the most severe form affecting visceral continent. In the Old World, leishmaniasis is reported
organs like spleen and liver and can prove fatal if left in some parts of Asia, the tropical regions and northern
untreated. CL and MCL forms are comparatively less part of Africa, southern Europe, and Middle East. In
severe, with the former manifesting self-healing ulcers the New World, it is prevalent in some areas of Mexico,
and the latter resulting in disfiguring lesions of oro- South, and Central America. The estimated number of
pharyngeal mucosal linings. The disease is transmitted CL per year still may range from 0.7–0 to 1.2 million.
by female Phlebotomine sp. and Lutzomyia sp. sand There is a decline in the number of estimated cases of
flies. However, transmission of the disease is rare by the visceral form of the disease and may range from
syringe sharing, blood transfusions, or from mother to over 400,000 cases to less than 100,000 cases [3].
foetus. The World Health Organisation (WHO) enlists In the current review, we discuss about the various
leishmaniasis as the second most severe NTD next to virulence factors of the different species of Leishmania
malaria and estimates over a million fresh cases and the strategies exploited by the parasites to over­
annually [3]. The existence of leishmaniasis can be come the immune defense mechanisms of the host for
dated back to as early 2500–1500 B.C. based on archae­ successful infection establishment. The review also
ological indications including pictures, mummified highlights the current scenario of diagnosis, limitations
bodies, and statues (Elisama et al, 2014). The detailed of frontline drugs, and the therapeutic potential of
description of CL (termed as “Apello Boil”) was given immunomodulators in controlling the menace of
by Alexander Russel. In 1903 Sir William Leishman and leishmaniasis.

CONTACT Nahid Ali [email protected]

© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
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904 A. K. GUPTA ET AL.

Life cycle, vector, and epidemiology parasite burden in these visceral organs.
Hypersecretion of adrenocorticotropic hormone in VL
Leishmania is a group of protozoan parasites belonging
patients leads to skin blackening of the patients, giving
to the Class Kinetoplastae and Order Trypanosomatida,
the disease its local name in India as Kala-azar [8].
which avails a digenetic life cycle involving an insect
Although majority of the cases are asymptomatic dur­
vector and a mammalian host. Leishmania sp can be
ing the initial phase of infection, symptoms may
characterized by the two prevailing forms, the elon­
develop even years later when the patients become
gated (10–20 μm) motile promastigote form and the
immunocompromised [9]. The emergence of HIV/VL
oval-shaped (3–7 μm in diameter) non-motile amasti­
co-infection has lately been a cause of great con­
gote form. The promastigote form exists in the sand fly
cern [10].
vector, where it undergoes various differentiation steps
and transforms into the infective metacyclic promasti­
gote form. These metacyclics are transmitted to mam­ Post kala-azar dermal leishmaniasis
malian hosts during the bite of the sand fly [5].
Treated VL patients often develop a complication of the
Amastigotes are the forms within the mammalian
skin, which acts as a reservoir of the parasite called
host, especially in phagocytic cells where they survive
PKDL. PKDL is characterized by macules, macupa­
as intracellular parasites. When a sand fly vector bites
pules, and nodular rashes in recovered VL patients.
an infected mammal, it ingests the amastigotes, which
The rashes usually develop around the mouth and
transform into the flagellated promastigote form on
gradually spread to other parts of the body. It is pre­
reaching the midgut of the insect. Eventually, the pro­
valent in areas where L. donovani is the causative VL
mastigotes move to the alimentary tract of the insect
agent, like in Sudan and India with 50% and 5–10%
where they survive extracellularly and multiply by bin­
cases, respectively. Although PKDL is usually not seen
ary fission. The promastigotes then migrate towards the
in people infected with L. infantum, it is often reported
salivary glands and oesophagus and are later trans­
in immune-compromised patients [11]. The manifesta­
mitted along with the insect saliva to the mammalian
tions of PKDL in India have been reported to occur 2–
host during the next blood meal. The anticoagulant
3 years post VL, while this interval is just 0–6 months
present in the saliva of the sand fly helps in the trans­
in Sudan [12–14]. Recent reports associate PKDL with
mission by preventing the blood from coagulating at
Th1 immune response, especially the production of
the site of insect bite. Post entry into the host, the
interferon γ, along with IL-10 in the peripheral blood
promastigotes are readily taken up by macrophages or
of treated VL patients.
dendritic cells within which they revert back to the
amastigote form and proliferate. This is followed by
the eventual egress of the amastigotes from the host Cutaneous and mucocutaneous leishmaniasis
cells due to the host immune response-mediated cyto­
CL is the most common form of leishmaniasis, and
lytic environment [6]. The amastigotes released are
unlike VL, it is less severe and usually self-healing.
either phagocytosed by other macrophages or taken
The clinical manifestations of the disease include
up by the sand fly during a blood meal. Thus, the
chronic ulcers at the sites of insect bite, which often
parasite continues its life cycle and culminates in infec­
leave life-long scars, leading to social stigmas, cosmetic
tion of surrounding cells and tissues in CL and organs
morbidities, and psychological effects. The extent of
rich in macrophages like the bone marrow, liver, and
symptoms varies depending on the leishmanial species
spleen in VL [7].
causing the infection and the immune state of the host.
L. major, L. aethipica, or L. tropica are the species of
Leishmania responsible for CL cases in the
Visceral leishmaniasis
Mediterranean region, America, central Asia, and
VL also called Kala-azar or black fever is the most Middle East. In South America, the common causative
severe form of leishmaniasis where visceral organs like agents of CL include L. braziliensis, L. guyanensis,
bone marrow, spleen, and liver are affected and can be L. panamensis, L. mexicana, and L.amazonensis [15].
fatal if not treated. The causative agents of VL in the Apart from human CL, canine CL is common in South
Old World include L. donovani and L. infantum, while America, and L. braziliensis and L. chagasi are the usual
the major causative agent in the New World is causative agents [16,17]. According to WHO, over 95%
L. chagasi. Clinical symptoms of the disease include new cases of CL in 2017 were reported from just six
fever, weight loss, anemia, hyper-gammaglobulinemia, countries, Brasil, Iran, Colombia, Afghanistan, Syria,
and hepatosplenomegaly mainly due to increased Iraq, and Algeria.
 VIRULENCE 905

MCL is a rare and severe variant of CL caused by Lipophosphoglycans

L. braziliensis, L. amazonensis, and L. mexicana. It is
Procyclic and metacyclic promastigotes differ in the
characterized by mucosal lesions, which lead to partial
thickness of their surface glycocalyx, which is mostly
or complete destruction of mucosal linings of the nose,
composed of glycosylated proteins and glycans.
throat, and mouth. These symptoms can be attributed
Lipophosphoglycans (LPGs) are the dominant structure
to the hyperallergic immune response targeting host
component of Leishmania surface glycocalyx [19]. LPG
tissues. Like VL, MCL is life-threatening with massive
is synthesized in promastigotes, and its ultrastructure is
lesions, which lead to permanent disfigurement requir­
composed of repetitive disaccharide units and phos­
ing early diagnosis and rapid treatment. Cases of MCL
phate, which are linked with a glycosyl phosphatidyli­
are endemic in regions of Latin America, specially
nositol (GPI) anchor. Side chain composition and core
Bolivia, Brasil, Ethiopia, and Peru.
positioning of LPG in surface glycocalyx largely varies
between promastigotes and amastigotes. Compared to
procyclics, LPG in metacyclics is longer in length and
Virulence factors of parasite
branched and completely absent in amastigotes [20,21].
Evasion of macrophage immune sentinel is a well- Therefore, the main requirement of LPG is confined
established strategy in obligate parasites. Leishmania solely during parasite entry and initial infection stages.
sp. has evolved with a plethora of membrane-bound On entry, the first phagocytic cell that Leishmania
or secreted virulence factors, which help in breaching encounters is the poly morpho-nuclear cells (PMNs)
the host immune barrier (Figure 1 and Table 1). Below or neutrophils, where it resides as promastigote. These
is the summarized overview of some well-illustrated PMNs help in transferring the promastigotes safely
leishmanial virulence factors, which are portrayed inside macrophages by forming apoptotic bodies. This
either as potential therapeutic targets or as vaccine method is well-known as “Trojan Horse” mode of
candidates. transfer, where LPG plays a crucial role [22]. LPG

Figure 1. Role of leishmanial virulence factors in entry and trans-differentiation of parasites. (1) Entry of promastigotes
through skin via bite of sand fly, (2) uptake of promastigotes by neutrophils, (3) safe transport of promastigotes from neutrophils to
macrophages by “Trojan Horse” mechanism, (4) formation of parasitophorus vacuoles (PVs) by prevention of phago-lysosomal fusion,
(5) trans-differentiation of promastigotes to amastigotes inside PVs (LPG: lipophosphoglycan; HSPs: heat shock proteins; and GPI:
glycosyl phosphatidyl inositol). Image created through paid version of Biorender.
906 A. K. GUPTA ET AL.

Table 1. List of genetically modified Leishmania sp. virulence factors.

Name of virulence Genetically modified type
factors (heterozygous/null mutation) Method of genetic modification Parasite species
Lipophosphoglycan lpg-/- Targeted gene disruption Leishmania major [25,31]
(LPG)
GRP94 lpg3-/- Targeted gene disruption Leishmania donovani [45]
Arginase arg-/- Targeted gene disruption Leishmania donovani [48]
EF1α - - -
GP63 gp63-/- Selective knock down by anti-sense RNA, Leishmania amazoniensis [74],
Targeted gene disruption Leishmania major [18]
CPC-A/B/C Δcpa, Δcpb, Δcpc Targeted gene disruption Leishmania mexicana [102]
Oligopeptidase opb-/- Targeted gene disruption Leishmania donovani [109],Leishmania
B (OPB) major [110]
Deubiquitinase DUB2-/- DiCre inducible gene deletion system Leishmania mexicana [114]
HSP100 HSP100-/- Targeted gene disruption Leishmania donovani [117],Leishmania
major [118]
HSP78 HSP78± and conditional HSP78-/- Targeted gene disruption, CRISPR-Cas9 Leishmania donovani, Leishmania
mexicana [120]
sHSPs HSP23-/- Targeted gene disruption Leishmania donovani [122]
A2 A2-deficient Selective knock down by anti-sense RNA Leishmania donovani [129]
PTP1 PTP1-/- Targeted gene disruption Leishmania infantum [133]

assists in the entry of Leishmania sp. promastigotes glycerol (DAG)/protein kinase C (PKC) signaling [32].
inside macrophages via binding with complement Purified LPG can reportedly block PKCα signaling of
receptor CR3 and integrin receptor p150/95 [23]. BALB/c and C57BL/6 mice macrophages [33]. Another
After phagocytosis in a macrophage, LPG–/– promasti­ major breakthrough in Leishmania infection biology
gotes are rapidly cleared compared to WT L. donovani. came with the discovery of the potential role of LPG
Moreover, external administration of purified LPG pre­ in delaying the appearance of late endosomal markers,
vented the early clearance of LPG-deficient promasti­ Rab7 and LAMP-1, on parasitophorus vacuoles (PV)
gotes. Thus, LPG is a highly essential surface antigen, and F-actin accumulation [34]. LPG not only prevents
playing a protective role during early trans- vacuolar acidification but also safeguards the parasites
differentiation of promastigotes to amastigotes. from reactive oxygen intermediates (ROIs) and lysoso­
LPG–/– L. major parasites displayed compromised viru­ mal enzyme-mediated damages [35]. The strong nega­
lence in human dendritic cells, murine macrophage cell tive charge and the presence of disaccharide repeats like
lines, and murine models and a poor survival rate in galactose–mannose in LPG mainly help in inhibiting
sand fly gut. Virulence and survival of LPG–/– parasites lysosomal enzymes [36]. Additionally, LPG can block
was restored upon cosmid-based complementation of the NADPH oxidase assembly in phagosomes, which
LPG encoding exons [24,25]. In L. major metacyclics, significantly affects the activation of innate immunity
LPG is highly branched and complex compared to during parasite infection establishment [37]. Although
procyclics. Resistance against complement receptor is LPG is absent in amastigotes, LPG predesigns an
positively correlated with the branching of LPG [26]. immune-suppressed situation during parasite entry so
L. major LPG prevents accumulation and lysis by C5b- that the amastigotes can easily survive and proliferate
C9 subunits [27]. In addition to complement and integ­ within. Interaction of parasite surface LPG with macro­
rins, LPG helps in mannose receptor-mediated entry of phage suppresses IL1β and IL12a secretion, as well as
parasites via its own mannan residues [28]. LPG helps IFN-γ-mediated nitric oxide (NO) generation [25,38].
in silent phagocytosis of parasites via C-reactive pro­ Besides Ca2+, LPG alters other secondary messengers of
teins (CRPs), without triggering the usual CRP- host macrophage like inositol lipids and inositol phos­
mediated inflammatory signaling of macrophages [29]. phate, which prevent phagocytosis and NO production
Upon entry, the parasite prevents fusion of phago­ [32,39]. In addition to the secondary messengers, LPG
some and lysosome to maintain a comparatively benign also prevents phosphorylation of ERK1/2 and subse­
microenvironment of phagosome where it can trans- quent activation of NF-κB and AP-1 module in order
differentiate from pH-sensitive promastigotes to pH- to inhibit NO generation [40]. LPG may not be essen­
resistant amastigotes [30]. The major role of LPG as tial for all Leishmania sp. for their virulence [41], but in
a leishmanial virulence factor was first established when some species of Leishmania, LPG seems to play
Spath et al. generated lpg–/– L. major parasites, which a crucial role for virulence and intracellular survival
showed attenuated infectivity in in vitro and in vivo (Figure 1).
infection models [31]. LPG mainly prevents this fusion LPG was identified as a potential vaccine candidate
by chelating calcium ions (Ca2+) and inhibiting diacyl for VL and is the first leishmanial antigen, which was
 VIRULENCE 907

conjugated with poly-electrolytic delivery vehicle the survival of the amastigotes. Parasite arginase is
named polyacrylic acid (PAA). LPG-PAA complex mainly localized in the glycosomes of both the promas­
showed minimal toxicity in J774 and mouse peritoneal tigotes and amastigotes and gets trafficked inside the
macrophage cells and exhibited enhanced anti- host whenever there is a lack of host arginase pool [47].
leishmanial efficacy [42,43]. Purified L. mexicana LPG Arginase not only helps in maintaining the L-arginine
administration in mice confirmed that LPG can induce pool inside promastigotes but also helps in the survival
PD-1 expression on the surface of CD8+ T cells and of amastigotes inside macrophages, contributing to
PD-L2 expression upon the macrophage surface, which parasite virulence. The potential of parasite arginase
induce immune-suppressive signals during vaccination as a virulence factor has been further confirmed by
[44]. However, LPG achieved success rate in vaccina­ infectivity assay with arg-/- L. major parasites.
tion due to low immunogenicity. Knockout parasites showed impaired survival potential
both in vitro and in vivo and resulted in decreased
intracellular parasite numbers [48].
GRP94
Genetic complementation of LPG-defective L. donovani
EF1α
parasites led to the identification of a unique and trun­
cated form of LPG, which contains only Manα1-PO4 The quest for other leishmanial virulence factors med­
residue of first repeat unit of LPG disaccharide. This iating regulation of macrophage signaling led to the
subtype of LPG was termed as LPG3, and it shared advent of another essential virulence factor, elongation
structural homology with mammalian endoplasmic factor 1α. Eukaryotic EF1α is a GTP-dependent transla­
reticulum (ER) chaperone GRP94 [45]. Like mamma­ tion factor, which mainly catalyzes binding of amino-
lian homolog, parasite LPG3/GRP94 is also localized in acyl tRNAs with ribosome. Structural modeling
the parasite ER and regulates chaperone-like functions, demonstrated that compared to human EF1α, 12-
i.e. protein assembly, secretion, antigen presentation, amino acid long-loop region is absent from leishmanial
etc. [46]. Interestingly, null mutation of GRP94- EF1α. This opened a new avenue for structure-based
induced pleotropic defects such as downregulation of drug targeting, like targeting of this particular domain
surface GPI-anchored proteins with no effect on either of parasite EF1α [49]. EF1α mainly gets exported from
N-glycosylated protein synthesis or proliferation rate of PVs and binds to host SHP-1 phosphatase, thereby
the promastigotes. Expression of GRP94 is not depen­ leading to macrophage inactivation. EF1α lacks
dent on stress and is mainly regulated developmentally. N-terminal secretory peptide, suggesting that EF1α is
Orthologs of L. infantum GRP94 were found to be secreted out of the PVs via a non-classical ESCRT-III
highly immunogenic, indicating the role of GRP94 in pathway [50]. Interestingly, only leishmanial EF1α, and
regulating the immune responses of host during infec­ not human EF1α, can block macrophage SHP-1 activ­
tion. Altogether, LPG3 plays a completely different and ity, which offers further areas of structural investiga­
exclusive role in leishmania metabolism compared to tions. Compared to GP63, EF1α offers higher potency
its mammalian homolog [45]. to block the SHP-1, preventing NO generation in
response to IFN-γ stimulation [51]. EF1α is a part of
the leishmanial secretome and functions as a cargo for
Arginase
exosomal export of other leishmanial antigens from
Besides being part of the Krebs-Henseleit cycle, PVs to macrophage cytosol [52,53]. Besides being
L-arginine aminohydrolase or Arginase plays non- a potential drug target, EF1α is a potential vaccine
canonical role in infection persistence and virulence candidate. Sabur et al. reported that cationic liposomal
of Leishmania sp. Arginase is a metalloenzyme, which EF1α can trigger delayed-type hypersensitive (DTH)
hydrolyses L-arginine into L-ornithine and urea, which response, Thelper cell proliferation, augmenting IFN-γ
contributes to the ureotelic behavior of Leishmania. In response, and long-term protective memory response
order to survive inside the macrophage and transform of both CD4+ and CD8+ T cells in L. donovani-
into amastigote form, it is crucial for Leishmania sp. to challenged BALB/c mice [54].
overcome the toxic effect of host nitric oxide. The
parasite, apart from exploiting the host arginase, itself
Proteases
possesses its own arginase. Parasite arginase converts
host L-arginine into L-ornithine [47] and helps in Proteases are a class of enzymes that can digest the
bypassing the L-arginine pool from nitric oxide genera­ target proteins or peptides, manifesting important
tion toward L-ornithine production, which is used for roles in the life cycle of any organism. These proteases
908 A. K. GUPTA ET AL.

are classified based on the amino acids present in their receptor signaling of macrophages. C3bi thus not only
active site, like serine-, threonine-, aspartyl-, metallo-, hampers the complement-mediated lysis of parasites but
and cysteine proteases. In Leishmania sp., aspartyl-, also augments safe internalization of parasites via C3bi
serine-, metallo-, and cysteine proteases have been opsonization into macrophages [68,69]. In addition to
extensively studied as virulence factors [55–57]. CRs, GP63 also assists in the parasite adherence to macro­
Expression of active aspartyl protease in the soluble phage through fibronectin receptor (FR) [70]. GP63 can
fraction of L. mexicana promastigotes was found to be degrade fibronectin, thus helping in downregulating ROS
essential for parasite proliferation, but its role in host generation and supporting parasite survival in macro­
modulation is still not well deciphered [58]. phages [71]. GP63 is one of the major virulence factors
that helps in the degradation of extracellular matrix of
subcutaneous tissue and helps in tissue penetration and
GP63
dissemination of L. mexicana [72].
GP63, discovered in 1980, is a 60–66 kDa molecular The role of GP63 in suppressing macrophage
weight leishmanial protease and was considered as immune signaling is controversial, but it aids in infec­
a major surface antigen (MSA) [56]. Later, due to its tion persistence. Interestingly, GP63-coated PVs are
ability of binding with Concanavalin A (Con A) and resistant toward phago-lysosomal degradation [73].
high glycosylation, it was renamed GP63. GP63 is also Antisense RNA-silenced GP63 L. amazonensis exhib­
known as leishmanolysin. Besides Leishmania, GP63 ited lower parasite burden, confirming a major role of
shares structural homologs in Trypanosoma sp. and GP63 in intracellular amastigote survival [74].
Trichomonasvaginalis [59]. GP63 has a wide range of Additionally, GP63 can cleave the SNARE-Vamp8 pro­
substrate specificity, including gelatin, hemoglobin, tein in order to prevent phagosomal maturation and
albumin, fibrinogen, and casein. It mainly cleaves at antigen cross-presentation to CD8+ T cells [75]. GP63
the junction of hydrophilic and hydrophobic amino is directly involved in leishmanial hijacking of diverse
acid residues at positions P1 and P’1, with basic host macrophage immune signaling machineries.
amino acid residues at positions P’2 and P’3 [60]. MARCKS (myristoylated alanine-rich C kinase sub­
GP63, a Zn-dependent metalloprotease, belongs to the strate) are the major inflammatory mediators that nor­
metzincin class. Presence of the sequence motif mally get up-regulated in LPS-stimulated macrophages.
HExxHxxGxxH and a pro-peptide at its N-terminal MARCKS results in the activation of MARCKS-
end makes it a zymosan/pro-enzyme-like molecule regulated proteins (MRP), which function as the
that remains inactive after its translation [59]. During major substrate of PKCs. These MRPs are proteolyti­
trans-differentiation from promastigotes to amasti­ cally cleaved by GP63 [76,77]. L. major-infected macro­
gotes, expression of GP63 drops [61]. However, in phages resulted in exhausted levels of MRP, which was
amastigotes, the low expression of GP63 is compen­ restored on treatment with GP63 inhibitors [76].
sated by the low expression of LPG; hence, GP63 is Amastigotes generally lack LPG and safeguard them­
no longer buried [20]. Like other secretory proteins, selves from ROI-mediated damage via GP63-mediated
GP63 is also processed in the endoplasmic reticulum, prevention of PKC activation [77]. Protein tyrosine
and nearly 75% of GP63 is either expressed on the phosphatases (PTPs) like SHP-1 get activated immedi­
parasite surface or is a part of the lipid rafts [62,63]. ately upon Leishmania entry, which negatively regulate
Extent of glycosylation, anchoring with glycosyl phos­ the activation of inflammatory JAK2/STAT1α pathway
phatidylinositol (GPI) link, Zn-chelation, and auto- [78]. GP63 after entering the macrophages through
proteolysis are the main factors that regulate secretion lipid rafts can transactivate PTPs (SHP-1, TCPTP, and
of GP63 [64,65]. gp63–/–L. amazonensis confirmed that PTP1B) by cleaving their C-terminal portion [79].
GP63 can be secreted either via vesicles or directly. Besides JAK/STAT signaling, L. donovani negatively
Direct secretion from a cell surface is dependent upon regulates TLR4-signaling-mediated NO generation by
autoproteolytic cleavage of the inactivation peptide. In SHP-1. SHP-1 inactivates IRAK-1 by tyrosine depho­
L. chagasi and L. donovani, large micelle-based and sphorylation and blocks TLR4 pathway-mediated acti­
exosome-based secretion of GP63 takes place. Thus, vation of NO by IRAK1/IRAK4 module [80].
despite N-terminal secretory signal peptide, GP63 can In addition to STAT1α, Leishmania GP63 exerts
get secreted via the ESCRT-III-dependent non- control over other major transcription factors of
conventional pathways [66,67] (Figure 1). macrophages like NF-κB and AP-1. It enters the
GP63 contributes to Leishmania virulence by proteo­ nuclear matrix via lipid rafting and ceramide augmen­
lytically cleaving C3b into C3bi. C3b is essential for the tation of the nuclear membrane. GP63 specifically and
recruitment of complement lysis machinery via the CR1 partially degrades NF-κB subunit p65RelA and
 VIRULENCE 909

produces p35RelA, which heterodimerizes with of action as papain proteases and are subdivided into
p50RelB and induces disease-promoting chemokines three types, CPA, CPB, and CPC. Among these, CPA
like macrophage inflammatory protein (MIP) 1α and and CPB are cathepsin L-like enzymes and CPC is
MIP1β [81,82]. Like JAK2/STAT1, AP-1 (composed of a cathepsin B-like enzyme [93–95]. Interestingly, in
C-jun and C-fos) is also essential for IFN-γ-mediated L. donovani and L. major, a single nucleotide poly­
NO production by macrophages [40]. Leishmania sp. morphism (SNP) has been observed in the genes
induces GP63-mediated proteolytic degradation of encoding CPs, which determines whether the parasite
C-Jun subunit of AP-1. In addition, it down-regulates infection will be dermatropic or viscerotropic [95,96].
IFN-γ signaling, as well as subsides IFN-α/β transcrip­ The role of CPs in Leishmania virulence was perceived
tion by targeting mTOR. GP63 cleaves mTOR, leading from murine models infected with parasites belonging
to dephosphorylation of 4E-BP1, which hampers the to the L. mexicana complex such as L. mexicana,
CAP-dependent translation of IFN-α/β in macrophages L. pifanoi, and L. amazonensis [97–99]. Besides this,
infected with L. major promastigotes [83]. a positive correlation was observed between the expres­
Inflammasome activation plays a crucial role in sion level of CPs and parasite virulence in hamsters
destroying intracellular amastigote burden. infected with L. infantum and human cell lines infected
Inflammasome complex is mainly initiated by NOD-like with L. chagasi [57,100]. Expression of CPs was signifi­
receptor protein 3 (NLRP3), which gets two subsequent cantly enriched in L. amazonensis amastigote extract
signals – i) TLR agonists like LPS and ATP and ii) intra­ compared to promastigotes, which further strengthened
cellular ROS [84]. Activation of NLR3 receptor complex the fact that the CPs play a potential role in the survival
leads to the production of IL-1β and IL-6, which assist in of amastigotes inside hosts [57]. Additionally, L. tropica
the clearance of parasites. Exosomal GP63 of L. major parasites, treated with CP inhibitor, exhibited dimin­
parasites reportedly blocks ROS production by preventing ished growth rate, pathogenicity, and survival [101].
PKC activation and degrades NLP3 inflammasome com­ Infectivity analysis with Δcpa, Δcpb, and Δcpc
plex to prevent activation of IL-1β in both murine and L. mexicana parasites demonstrated that parasite viru­
human infection models [85]. lence was severely hampered after deletion of cpb gene
Interestingly, GP63 becomes a standalone vaccine can­ compared to cpa and cpc gene. Interestingly, parasite
didate because it is essential for parasite survival, highly virulence was completely restored by not single but
immune reactive surface antigen, and low mutagenic [86]. complementation with multiple copies of cpb in cosmid
Amino acid sequences of human T cell epitope have been vector [102]. Δcpb L. mexicana parasites were report­
identified in L. major, L. donovani, and L. chagasi GP63 edly found to be unable to induce IL-4 expression in
exon sequences [87–89]. These epitopes can potentially BALB/c mice, and a Th1 response was mounted limit­
mount Leishmania-specific CD8+ T cell response and ing their expansion. However, insertion of cpb gene
elevate IFN-γ levels, which can offer excellent protection recovered the virulence and the capacity of the parasite
[87]. Whole exon of L. major GP63 became the first to induce IL-4 production, indicating that L. mexicana
candidate for DNA vaccine [86]. Later on, polytope infectivity in BALB/c mice is directly related with its
DNA vaccine with multiple T cell epitopes of GP63 and capacity of IL-4 induction. On the contrary, in C57BL/6
HSP70 from Mycobacterium tuberculosis adjuvant proved mice, Δcpb L. mexicana parasites were unable to induce
successful in L. donovani-infected BALB/c mice model the suppression of IL12p40 and STAT4, resulting in
[90]. GP63 was also employed as a candidate of choice Th1 response-mediated parasite clearance [102]. CPB
for novel gunshot emulsification-based immunization assists in infection establishment of L. major parasites
approach and provided better protection against in C57BL/6 mice via suppression of IFN-γ expression
L. mexicana in infected BALB/c mice compared to and in macrophages and dendritic cells via suppression
Soluble Leishmania Antigen (SLA) [91]. Additionally, of IL-12 production [103]. Being a potential protease,
recombinant Ldgp63 containing cationic liposome acted CPB mainly cleaves inflammatory transcription factors
as a stable and potent antigen, which induced long-term like NF-κB p65 subunit, STAT-1 and AP-1, which helps
memory responses in BALB/c mice against L. donovani the parasite in the prevention of IL-12 and NO produc­
infection [92]. tion by the host. Unlike gp63, CPB completely cleaves
p65. Besides transcription factors, CPB cleaves MHC-II
protein inside the PVs, which helps in preventing anti­
Cysteine proteases
gen presentation and activation of the Th1 immune
Cysteine proteases (CPs) of Leishmania sp. are being response [104]. Subcutaneous introduction of recombi­
studied as effective drug target and vaccine candidates. nant leishmanial CPB in BALB/c mice footpads aug­
CPs that are found in Leishmania sp. have similar mode mented IL-4 and IL-5 levels with concomitant cleavage
910 A. K. GUPTA ET AL.

of CD25 receptor [105]. Structural insights of LmxCPB we have assembled few such crucial proteins of para­
confirmed that it has a COOH-terminal extension sites, which are recently getting highlighted as potential
(CTE), which gets hydrolyzed before its secretion. virulence factors and chemotherapeutic targets or vac­
This CTE domain also possesses immune regulatory cine candidates.
function in the host. Later, it was established that only
the CTE portion of CPB protein is capable of inducing
Deubiquitinases (DUBs)
Th2 cytokines. L. pifanoi and L. amazonensis amasti­
gotes, pre-incubated with anti-CTE antibodies, showed During trans-differentiation of parasites, reversible
decreased proliferation inside macrophages and post-translational modifications (PTMs) like ubiquiti­
immune suppression capabilities [97,106,107]. These nation/deubiquitination play significant roles.
data indicate that not only the active site but also the Ubiquitination is a highly conserved process through­
CTE domain of CPB is crucial for Leishmania infection out evolution and mainly helps in maintaining the
establishment and host immune modulation. Besides protein balance in the eukaryotic cells. In L. mexicana
CPB, CPC has been well studied as a potential DNA promastigotes, ubiquitination takes place with the help
vaccine candidate against VL. CPC expressing DNA of 2 E1 ubiquitin-activating enzymes, 13 E2 ubiquitin-
vector (pVAX1-cpc) induced strong immune protective conjugating enzymes, and 79 E3 ubiquitin ligase-
Th1-biassed response in L. donovani-challenged BALB/ mediated ubiquitin-proteasome system (UPS)-based
c mice in association with substantial reduction of tagging and degradation of target parasite proteins.
parasite burden [108]. Taken together, besides being UPS pathway plays crucial roles in parasite autophagy,
a vital virulence factor, CPs can be potential vaccine DNA repair, and protein trafficking. However, the key
candidates for leishmaniasis. player of UPS system is deubiquitinases (DUBs), which
remove the reversal ubiquitin group as and when
required and add another level of fine-tuning in the
Serine protease
parasite life cycle regulation. Till date, 20 DUBs were
Oligopeptidase B (OPB), a serine protease (SP) of 115 identified in L. mexicana, which mainly belong to seven
kDa molecular weight, was initially considered to have structural super families, ubiquitin-specific proteases
a promising role in virulence. OPB gets upregulated (USPs, family C19), C-terminal hydrolyases (UCHs,
during amastigote stage differentiation and helps in family C12), ovarian tumor proteases (OTUs, family
the protection of amastigotes by covering with enolase C65), JAB1/MPN/MOV34 metalloenzymes (JAMM/
and plasminogen for their proliferation. Role of OPB in MPN+, family M67), Josephins (family C86), MINDY
host macrophage global gene dysregulation was estab­ (family C115), and ZUFSP (zinc finger with UFM1-
lished by infecting them with OPB-deficient specific peptidase domain protein, family C78) [113].
L. donovani parasites [109]. Compared to WT strain By employing bar-seq CRISPR-Cas9 technology,
that induced changes in 23 macrophage genes, OPB–/– Damianou et al. identified DUBs 4, 7, and 13 as essen­
L. donovani induced 495 genes. At the same time, OPB- tial during amastigote stage development of
deficient L. major promastigotes showed impaired L. mexicana promastigotes. Additionally, by chemical
development of metacyclic promastigotes and, thus, probing, they deciphered essentiality of DUB 3, 5, 6, 8,
the inability to infect macrophages [110]. Activity and 10, 11, and 14 for parasite survival during in vivo
expression of another leishmanial SP, subtilisin pro­ infection in mice. With the help of DiCre inducible
tease (SUB), also increased in the amastigotes stage of gene deletion system, Damianou et al. also demon­
L. donovani [111]. Mice injected with L. amazonensis strated the importance of DUB2 in the establishment
soluble fraction containing active SP showed enhanced of infection in macrophages and animals [114]. DUB2
sensitivity toward parasite infection. On the contrary, mainly cleaves off di-ubiquitine chain in a broad link­
when animals were injected with SP inhibitor-treated age-specific manner. The association of another DUB,
extract, the susceptibility of the animals toward infec­ Otubain (OTU), with host immune signaling was first
tion diminished, suggesting a direct role of parasite SP established during L. infantum infection by Azevedo
invirulence [112]. et al. OTU mainly cleaves K48-linked tetra-ubiquitin
As Leishmania parasites persist in both sand fly chains of target proteins [115]. Recombinant LiOTU
vector and mammalian hosts, they are well-equipped can trigger inflammatory responses in host macro­
with some unique group of chaperons and post- phages via lipid droplet biogenesis, IL-6, and TNFα.
translational modifiers, which assist in the transforma­ Interestingly, a recent report highlighted a highly sen­
tion of the parasite from sand fly stage to mammalian sitive diagnostic potential of L. donovani OTU for
stage and vice versa. Therefore, in the following section, endemic VL samples [116]. These reports collectively
 VIRULENCE 911

suggest the fervent role of DUBs in the virulence of Small HSPs

parasite.
Small molecular weight HSPs (sHSPs) are mainly com­
posed of a conserved α-crystallin domain (ACD), which
folds in 7–8 stranded β-sandwich structure and exists in
Heat shock protein (HSPs) a dimer form. sHSPs are highly divergent compared to
high molecular weight HSPs. sHSPs bind to a broad
During transmission from sand fly to mammalian
range of target proteins and function like holdase [121].
hosts, the parasite needs to acclimatize to the almost
sHSPs mainly assist the ATP-dependent chaperons like
10°C temperature upshift and the acidic pH (4.5–5.5)
HSP100 to fold the target proteins. HSP20, P23, and
within the macrophage phagosomes. To overcome this
HSP23 are the well-characterized sHSPs from
challenge, the parasite induces the production of
Leishmania sp [122–124]. HSP20 has been found to
a plethora of leishmanial heat shock proteins (HSPs),
play an important role as a potential immunogenic
which safeguards its proteins from heat-induced
antigen during canine leishmaniasis. However, its role
damages. Thus, parasite HSPs are key players during
as a protective DNA vaccine is still questionable. P23
mammalian stage development of Leishmania sp
and HSP23 from L. braziliensis function as HSP90 co-
(Figure 1).
chaperones, and null mutants of P23 produced gelda­
namycin (HSP90 inhibitor)-sensitive parasites [125].
Further analysis of HSP23–/– parasites identified
HSP100 HSP23 as a heat-inducible chaperon, which is highly
expressed in amastigote-like conditions and essential
HSP100 is an AAA+ casinolytic protease B (clpB)
for amastigote stage differentiation. HSP23–/– parasites
family protein, and through gene manipulation studies,
display enhanced sensitivity toward trivalent antimony
it was found to play a non-canonical role in leishmanial
Sb(III). HSP23 reportedly plays a potential role in
virulence. HSP100-/- L. donovani and L. major para­
resistance against trivalent antimony Sb(III) and metal­
sites showed no growth impairment in axenic culture
loid-based anti-leishmanials, highlighting a potential
and had no sensitivity toward heat stress. However,
connection of the sHSP with resistance generation
both the species failed to thrive within mice models
against antimonial drugs [122].
and failed to transform into amastigotes [117]. HSP100
is an amastigote-specific protein secreted from the
parasite’s flagellar pockets via temperature-induced
A2
exosomal secretion pathway. HSP100 contributes to
parasite virulence by playing a major role in exosomal A major rate-limiting step of Leishmania infection
trafficking pathway [118]. establishment inside host macrophage is the trans-
differentiation of parasites from promastigote to amas­
tigote. Formation of amastigote is crucial for the toler­
ance and survival of parasites inside the harsh
HSP78
phagosomal vacuoles. Amastigotes specifically possess
HSP78 is another clpB protease family member, which some crucial virulence factors that are absent in the
is an ATP-dependent amastigote-specific protein that promastigote form of the disease. A2 is one such amas­
assists in the management of heat and pH stress [119]. tigote-specific protein that was first discovered in
HSP78-/- L. donovani parasites are non-viable, and L. donovani by karyotype analysis. However, interest­
conditional knock out of HSP78 in L. donovani con­ ingly, A2 gene is mostly expressed in parasites causing
firmed essentiality of the protein for promastigote VL but not CL. Moreover, antibody response against
growth. Moreover, partially depleted HSP78 A2 gene was reported in human VL patients and
L. donovani promastigotes showed impaired infectivity infected dog sera but not in CL-infected individuals
in macrophages and BALB/c mice. HSP78 plays [126], indicating a prospective role of A2 in viscereliza­
a crucial role in suppressing pro-inflammatory tion of Leishmania parasites in mammalian organs
responses and cytokines of macrophages, along with [127]. A2 gene encodes for a total seven isotypes of
nitric oxide. In a pioneering study in this regard, Das proteins with molecular weights ranging from 45 to 100
et al. reported the regulatory role of HSP78 in the kDa. A2 mRNA and protein expression are completely
establishment of Leishmania infection in hamsters. absent in promastigote but abundantly expressed in
ATP analogue, Ap5A, helped in identifying HSP78 as amastigote-like conditions, i.e. parasites cultured at
a potential chemotherapeutic target [120]. 37°C and pH 4.5 [128] . A2 protein amino acid
912 A. K. GUPTA ET AL.

sequence shares unique repetitive stretch of 10 amino Leishmania sp. mediates modulation of various host
acids, which is homologous to that of S antigen of processes like apoptosis and arsenals, like generation
Plasmodium falciparum V1 strain. Interestingly, of ROS and RNS, and alters Th1/Th2 cytokine balance
S antigen is another stage-specific virulence factor of and TLR-mediated signaling mechanisms. Leishmania
P. falciparum, which is responsible for malaria infection sp. parasites also possess the capability of modulating
in human host [128] . Antisense RNA-mediated A2- important host signaling pathways like MAPK and
silenced L. donovani amastigotes determined that A2 altering host miRNA pool to its favor. Leishmania sp.-
deficiency severely hampered virulence and survival of secreted virulence factors interestingly either abrogate
amastigotes in both macrophages and BALB/c mice the functioning of host proteins/factors responsible for
[129]. Besides, over-expressing A2 gene in L. donovani the activation of immune response or exploit host-
and L. major exhibited enhanced organ parasite burden negative regulatory proteins controlling immune func­
in experimental animal model [130]. Immunization of tioning. In this section, we focus on few important
mice with recombinant A2 protein or DNA vaccine strategies exploited by the parasite to alter the macro­
significantly mounted protective immune response phage niche to a disease-conducive state.
against L. donovani challenges [131]. This suggests
that besides being a vital virulence factor, A2 possesses
Suppression of reactive oxygen and nitrogen
promising attributes to become a vaccine candidate.
species generation
When a pathogen enters a host cell, one of the major
PTP1
challenges that it faces is the burst of ROS and RNS.
Phosporlylation and dephosphorylation of proteins Production of ROS and RNS by host cells to destroy
involved in significant biochemical pathways of invading pathogens or phagocytosed foreign materials
Leishmania sp. play a crucial role during trans- are under tight regulation to ensure minimum collat­
differentiation from promastigotes to amastigotes. eral damage to the host. The process involves tightly
Leishmania genome database mining identified a group controlled steps catalysed by various enzymes like
of protein phosphatases of parasites named protein tyr­ NADPH oxidase 2 (NOX2) and nitric oxide synthase
osine phosphatases (PTPs) [132]. Tyrosine phosphatases (NOS). The production of NO is catalysed by NOS by
are involved in the removal of phosphate group from reacting with terminal nitrogen of the guanidium group
target protein, thereby regulating many essential life- of L-arginine. Early findings in this regard suggest that
cycle stages like cell cycle, differentiation, disease estab­ activated macrophages can inhibit L. major in an
lishment, etc. Genetical manipulation studies confirmed L-arginine-dependent manner [134]. This finding was
that although LdPTP1 gene is not essential for the survi­ later confirmed by studies indicating that usage of
val of L. donovani promastigotes, LdPTP1 null parasites L-arginine analogue, L-N-monomethyl arginine, and
are unable to persist in BALB/c mice. Due to the remark­ inhibitors of NO pathway reversed the antileishmanial
able structural homology between human PTP1B and effect of IFN-γ or LPS-activated macrophages [135].
L. inflantum PTP1 active sites, in silico studies identified Their work also demonstrated that in vivo administra­
parasite PTP1 as a promising therapeutic target [133]. tion of L-arginine analogues in the Leishmania-resistant
CBA mice rendered them susceptible to infection. In
order to evade NO production, most leishmanial para­
Subversion of host defense machineries by
sites exploit the host protein SHP-1 so as to interfere
leishmania
with JAK2, ERK1/2, and the transcription factors, AP-1
Leishmania parasites, like any other pathogen post and NFκB. Moreover, SHP-1-deficient macrophages on
entry, is readily taken up by host macrophages and infection with L. donovani could activate NO produc­
other phagocytes. However, unlike most pathogens, tion [136], suggesting the importance of host SHP-1 for
Leishmania sp. tends to survive within the macrophages parasite survival. However, interestingly, reports by
and have developed strategies to overcome the Spath et al. suggest that the intracellular survival of
unfriendly intracellular environment designed for elim­ L. major was not dependent on SHP-1 (PMID:
ination of pathogens and foreign materials. Thus, leish­ 18682252). Leishmania-mediated SHP-1 activation can
maniasis is a good infection model for studying host– be attributed to the parasite’s elongation factor-1α (EF-
parasite interaction. The parasite has viciously evolved 1α) [51]. The group, however, reported the results 16 h
various survival strategies to enter host macrophages post infection and could not explain the activation of
and proliferate within by overriding the various defense SHP-1 at early time points of infection, nor the shuttle
weaponries of the host cells. Various reports suggest mechanism of EF-1α from the phagolysosomes. Later
 VIRULENCE 913

studies by Gomez et al. suggested activation of host miR-294 and miR-721, leading to increased NOS2 pro­
SHP-1 to be mediated by the metalloprotease of the duction [141] (Figure 2).
parasite, gp63, known to traverse through lipid rafts to ROS or ROIs possess parasite killing capability, yet
gain access to the host cytosol [137]. Another mechan­ their effects can be considered transient and limited to
ism of downregulating NO production can be wit­ host protection only during the early phases of infec­
nessed in L. amazonensis, which upregulates the tion [142]. However, for successful establishment of
expression of host arginase and polyamines [47]. infection, the parasite must overcome the effects of
Upregulation of arginase was reported in both suscep­ ROS. L. donovani has been widely reported to inhibit
tible and resistant laboratory mice on infection with ROS generation in infected macrophages [143,144].
L. major, which correlated with reduction in the L. chagasi infection has also been documented to reg­
expression of NOS2 [138,139]. Upregulation of host ister decreased superoxide production in murine and
arginase also provides polyamines for parasite salvage human macrophages [145]. L. amazonensis and
and proliferation [139]. This can be justified by the L. brasiliensis have been reported to exploit the host
finding that the arginase of the parasite, being an anti-oxidant enzyme superoxide dismutase 1 (SOD1) as
important enzyme for polyamine biosynthesis, is neces­ a survival strategy [146]. Similar to other intracellular
sary for the survival of L. donovani promastigote but pathogens like Mycobacterium abscessus and Salmonella
not the intracellular amastigote form [140]. This indi­ typhimurium [147,148], various leishmanial species like
cates a probable vicious strategy by the parasite of L. chagasi, L. pifanoi, and L. donovani have also been
upregulating the host arginase not only to evade NO reported to exploit another host antioxidant enzyme-
production but also to sustain its own biosynthetic hemeoxygenase 1 [149–151] (Figure 2). L. pifanoi infec­
processes. In this regard, another role of the parasite tion induced HO-1 levels at a very early stage of infec­
arginase can be witnessed in L. amazonensis-infected tion, suggesting the necessity of cellular ROS evasion by
macrophages where the former led to the inhibition of the parasite for successful establishment of infection.

Figure 2. Leishmania suppresses ROS and RNS generation for successful survival within the host cell. In order to tackle the
burst of ROS and RNS on infection within the host, Leishmania has evolved various virulence factors as well as mechanisms. LPG of
Leishmania sp. inhibits PKC activation, which is necessary for the formation of NADPH oxidase complex, thus, blocking ROS
generation. For suppression of mitochondrial ROS generation, the parasite viciously exploits the mitochondrial membrane protein,
UCP2. Apart from these strategies, the parasite has also been reported to upregulate host antioxidants like HO-1 and SOD1. In order
to suppress NO production, the parasite exploits the host PTP like SHP-1 by virtue of virulence factors like EF-1α and GP63. SHP-1
blocks JNK and ERK activation, required for NO production. Leishmania also upregulates host arginase-1, which inhibits the harmful
effects of NO. Apart from facilitating the parasite to overcome the effects of NO, host arginase also provides polyamines for parasite
salvage (ROS: reactive oxygen species; RNS: reactive nitrogen species; LPG: lipopohosphoglycans; UCP2: uncoupling protein 2; HO-1:
hemeoxygenase 1; SOD-1: superoxide dismutase-1; PTP: protein tyrosine phosphatase; SHP-1: Src homology 2 domain-containing
protein tyrosine phosphatase 1; NO: nitric oxide; and EF-1α: elongation factor-1α). Image created through paid version of Biorender.
914 A. K. GUPTA ET AL.

NAD(P)H oxidase is the major contributor of cytosolic associated with a burst of pro-inflammatory cytokines
ROS in macrophages, and the different subunits of the [161], and Leishmania infected macrophages have been
enzyme complex are assembled on stimulation. Reports reported to be unresponsive to LPS treatment [162],
suggest inhibition of protein kinase C-mediated phos­ thus indicating the importance of TLR activation dur­
phorylation of p47 and its interaction with p67 by ing infections by the parasite. TLRs play diverse roles
L. donovani amastigotes [152,153] (Figure 2), but strik­ from parasite clearance to aggravated pathology and are
ingly, lipophosphoglycan (LPG) of L. donovani promas­ often species-specific in response to the various
tigotes blocks the assembly of the enzyme without Leishmania-expressed ligands [163,164]. Extensive stu­
involving p47 [154]. dies in this regard suggest the involvement of various
Besides the cytosolic ROS production by NADPH leishmanial molecules in the activation of TLRs, spe­
oxidase, mitochondria are a major contributor of intra­ cially TLR2, TLR4, and TLR9. TLR2-mediated recogni­
cellular ROS. The generation of mitochondrial ROS tion of LPG of L. major, L. mexicana, and L. aethiopica
takes place due to premature leakage of electrons in leads to increased ROS and NO production [165–167]
the electron transport chain, which increase during and favors a protective immune response.
physiological and pathological conditions [155,156]. Simultaneously, contrary reports exist suggesting
Extensive studies implicate mitochondrial ROS genera­ TLR2 activation favoring persistence of
tion with innate immunity and antipathogenic activity L. amazonensis and L. braziliensis infection [168,169].
[156]. L. donovani has been reported to subvert the This differential response can be attributed to the dif­
outburst of mitochondrial ROS by upregulating uncou­ fering thickness of the LPG coat of the mentioned
pling protein 2 (UCP2), an inner mitochondrial mem­ species [169]. Association of TLRs and MyD88-
brane protein [143,144] (Figure 2). Leishmania- mediated pathway during infection by Leishmania was
mediated upregulation of UCP2 has been reported to first studied in 2002, reporting decreased IL-1α expres­
de-polarise mitochondrial membrane potential, even­ sion in MyD88−/− mice upon infection by L. major
tually leading to altered electron transport and ROS [170]. The following study in C57BL/6 mice by
formation [157]. Muraille et al. exhibited the importance of MyD88 in
Apart from exploiting host-negative regulatory pro­ controlling cutaneous lesions by Leishmania along with
teins, Leishmania sp. themselves are armed with certain enhanced IL-4 and reduced levels of IFN-γ and IL-12
characteristics that provide resistance to the harmful [171]. Administering anti-IL4 antibodies led to an
effects of ROS and RNS. The layer of LPG, especially increase in the levels of IFN-γ and drove the cytokine
on the amastigotes form of the parasite, provides pro­ balance toward disease resolving Th1 state [172]. The
tection from ROS and RNS. It has been reported to probable association of LPG with MyD88 and TLR2
delay the assembly of the NADPH oxidase 2 on the was reported by de Veer et al., indicating the essenti­
surface of the phagolysosomes [158]. The metallopro­ ality of MyD88 in the clearance of L. major infection
tease gp63 has also been implicated with the inhibition [166]. The role of TLR2 in visceral leishmaniasis was
of various macrophage signaling requisite for the sti­ reported in an in vivo model using TLR2 ligand, arabi­
mulation of NADPH oxidase and iNOS [158]. nosylated lipoarabinomannan, which registered
enhanced NO and proinflammatory cytokine produc­
tion. This led to a drastic decrease in the organ parasitic
Modulation of toll-like receptor-mediated
burden and a strong Th1 response [167]. TLR2 was also
signaling
reported to be upregulated by 65 kDa and 98 kDa
TLRs, often considered the sentinels of the host, are antigens from L. donovani amastigotes [173]. While
capable of recognising pathogen-associated molecular studies by de Veere et al. and Debus et al. showed no
patterns (PAMPs) by virtue of PRRs (pattern recogni­ effect of LPG and GPIL on TLR4 [166,172], later stu­
tion receptors) similar to Nod-like receptors (NLRs), dies by Kropf et al. demonstrated the importance of
RIG-I-like receptors (RLRs), and cytosolic DNA sen­ TLR4-mediated iNOS activation on L. major clearance
sors (CDs) [159]. TLRs behave as bridges between the [174]. TLR4-mutated mice have been reported to heal
innate and adaptive immunity of the host [159]. Post cutaneous lesions caused by L. major [175]. In vivo
recognising PAMPs, TLRs induce a cascade of signaling studies with L. chagasi suggested infection-mediated
pathways, leading to the nuclear localisation of NFκB upregulation of TLR-2 and TLR-4 along with increased
and expression of antipathogenic products like pro- expression of IL-17, TNF-α, and IFN-γ during early
inflammatory cytokines and type 1 interferons [160] hours of infection [176]. L. panamensis infection in
by macrophages and dendritic cells. However, macro­ human primary macrophages also suggested upregula­
phage-mediated phagocytosis of Leishmania is not tion of levels of TLR1, TLR2, TLR3, and TLR4.
 VIRULENCE 915

However, interestingly, the study associated TLR3 and L. amazonensis and L. brazilensis by inducing NO pro­
TLR4 activity with increased TNF-α levels [177]. TLR4 duction [189]. Interestingly, L. donovani exploits the
has also been shown to sense glycospinghophospholi­ host-negative regulatory proteins A20 and UCP2 to
pid antigen of the parasite and stimulate inflammatory suppress the formation of NLRP3 inflammasomes
signaling intracellularly, leading to parasite clearance (Figure 3). The study reveals that A20, a host deubi­
[178]. In a recent study by Polari et al., L. brasieliensis quitinase, blocks the NFκB activation, while UCP2, an
infection was implicated with enhanced expression of inner mitochondrial protein, suppresses mitochondrial
TLR2 and TLR4, which triggered TNF-α and IL-10 ROS generation in order to suppress the two activation
production in monocytes isolated from CL patients steps of inflammasome formation [144]. However,
[179]. TL4, on ligand binding, activates a downstream strikingly different results have been reported in
signaling cascade involving signalosome complex, L. major infection, which aggravated post activation of
which includes MyD88, TRAF6, IRAK1/4, and ubiqui­ NLRP3 inflammasomes by inducing IL-18-mediated
tin conjugating enzymes and eventually culminates in Th2-based disease propagative response [190]. Reports
the activation of the NFκB pathway [180,181]. concerning the role of CLRs in Leishmania infection
However, assemblage of the signalosome complex is suggest that stimulation of Dectin-1 and mannose
interrupted during Leishmania infection [182]. receptors induces an antiparasitic oxidative stress, lead­
Moreover, inhibition of the NFκB pathway is reported ing to clearance of L. infantum infection [191].
by upregulating the host deubiquitinase, A20 [144,183] However, the same study reports an opposite role of
(Figure 3). A20 deubiquitinates TRAF6 and hinders its the CLR, SIGN3, which promotes parasite resilience by
association with TAK1, thus blocking TAK-1-mediated inhibiting LTB4-IL1β axis. Moreover, another report
NFκB activation. suggests that L. major exploits Mincle to target ITAM
Apart from TLR2 and TLR4, TLR3 has also been signaling in order to block an adaptive immune
reported to play an important role in Leishmania sp. response [192]. Despite these studies, a lot still remains
infection. Flandin et al. in their study suggest involve­ to be explored for better understanding of the role of
ment of TLR2 and TLR3 in the clearance of L. donovani NLRs and CLRs in Leishmania infection.
via increased production of host NO and TNF-α [184].
The work further exhibits the importance of TLR3 in
Modulation of host signaling pathways
leishmanicidal activity of the IFN-γ-primed macro­
phages. Interestingly, stimulation of TLR3 during For successful establishment of infection and prolifera­
L. guyanensis infection induces hyperinflammatory tion within the host macrophage, Leishmania sp. sub­
response along with an increase in the parasitic burden verts the various host defense machineries. This can be
and exacerbated disease symptoms [185]. This study attributed to the capability of the parasite to alter and
associates the activation of TLR3 with the dsRNA of take over important host signaling pathways. This leads
the endosymbiotic virus, LRV1, associated with the to the inhibition of host defense mechanisms and also
parasite. TLR9, on the other hand, is known to system­ subverts the various leishmanicidal functions, which
atically resist Leishmania infection by inducing the are stimulated post activation of macrophage.
secretion of IL-12, which ultimately leads to IFN-γ Leishmania has developed strategies so as to either
production by natural killer (NK) cells [185–187]. inhibit or suppress proteins that stimulate immune
A therapeutic study against L. major suggested the activation or upregulate negative regulatory proteins
probable involvement of CpG-TLR9 axis in parasite of the immune system and their functions.
clearance. The study reports on the stimulation of Protein kinase C (PKC) family comprises of 10
proinflammatory cytokines, especially IL-12 mediated serine/threonine kinases and were initially described
by TLR9 activation by CpG [186]. Liese et al. in their as Ca2+ and phospholipid dependent [193]. PKC sig­
study found that L. major-infected TLR9-/- mice regis­ naling has been implicated with regulation of macro­
tered higher parasitic burden [188]. Another study phage activation and production of IFN-γ and TNF-α
reveals that L. infantum infection of dendritic cells led [194,195], eventually leading to the generation of NO
to TLR9-mediated production of IFNγ and increased and ROS [196]. Leishmanial LPG blocks PKC activity
levels of IL-12 [187]. by binding to the regulatory domain of PKC, which
Studies involving the roles of NLRs and CLRs in possesses the diacylglycerol, Ca2+, and phospholipid
context of Leishmania sp. infection are currently binding sites [197]. However, interestingly, amastigote
being looked upon. Lima-Junior et al. suggested pro­ forms of L. donovani that are devoid of LPG can also
duction of IL-1β via activation of NLRP3 inflamma­ inhibit PKC activity in monocytes [152], indicating
somes to provide protection against infection to the presence of other mechanisms exploited by the
916 A. K. GUPTA ET AL.

Figure 3. Leishmania overrides important MAPK and TLR signaling for successful survival. Stimulation of TLRs triggers
a signaling pathways that ultimately culminates in the activation and nuclear translocation of the transcription factor such as NFκB.
Activation of MAPKs such as p38, JNK, and ERK1/2, through phosphorylation by upstream kinases, leads to the activation of the
transcription factors-AP-1 and IRFs. Activation of NFκB, AP-1, and IRFs transcription factors activate the expression of pro-
inflammatory genes and genes involved in host immune defense like IL-12, IL-1β, NLRP3, etc. In order to regulate TLR-mediated
NFκB activation, the parasite upregulates the host deubiquitinases such as A20, which removes the necessary ubiquitination from
TRAF6 and blocks its interaction with TAB/TAK complex. In order to block MAPK activation, the parasite exploits host PTPs like SHP-1,
MKP-1, and PP2A. Furthermore, cysteine proteases of some species of Leishmania degrade ERK1/2 and JNK (NLRP3: Nod-like receptor
protein 3; AP-1: activator protein 1; IRFs: interferon regulatory factors; SHP-1: Src homology 2 domain-containing protein tyrosine
phosphatase 1; MKP-1: MAPK phosphatase 1; and PP2A: protein phosphatase 2A). Image created through paid version of Biorender.

parasite to inhibit PKC activity. Olivier et al. reported eventually result in the phosphorylation-mediated acti­
that GIPLs from L. major amastigotes can inhibit vation of signal transducer and activator of transcrip­
PKC activity [198]. Another report suggests that tion (STAT), leading to nuclear translocation of the
L. donovani induces ceramide generation in murine former and transcriptional activation or repression of
macrophages to alter PKC activity as a survival strat­ various genes [200], including the previously discussed
egy [199]. iNOS gene responsible for the production of NO.
Apart from the PKC family, Leishmania infection L. donovani promastigotes exploit the host SHP-1 to
exhibits negative regulation of Janus Kinase 2 (JAK2), inhibit IFN-γ-induced JAK-2 phosphorylation and NO
a member of the Janus tyrosine kinase family. production [78]. Interestingly, IFN-γ-mediated
Activation of Janus Kinase signaling is implicated with STAT1α activation was also found to be abrogated
important cell functioning like proliferation, migration, upon infection by L. donovani in SHP-deficient macro­
apoptosis, differentiation, and immune stimulation phages, indicating the presence of other mechanisms
[200]. JAK signaling involves receptor binding of cyto­ employed by the vicious parasite to subvert STAT1
kines or growth factors, multimerization of the recep­ activation [136]. This is probably due to enhanced
tors, and activation by transphophorylation. These proteasomal degradation of STAT1 in Leishmania-
 VIRULENCE 917

infected macrophages [201]. Moreover, additional cause degradation of JNK [215] (Figure 3). p38 MAPK
reports suggest attenuated levels of IFN-γ receptor is also known to be inhibited during leishmanial infec­
alpha subunit [202] and increased transient expression tion [143,216], and its activation using anisomycin
of suppressor of cytokine signaling 3 (SOCS3), which enhances parasite killing [217]. Ball et al. in their
have been known to negatively regulate IFN-γ signaling study reported the exploitation of the host inner mito­
[203]. Further studies in this regard suggest L. donovani chondrial membrane protein, UCP2, to suppress ROS-
infection inhibits the nuclear translocation of IFN-γ- mediated activation of p38 MAPK [143]. An interesting
induced nuclear transport of STAT1α by blocking the study reported differential role of ERK and p38 MAPKs
interaction between STAT1α and importin-α5 [204]. in regulating LPS-induced expression of iNOS and IL-
Mitogen-activated protein kinases (MAPKs) belong 12 [218]. p38 promoted the expression of IL-12 while
to a group of serine/threonine kinase family including ERK dampened LPS-induced IL-12 expression. The
p38 MAPK, c-jun terminal kinase (JNK), extracellular study further reports that synthetic Leishmania LPG
signal-related kinases 1 and 2 (ERK1/2) and play targets ERK MAPK to subvert the production of IL-12
a major accessory and effector role in host cells for as a survival strategy. However, a contradictory report
production of NO and proinflammatory cytokines suggests that Leishmania LPG promotes pro-
[205]. Activation of these pathways takes place post inflammatory and endotoxin-like response and stimu­
phosphorylation of Ser/Thr and Tyr residues present lates the production of IL-12 and NO. This is achieved
on their regulatory domain by MAP/ERK kinase by LPG-mediated activation of p38 and ERK MAPK,
(MEK) [206]. MEK is itself activated by upstream leading to activation of AP-1 [219]. Apart from the
kinase-MEK kinase (MEKK) [207]. After activation, above mechanisms exploited by the parasite, Halle
the kinases phosphorylate an array of intracellular pro­ et al. demonstrated that the leishmanial metallopro­
teins and transcription factors like NFκB, AP-1, and tease, GP63, also inactivates p38 MAPK, possibly by
IRFs, triggering the stimulation of a diverse signaling degrading the upstream adaptor TAB1 [220].
cascade eventually regulating expression of various
genes [208–210]. Leishmania as a survival strategy has
Host cell apoptosis and leishmania
been widely reported to modulate the alteration of
MAPK (Figure 3). L. donovani has been reported to Apoptosis is a natural phenomenon in multicellular
impair PMA-dependent activation of MAPK in infected organisms whereby a host cell undergoes programmed
macrophages to subvert the expression of c-FOS and cell death. It is an important component of various cell
ELK-1 [211]. L. donovani infection also enhanced the functioning like normal turnover of cells, hormone-
activity of host protein tyrosine phosphatase (PTP), mediated atrophy, immune system functioning, and
SHP-1, which inhibits MAPK pathway [78,143] development of embryo [221]. Since intracellular
(Figure 3). These studies explain the findings that pathogens tend to survive within the cellular niche of
SHP-1-deficient macrophages exhibit JAK2 and the host, apoptosis serves as the ultimate resort for
ERK1/2 activation on treatment with IFN-γ in infected cells so as to completely eliminate the former
L. donovani-infected state [136]. L. amazonensis amas­ by destruction of its own self. Hence, most intracellular
tigotes, on the other hand, have been shown to block pathogens have developed strategies to ensure inhibi­
LPS-mediated activation of ERK1 [212]. Activation of tion of apoptosis of host cell as a surviving strategy.
other phosphatases like MKP1 and PP2A targeting The anti-apoptotic host protein myeloid cell leuke­
ERK1/2 MAPK activation during Leishmania infection mia-1 (MCL-1), which is a member of the Bcl-2 family,
has also been reported [213]. Apart from phosphatases, has been implicated with various intracellular infections
Leishmania infection has been implicated with elevated [222,223]. Reports suggest enhanced RNA and protein
endogenous ceramide in host macrophages [199]. levels of MCL-1 during infection by virulent but not
Ceramide being an important intracellular lipid med­ attenuated strains of Mycobacterium tuberculosis [222].
iator regulates important cell functions like apoptosis Similarly, Leishmania infection has also been associated
and senescence [214]. Ghosh et al. in their study with increased MCL-1 expression. In a recent study,
demonstrated intracellular ceramide-mediated depho­ Das et al. demonstrate that unmethylated CpG motifs
sphorylation of ERK so as to dampen AP-1 and NFκB in L. donovani DNA regulate TLR9-mediated delay in
activation in L. donovani-infected macrophages [199]. the programmed cell death of the host macrophage
Strikingly, L. mexicana amastigotes inhibit host ERK1/2 [224]. Significant upregulation of MCL-1 during infec­
signaling not by phosphorylation but rather by enhan­ tion by L. donovani was also reported by another
cing their degradation. This is achieved by the parasite’s group, which further suggests infection-induced MCL-
cysteine peptidases, which also have been reported to 1 to be localised in the host mitochondria. The study
918 A. K. GUPTA ET AL.

reports that parasite-induced MCL-1 associates with RNA-binding protein complex (Dicer1-TRBP) [231].
Bcl-2 homologous antagonist/killer (BAK) and inhibits The functional and mature miRNA strand of this
its homo-dimerization and eventual release of mito­ duplex then couples with RISC-Argonaut 2 (AGO 2)
chondrial membrane cytochrome c [225]. Recently, to form micro-ribonucleoprotein (miRNP) complex
a significant level of Bcl-2 was reported in the periph­ [232]. miRNP interacts with the “seed” sequence of
eral blood of VL patients. The study further exhibited the 3’ untranslated region (UTR) of target mRNA
that Bcl-2 inhibition led to increased NO-mediated with partial or complete sequence complementarity to
parasite clearance [226]. Furthermore, the parasite has accomplish their translational suppression or degrada­
also been reported to indirectly inhibit host apoptosis tion, respectively [233] (Table 2).
by improving the stability of PTPs in a SOCS- After infecting the mammalian host, Leishmania sp.
dependent manner. PTPs in turn inhibit the activation enters into the phagocytic cells of the hematopoietic
of the caspase cascade necessary for triggering apopto­ lineage like neutrophils, macrophages, and dendritic
sis of host cells [227]. cells (DC) where it trans-differentiates from promasti­
Further, the parasite also exploits the multifaceted gote to amastigote form [243]. In order to downregu­
regulator, AKT signaling pathway, to inhibit host late the activation of initial inflammation and establish
defense mechanisms like production of inflammatory a successful immune-suppressive microenvironment,
cytokine and host cell apoptosis. Phosphoinositide-3 a complex host–parasite interaction prevails, which
kinases (PI3K) on interaction with membrane receptors includes alteration of immune regulatory miRNA
phosphorylate inositol phospholipid producing the sec­ expression profile [244]. Like Thelper (Th) cells, macro­
ondary messenger, inositol-3,4,5-triphosphate, which phages are also plastic in nature, which can shuffle
recruits and activates AKT (protein kinase B). between pro-inflammatory “classically activated” (M1)
A recent study by Gupta et al. suggests that form and anti-inflammatory “alternatively activated”
L. donovani infection triggers AKT activation to inhibit form (M2) depending upon the environmental cues
GSK-3β. Inhibition of GSK-3β leads to the activation of present. Macrophages stimulated with endotoxins like
the anti-apoptotic, β-catenin, and concomitant inacti­ lipopolysaccharide (LPS) and Th1 cytokines like inter­
vation of the pro-apoptotic Forkhead box protein O1 feron-γ (IFN-γ) differentiate into M1 type. On the
(FOXO-1) [228]. However, the surface molecule of the contrary, stimulation with Vitamin D3, macrophage
macrophage with which the parasite interacts triggering colony stimulating factor (M-CSF), Th2 cytokines like
the PI3K/AKT pathway is still not known. Interestingly, interleukin 4 (IL4), and interleukin 13 (IL13)
reports in Schwann cells suggest that L. major does not activates M2 type of macrophages [245]. M1 macro­
trigger the PI3K/AKT pathway [229], indicating the phages are characterized by enhanced antigen-
presence of other strategies exploited by this parasite. presenting properties, transcription factors like NF-κB,
PU.1, and capacity to secrete pro-inflammatory cyto­
kines like IL1β, IL6, IL12, tumor necrosis factor-α
Modulation of host non-coding RNAs as
(TNFα), etc. The M1 cytokines engage Th1-adaptive
a survival strategy
arm, which cross-induces parasite clearance from
MicroRNAs (miRNAs) are 18–22 nucleotide long non- macrophages by triggering nitric oxide and oxidative
coding RNAs (ncRNAs) that post-transcriptionally reg­ burst [246]. On the contrary, L. donovani triggers M2
ulate the turnover, translation, and expression of polarization via activating mTOR, PPAR-γ, and CD163
a specific group or cluster of mRNAs. Monocistronic [247–249]. M2 macrophages activate the Th2 arm by
or polycistronic (miRNA cluster) form of miRNAs can IL10, TGF-β, IL-4, etc. These Th2 cells eventually assist
get transcribed from host exon or intron sequences in in the initiation of polyamine biosynthesis pathways in
a RNA polymerase II-dependent manner. miRNAs can infected macrophages in an arginase 1-dependent man­
be encoded either from their own promoters or can ner, which favors parasite growth [246].
utilize promoters regulating other mRNA expression (if In the last few years, small RNA profiling of various
miRNAs are intragenic). After transcription, miRNA macrophages infected with Leishmania sp. uncovered
obtains a folded double-stranded hairpin-like confor­ that L. donovani, L. major, L. amazonensis, and
mation known as pri-miRNA and gets processed by L. infantum parasites can potentially alter macrophage
class 2 RNase III DROSHA and DGCR8 to form pre- immuno-miRs expression to favor their own prolifera­
miRNA inside the nucleus [230]. Pre-miRNAs get tion and survival within macrophages [250–252]. Li
exported from nucleus to cytosol in Exportin 5- Ran et al. reported that miR-9, miR-146a/b, miR-181a,
GTPase-dependent fashion and processed into miRNA miR-124, let-7c, and miR-210 are responsible for pro­
duplex by Dicer1-transactivation response element moting M2 phenotype, whereas miR-130a/b, miR-125a/
 VIRULENCE 919

Table 2. List of host non-coding RNAs modulated by Leishmania sp.

Name of non-coding Possible virulence
RNAs Target genes factor involved Function during Leishmanial infection
miR-9 PPAR-δ ... ... . Suppresses M1 function [234]
miR-146a-5p TRAF6, IRAK1 ... ... . Promotes M2 polarization by targeting TLR4 pathway [235,256]
miR-181a C/EBP-α, KLF6 ... ... . Promotes M2 polarization and suppresses M1 function [236]
miR-124 C/EBP-α ... ... . Suppresses inflammatory response [237]
let-7c C/EBP-δ ... ... . Suppresses inflammatory response [238]
miR-210 NFκB ... ... ... Suppresses M1 polarization [239]
miR-130a/b PPAR-γ ... ... . Promotes M1 and inflammatory response [240]
miR-26a PPAR-γ ... ... . Promotes M1 and inflammatory response [241]
miR-21 SMAD7, PU.1 ... ... . Targets JAK-STAT signaling and suppresses inflammation [250]
miR-720 GATA3 ... ... . Promotes M1 and inflammatory response [242]
miR-511 TLR4 ... ... . Promotes anti-inflammation and survival of parasites [25]
miR-122 Cationic amino acid gp63, host c-Myc Promotes parasite survival via lowering serum cholesterol [257,260]
transporter 1 (CAT1)
miR-294-3p NOS2, TNFα leishmanial Arginase Promotes parasite survival via suppressing NO and ROS production
[260,262]
miR-30e NOS2 ... ... ... Promotes parasite survival via suppressing NO production [260]
miR-302 NOS2 ... ... ... Promotes parasite survival via suppressing NO production [262]
miR-721 NOS2 ... ... ... Promotes parasite survival via suppressing NO production [260]
miR-210 p-50 subunit of NFκB ... ... ... Promotes M2 polarization and suppresses NF-κB pathway-mediated
activation of IL12a and TNFα [266]
miR-361-5p TNFα, Granzyme ... ... . Prevents TNFR-mediated clearance of parasites [267,268]
miR-193b TNFα ... ... . Prevents TNFR-mediated clearance of parasites [267]
miR-671 TNFα ... ... . Prevents TNFR and CD40-mediated clearance of parasites [267]
miR-548d-3p TNFα, Granzyme ... ... . Prevents TNFR-mediated clearance of parasites [267]
miR-346 TAP-1, RFX1 and BCAP31 ... ... . Subverts MHC I/II-mediated antigen presentation [270]
miR-466i MyD88 ... ... . Subsides TLR4 pathway and upregulate IL10 expression [271]
miR-30a Beclin (BECN1) ... ... . Prevents autophagy activation for parasite survival [274]
miR-574 STAT4 ... ... . Downregulates IFN-γ response from CD4 + T cells [275]
miR-6994-5p STAT1 ... ... . Downregulates IFN-γ response from CD4 + T cells [275]
miRNA-6994-5p IFN-γ ... ... . Downregulates IFN-γ response from CD4 + T cells [275]
miR-5128 IFN-γ ... ... . Downregulates IFN-γ response from CD4 + T cells [275]
miR-7093-3p IL12a ... ... ... Downregulates IL12 signaling cascade of Th1 cells [275]
miR-574-5p NFAT ... ... ... Downregulates IL12 signaling cascade of Th1 cells [275]
miR-7235 ZAP70 ... ... ... Downregulates IL12 signaling cascade of Th1 cells [275]
let-7a SOCS4 ... ... ... Downregulates IL12 signaling cascade of Th1 cells [250]
miR-155 Arginase 2 ... ... ... Promotes antigen presentation and parasite clearance via DC [261]
Alu RNA Not known yet gp63 Prevents infection establishment in phagosomal vacuole [276]
Signal recognition Not known yet gp63 Prevents infection establishment in phagosomal vacuole [276]
particle RNA
B1 RNA Not known yet gp63 Prevents infection establishment in phagosomal vacuole [276]

b, miR26a, miR-21, and miR-720 promote M1 pheno­ 26a in L. donovani-infected mouse BMDMs. Here
type [253]. Small RNA profiling of L. amazonensis- authors reported that miR-146a promotes M2 polariza­
infected murine macrophages revealed a mixed M1- tion by targeting TRAF6- and IRAK1-mediated activa­
and M2-type response during infection [254]. Geraci tion of NFκB module and IL12-iNOS axis [256]. These
et al. reported enrichment of miR-21 and let-7a, which findings indicate a possible correlation of Leishmania
are known to target JAK-STAT signaling mediators infection-mediated alteration of macrophage plasticity
SMAD7 and SOCS4, respectively, by small RNA profil­ and miRNAs.
ing of human monocyte-derived macrophages (human Besides inflammatory genes, parasites can modulate
MDM) and DC cells infected with L. donovani. They global landscape of host metabolism by directly target­
also found enrichment of miR-511 and miR-21, which ing miRNA biogenesis with different virulence factors.
target TLR4 pathway and PU.1 transcription factor, L. donovani delivers zinc metalloprotease gp63 in host
respectively [250]. Nimsarkar et al. consolidated che­ hepatocyte cytosol via exosomes, which cleaves miRNA
mical systems biology and synthetic biology approach processing enzyme Dicer1. Degradation of Dicer1 leads
to uncover unique juxtacellular export of miR-146a-like into inhibition of miRNP-mediated biogenesis of miR-
elements from L. major to macrophage cytoplasm, 122 in hepatocytes and contributes to lowering of
which target SMAD4, the negative feedback regulator serum cholesterol level and facilitates infection progres­
of TGF-β signaling cascade, and promote M2 profile sion in liver [257]. Besides being a serum biomarker for
[255]. Later Das et al. reported enrichment of M2 canine VL, miR-122 can become a potential therapeutic
polarizing miRNAs like miR-146a, miR-181a, and target for lowering liver parasite burden [258].
miR-125a and downregulation of M1 polarizing miR- Leishmania sp. can extraordinarily exploit host
920 A. K. GUPTA ET AL.

transcription machinery in order to regulate cellular In addition to human and murine VL models, in
abundance of miRNAs. In human MDMs, canine VL, L. infantum has been found to differentially
L. donovani exploits host c-Myc as a proxy virulence modulate miR-150, miR-451, miR-192, miR-194, and
factor in order to induce overall miRNA suppression. miR-371 titers in peripheral blood monocytes
Therefore, host c-Myc serves as a novel proxy host (PBMCs). These miRNAs are reportedly involved in
protein harnessed by L. donovani for its survival downregulating inflammatory response of macrophages
[259]. In addition to gp63, parasite arginase can utilize by targeting TNF-α, CD80, and IFN-γ [269].
host L-arginine source and prevent inflammatory nitric Macrophages are one of the professional antigen-
oxide generation. However, along with its own arginase presenting cells, which bridge pathogen infection with
1, parasites harness host macrophage arginase as well the adaptive arm of the immune response via class
via miR-122-mediated repression of cationic amino I and II major histocompatibility complexes (MHCs)
acid transporter 1 (CAT1) [260]. L. donovani infected [269]. L. infantum can strategically overcome MHC I/
macrophages, and DCs display lower burden of miR- II-mediated antigen presentation by targeting TAP-1,
155. miR-155 prevents parasite survival by repressing RFX1, and BCAP31 via miR-346 in human U937 and
arginase 2 in DCs and allows activation of T cells; THP-1 cell lines [270]. Another major APC is DCs,
hence, by downregulating miR-155, the parasites pro­ where TLR4 pathways play crucial roles for down­
mote their own survival in DCs [261]. In order to stream activation of inflammation upon parasite infec­
subvert nitric oxide-mediated clearance within macro­ tion. In SAG-resistant L. donovani infection,
phages, L. amazonensis reportedly upregulates miR- enrichment of miR-466i reportedly targets MyD88 to
294-3p, miR-30e, miR-302, and miR-721 levels, which subside TLR4 pathway and upregulate IL10 expression
target NOS2 mRNA and prevent NO generation [271]. Besides this, L. amazonensis targets TLR-
[260,262]. Besides nitric oxide, miR-294-3p is also dependent activation of NOS2 through enriching let-
involved in downregulating TNFα transcript, which 7e levels. Muxel et al. showed that TLR pathway med­
helps the parasites to evade ROS-mediated toxi­ iators like Traf6, Ppara, Mapk8ip3/Jip3, Tnfpaip3,
city [262]. Map2k4, Tbk1, and Tnf are upregulated upon inhibi­
Parasites attempt to diminish the chemokine tion of let-7e, indicating that parasite exploits the
response of macrophages by means of certain miRNAs for subsiding TLR-pathway-mediated clear­
miRNAs. For example, L. major infection causes sig­ ance [272]. TLR mediators limit parasite infection not
nificant downregulation of CCR2, CCL5, CXCL10, etc. only by activating inflammation but also autophagy
via up regulating let-7a, miR-25, miR-26a, miR-132, activation. C57BL/6 mice lacking endosomal TLRs like
miR-140, miR-146a, and miR-155 in human macro­ TLR3, TLR7, and TLR9 are highly susceptible to
phages [263,264]. Likewise, L. donovani and L. major infection due to impaired autophagy activa­
L. amazonensis infection induce hypoxic environment tion [273]. In addition, L. donovani can negatively
in the macrophages via activation of hypoxia-inducible regulate Beclin1 (BECN1) via miR-30a in human
factor 1α (HIF-1α) [265,266]. In L. donovani-infected MDMs and THP-1 macrophages [274].
macrophages, HIF-1α activates miR-210 expression, Small RNA profiling revealed that L. donovani can
which suppresses NF-κB pathway-mediated activation differentially regulate expressions of miRNA profile
of IL12a and TNFα by targeting p-50 subunit [266]. that are involved in the plasticity of CD4+ T cells.
These findings postulated an interesting survival strat­ Among the pathways affected by L. donovani are
egy of Leishmania parasites via hypoxia-mediated Notch, JAK-STAT, IFN-γ, and MAPK. Kumar et al.
miRNAs activation. miR-21 is another major regulator reported that L. donovani prevents IFN-γ secretion
of IL12a in L. donovani-infected murine macrophages from Th1 response by upregulating miR-7a-1-3p,
and DCs . In pentavalent antimonial-resistant localized miR-690, miR-7017-5p, miR-574-5p, and miR-7235-
cutaneous leishmaniasis (LCL) patients, L. braziliensis 5p. miR-574 and miR-6994-5p target STAT4 and
infection stimulates miR-361-5p, miR-193b, and miR- STAT1, respectively, and miRNA-6994-5p and miR-
671 expressions to prevent TNFα, CD40, and TNFR- 5128 target IFN-γ genes, which ultimately downregu­
mediated parasite clearance from cutaneous macro­ late IFN-γ signaling cascade in CD4+ T cells during
phages [267]. miR-361-3p and miR-548d-3p serve as L. donovani infection. Additionally, the authors
poor prognosis markers of active CL. During reported that by upregulating miR-7093-3p, miR-
L. (viannia) braziliensis infection, these miRNAs mainly 5128, miR-574-5p, and miR-7235, L. donovani targets
target granzyme (GZMB) and TNF pathway activation IL-12 R, NF-AT, and ZAP70, which together hamper
to support parasite survival [267,268]. IL12 signaling cascade of Th1 cells [275].
 VIRULENCE 921

Apart from miRNAs, recent reports suggested that SE elements are clusters of multiple enhancers, longer
L. donovani infection can negatively modulate small in length and occupied by extraordinary writer proteins
ncRNAs (Alu RNA, B1 RNA, and signal recognition like bromodomain 4 (BRD4), p300, RNA pol II, etc.
particle RNA) for successful establishment of infection The authors described that BRD4 plays a crucial role in
in phagosomal vacuoles of macrophages. These ncRNAs L. donovani infection-induced SE element formation at
have signature B-box element at their promoter regions, upstream of miR146a-5p promoter, which drives M2
which require recruitment of RNA pol III transcription polarization of macrophages [256].
factor TFIIIC for transcription. L. donovani gp63-based
activation of thrombin receptor PAR1 increases the cyto­
Approaches in leishmania diagnosis and the
solic Ca2+ levels and hence activates Ca2+-dependent
virulence factors
calpain protease, which degrades TFIIIC [276]. In addi­
tion to L. donovani, computational prediction identified Diagnosis of leishmaniasis can be done either directly
a role of ncRNAs in gene expression modulation during by observing the parasite in the infected patients’ sam­
L. amazonensis, L. braziliensis, L. infantum, and L. major ples through microscopy or by indirect methods of
infection [277]. detecting leishmanial genetic materials and proteins
such as virulence factor. The direct method of detecting
Leishmania parasites by the microscopic visualization
Role of host epigenetic factors in leishmania
of amastigotes in the infected tissue is the most rational
infection progression
way of diagnosis, and it is still accepted as a gold
Recent studies highlight subversion of host macrophage standard test in many endemic regions worldwide.
immune response by Leishmania largely by modulating Interestingly, newer studies suggest various leishmanial
histone dynamics of many major inflammatory genes virulence factors as antigens to possess diagnostic
by different histone modifiers known as “writer” pro­ potential in terms of antigens or antibodies in response
teins. Parmer et al. was the first to reveal an interesting to the factors. Antigens or antibodies are used in diag­
correlation between histone methylation and macro­ nosis mainly in the ELISA and immunochromato­
phage polarization during VL. They reported that graphic format.
L. donovani represses TNFα expression by Smyd2- The diagnostic performance of the test depends lar­
mediated H3K36 dimethylation of TNFα promoter gely on the protein used in the assay. The use of ELISA
and iNOS gene expression by Ezh2-mediated H3K27 with crude soluble proteins isolated from Leishmania
trimethyation of NOS2 promoter in J774 macrophages. promastigotes traditionally show varying sensitivity and
Moreover, L. donovani induces IL10 expression by specificity depending on the protein used [283–286].
Ash11-mediated H3K4 trimethylation of IL10 promo­ Cross-reactivity of total crude antigens with other simi­
ter [278]. L. amazonensis also exploits host histone lar infections like trypanosomiasis, toxoplasmosis, and
modification system to survive inside macrophages. tuberculosis has been observed in many cases. As
Histones deacetylases (HDACs) form complexes with a result, search for a specific leishmanial protein that
p50/p50-dimer that remove H3K9Ac acetylation and has high sensitivity and no cross-reactions with other
CBP/p300 from iNOS promoter and suppress its diseases is still in progress. In one of the studies,
expression. Thus, by upregulating HDAC1, L. donovani proteins of different molecular weights
L. amazonensis induces hyporeactive stage in infected such as 31, 34, 36, 45, 51, 63, 72, 91, and 97 kDa were
patients [279]. Similarly, in L. donovani-infected THP-1 purified and evaluated in a serological ELISA. It was
cells, HDAC1 was enriched, which subsequently observed that 34 and 51 kDa proteins showed 100%
removes H3K27 acetylation from macrophage defense sensitivity and no cross-reactivity with other diseases
gene promoters. Thus, Roy et al. proposed HDAC1 [287]. Advancement in molecular biology led to the
inhibitors as potential anti-leishmanial agents [280]. discovery of more defined recombinant proteins that
Hijacking host histone H3 modification is a strategy were easier in purification with no batch-to-batch var­
of Leishmania parasites to target inflammasome path­ iation, unlike crude proteins. A number of recombinant
way as well [281]. Histone H3K9/14 hypoacetylation Leishmania proteins have been cloned and studied for
and H3K4 hypo-trimethylation induced by serological diagnosis, such as rK9, rK26, rKRP42,
L. amazonensis help the parasite to subvert NLRP3 rKE16, rA2, rKDDR, and rK39. Among all, rK39,
and NF-κB activation [282]. Das et al. identified a kinesin-related protein of L. chagasi, is reputed as
a unique mode of miRNA regulation via super enhan­ the best recombinant protein for VL diagnosis, espe­
cer elements (SE) formation at miRNA promoters in cially in the context of the Indian subcontinent.
L. donovani-infected macrophages and BALB/c mice. Roughly, 88.6–99% sensitivity and 81–98.2% specificity
922 A. K. GUPTA ET AL.

have been observed for the diagnosis of VL caused by performance shows drastic variations among different
L. infantum and L. donovani. Using Indian sera, geographical locations of VL endemicity. Sensitivity
a recombinant protein cysteine protease C (CPC) rates of rK39-ICT performed in the Indian subconti­
proved to be a good diagnostic candidate with 98.15% nent were determined to be 97%, whereas in Brazil and
sensitivity as compared to 92.59% and 96.29% with East Africa, sensitivities go down to 85% and 67%,
glycoprotein 63 (GP63) and elongation factor 1-α respectively [290,291]. The disparity in rK39-ICT per­
(EF1- α), respectively. In a similar study, CPC with formance initiated the development of numerous pro­
urine showed a sensitivity of 96% than 90% and 84% teins for ICT that were isolated from regional
for GP63 and EF1- α, respectively [288]. Since the L. donovani species, such as rK9, rK26, rK28, rKE16,
antibodies continue to be present in the blood after and rKRP-42. Most of these recombinant proteins are
VL treatment, most of the leishmanial proteins cannot kinesin-related, and their performance varies in VL
be used as a test of cure. However, a recombinant diagnosis. Among non-kinesin proteins, Ld-OCP
protein, L. donovani–otubain cysteine peptidase (Ld- showed 100% sensitivity as compared to rK39-ICT,
OCP), showed complete clearance of antibodies in fol­ which showed 83.33% and 64.10% sensitivity with the
low-up patients after 6 months of treatment [116]. Indian and Brazilian sera, respectively [116].
Several proteins such as rHSP83, LiHyE, HSP70, Interestingly, as a test of cure, Ld-OCP did not recog­
HSP83.1, rLb8E, and rLb6H have been tested in CL nize antibodies in L. donovani-infected patients after 6
and MCL diagnosis, and promising preliminary results months of treatment; in contrast, rK39-ICT still reacted
were reported in ELISA [286]. with 86.66% follow-up serum samples [116].
To overcome the limitations of the invasive parasi­ In spite of the knowledge gathered on different
tological method and laboratory-based serological tests, leishmanial virulence factors and their use in drug
the point-of-care test has been developed for simple target identification, diagnosis is also an area where
and field-adaptable diagnosis. The World Health these factors have been used directly or indirectly.
Organization (WHO) has approved the ASSURED
(affordable, sensitive, specific, user-friendly, rapid and
Limitations of available antileishmanial
robust, equipment-free, and deliverable to end-users)
therapies
criteria of point-of-care tests used primarily for infec­
tious diseases diagnosis. Immunochromatographic test Therapy against leishmaniasis are limited and do not
(ICT) has proved to be a benchmark in the field diag­ meet the satisfactory standards, and most of the drugs
nosis of NTDs, including leishmaniasis due to its high used against leishmaniasis were repurposed from other
performance, low cost, simplicity, and promptness. ICT diseases [292]. Although patients respond to most of
in dipstick format with L. donovani antigen, LAg the available drugs by exhibiting apparent cure within 2
demonstrated 100% specificity with Indian and weeks, significant cases of relapse of leishmaniasis have
Brazilian VL sera, as well as Indian VL urine been reported within 6 months of treatment. However,
[284,285]. Further, in a multicentric study, serum- with the advancement in our knowledge of important
based dipsticks showed 97.10% sensitivity and 93.44% virulence factors and enzymatic pathways of
specificity in six countries, India, Sri Lanka, Nepal, Leishmania, additional selective drugs are being pro­
Ethiopia, Brazil, and Spain [289]. Similar to ELISA, posed. Synthetic and natural compounds targeting
leishmanial proteins were also used in ICT to detect important leishmanial enzymes/factors like topoisome­
antibodies present in the infected samples in a lateral rases [293], Hsp78 [120], and Aurora kinase [294] are
flow assay (LFA) format. An LFA is a rapid screening being studied. Moreover, the knowledge of the various
test where protein is coated on the nitrocellulose mem­ subversion strategies of the parasite to evade the host
brane. The binding of coated protein and sample anti­ immune defense has led to the advent of various nat­
bodies can be seen directly as a color band due to tracer ural immunomodulators as futuristic antileishmanial
molecules like colloidal gold. Using crude antigen, LAg therapy.
in LFA exhibited sensitivities of 96.49% and 95.12% Pentavalent antimonials are the first-line treatment
and specificities of 95% and 96.36% with Indian VL used extensively worldwide against both VL and CL.
sera and urine, respectively, infected with L. donovani. Among them, Sodium Stibogluconate and Meglumine
In a similar study, 88.57% sensitivity and 94.73% spe­ Antimonate are clinically used for over seven decades.
cificity were monitored with the Brazilian sera infected In spite of the extensive usage, the exact antileishmanial
with L. infantum [290]. Among recombinant proteins, mechanism of pentavalent antimonials is not well elu­
rK39-ICT has certainly been the widely used rapid test cidated even today. However, like most other drugs,
for VL diagnosis in the last decade. However, its antimonial therapy is also associated with various
 VIRULENCE 923

toxicities like cardiotoxicity, hepatotoxicity, and other associated toxicities [306,307]. Apart from suppressing
adverse effects like pancreatitis and arthealgia [295]. disease-conducive environment in the host,
The antimonial drugs showing poor oral absorption KalsomeTM10 also induced apoptosis-like cell death in
has to be administered intravenously or intramuscu­ the parasite [308], proving it to be a better alternative to
larly at a dose of 20 mg/kg for 20–30 days [292]. Apart Ambisome or other liposomal formulations of AmpB.
from this, the high cost of the drugs limited their use in Miltefosine (hexadecyl phosphocholine) was the first
developing countries. In order to tackle the cost, drug to be registered in India, which was orally admi­
a cheaper generic version Sodium Antimony nistered [309]. Miltefosine at a dose of 50–100 mg for
Gluconate (SAG) was developed by Albert David in 28 days exhibited around 94% cure [310]. Miltefosine is
India [296]. However, in the last two decades with the known to impair the synthesis of cell surface compo­
emergence of resistance among patients, the drug has nents of the parasite eventually, leading to programmed
lost its efficacy. The emergence of resistance may be cell death [311,312]. Miltefosine also possesses immu­
attributed to long drug exposure either due to elon­ nomodulatory effects on macrophages and has been
gated drug regimen or the long half-life of the drug. In reported to enhance phagocytosis [313]. A study by
order to overcome this problem, several liposomal for­ Gupta et al. reports that miltefosine treatment in
mulations of the drug are being tried and are showing infected hamsters shifted the cytokine balance to the
promising results [297]. Recent advances in improving disease, resolving Th1 type by inducing IL-12, TNF-α,
the efficacy of antimonials include combination thera­ IFN-γ, and iNOS production, simultaneously decreas­
pies [298]. ing the production of anti-inflammatory cytokines
Due to the dearth in the knowledge of validated drug [314]. Similar potency of shifting the Th1/Th2 cytokine
targets, most of the other antileishmanial drugs available balance towards Th1 by miltefosine was also reported
were a result of drug repurposing. The widely used in Indian PKDL patients [315]. However, like the other
antifungal drug amphotericin B (AmpB) is one of the two frontline drugs, miltefosine has limited usage due
first drugs repurposed in this regard. The antifungal to its high cost and associated toxicities like gastrotoxi­
drug Amphotericin B is a polyene macrolide, which is city, heptaotoxicity, and nephrotoxicity [309].
used in the deoxycholate form as a frontline antileish­ Moreover, miltefosine is teratogenic in nature; its
manial therapy in patients with antimony-resistant para­ usage by pregnant women is limited as it increases the
sites [292]. Since amphotericin B binds with sterols of chances of contraception and needs monitoring during
host cell walls, it inhibits the interaction with promasti­ treatment for at least 4 months after treatment.
gotes, thus hampering the internalisation of the parasite Furthermore, the long half-life of the drug increases
[299]. Later studies by Chattopadhyay et al. suggested the probability of drug resistance [292].
a novel antileishmanial mechanism of AmpB. They
reported AmpB-mediated targeting of the sterols like
Advent of immunomodulators: Futuristic
ergosterols present in the cell wall of the parasite leading
antileishmanial therapy?
to lysis of the cell [300]. AmpB with an antileishmanial
efficacy of >95% has been reported to be highly effective In order to tackle the limitations of available drugs,
against VL [301] and Indian PKDL [302]. However, extensive research focused on identifying leishmanial
AmpB infusions in patients were associated with adverse virulence factors as putative drug targets are being
toxicities like renal complications and hypokalemia carried out. The importance of HSPs, DUBs, and var­
[303]. Liposomal formulations of AmpB like ious kinases in the virulence and pathogenicity of the
Ambisome, Fungisome [304], amphotericin B lipid com­ parasite marks them as potent antileishmanial drug
plex, and amphotericin B cholesterol dispersion exhib­ targets. Apart from the leishmanial virulence factors,
ited better clinical results with improved drug stability the strategies exploited by the parasite, to evade the
and targeted delivery. Ambisome has already been defense mechanisms of the host, are also being con­
approved by US FDA and has been registered for use sidered as potent antileishmanial targets using immu­
as antileishmanial drug in India, Egypt, and Brazil [305]. nomodulators. Immunomodulators are compounds
However, cholesterol is an important constituent of the capable of altering the response of the immune system
formulation, and at the same time, it is known to facil­ and can behave either as immunostimulators or
itate the internalisation of the parasite in VL. In order to immunosuppressives [316]. Immunostimulators
overcome this hurdle, a modified lipid formulation, enhance the host response against infectious diseases
KalsomeTM10, with AmpB intercalated with sterols and or tumors by triggering pro-inflammatory cytokines or
phosphatidylcholine was introduced. KalsomeTM10 response [317]. Since Leishmania for successful survi­
exhibited improved parasite clearance and minimum- val subverts the host immune responses and defense
924 A. K. GUPTA ET AL.

machineries, use of such immunomodulators can antileishmanial potency is genipin. Genipin inhibits
prove a futuristic therapy against the disease. L. donovani-induced UCP2 levels and hence increases
Fucoidan, the sulphated polysaccharide derived from ROS generation in infected macrophages. Genipin
Fucus vesiculosus, has long been used traditionally as treatment also successfully drove the cytokine balance
an anti-thrombic and anti-malarial agent. Fucoidan to the disease resolving Th1 state. Genipin reduced
was reported to be effective in controlling both anti­ splenic and liver parasite burden, despite showing no
mony-susceptible and -resistant VL [318]. The antil­ direct effect on both the promastigotes and axenic
eishmanial effect was explained later by the study amastigotes [157]. Thus, the antileishmanial potency
demonstrating that fucoidan treatment mediates PKC- of genipin is by targeting the host-negative regulatory
dependent MAPK and NFκB activation, eventually protein, UCP2, which is exploited by the parasite for
leading to the production of NO and disease resolving ROS suppression. Like GA, combination of genipin
cytokines [319]. Cystatin is another immunomodula­ and SAG showed synergism against leishmaniasis, sug­
tor that inhibits cysteine proteases of Leishmania by gesting that combination therapy involving such
which the parasite ensures a disease-conducive Th2 immunomodulators and other frontline antileishma­
response in the host [320]. Cystatin treatment shifts nial drugs may be a promising futuristic antileishma­
the CD4+ cell differentiation from Th2 to Th1 and also nial approach.
upregulates NO production [321]. Later studies also
indicated that cystatin treatment could effectively cure
experimental VL by NFκB-mediated proinflammatory Concluding remarks
response in infected macrophages [322]. The list of Leishmania as a parasite has evolved various mechanisms
immunomodulators studied against leishmaniasis and strategies to overcome host defense machineries. The
include the triterperne derivative from the water- quest to find the effectors that makes the parasite capable
soluble extract from the roots of licorice −18-β- of subverting host defense mechanisms led to the discov­
glyccyrrhetinic acid (GRA). GRA treatment activated ery of various virulence factors like LPG, EF1-α, and
NFκB in experimental VL animal models along with GP63. LPG and GP63 have been reported to play impor­
concomitant upregulation of NO production and Th1 tant roles in evading the primary defense artilleries of the
cytokines [323]. Later studies by this group determined host like ROS and RNS production, inhibiting NFκB
that GRA treatment also reversed the host phospha­ activation, overriding different host signaling, and shift­
tase/kinase balance, which is altered by the parasite as ing the host immune response to the disease conducive
a survival strategy [324]. Another bioactive component Th2 state. The parasite as a survival strategy also exploits
from the roots of licorice, glyccyrrhizic acid (GA), was certain host-negative regulatory proteins like A20, UCP2,
also shown to exhibit antileishmanial properties. HO-1, Arginase, c-Myc, and SHP-1, but the mechanism
Treatment of L. donovani-infected macrophages with or the factors responsible for these are not very well
GA inhibited COX2-mediated prostaglandin E2 release elucidated, and further works are required to answer
in the host and increased expression of pro- these questions. With passing time and increased research
inflammatory cytokines like IL-12 and TNF-α [325]. in this area, novel virulence factors are coming into light
The authors suggested that GA treatment, in combina­ like Hsp78, LACK, various leishmanial kinases, and deu­
tion with SAG, was effective against SAG-resistant biquitinases. Understanding of the leishmanial virulence
L. donovani. GA treatment inhibited expression and factors and host immune subversion strategies has led to
efflux activity of host ABC transporter multidrug resis­ advanced diagnostics of the disease and has suggested
tance-associated protein-1 (MRP-1) and host futuristic drug therapies, which is the need of the hour
P-glycoprotein (P-gp), which were responsible for in order to tackle the menace of leishmaniasis.
efflux of antimony from SAG-resistant infected host
cells [326]. Interestingly, a recent study reports an
alternative antileishmanial mechanism of GA where Disclosure statement
GA treatment abrogated L. donovani growth by inhi­
No potential conflict of interest was reported by the
biting L. donovani HMG-coA- reductase (HMGR), author(s).
depleting membrane ergosterol levels [327]. A similar
effect of GA has been reported very recently in CL.
Treatment with GA and hydroalcoholic extract of Funding
Glycyrrhiza glabra inhibited the growth of promasti­ This work has, in part, received funding from UK Research
gotes and amastigotes [328]. Another immunomodu­ and Innovation via the Global Challenges Research Fund
lator that has been very recently reported to possess under grant agreement “A Global Network for Neglected
 VIRULENCE 925

Tropical Diseases” grant number Global Challenges Research 16. Reithinger R, Davies CR. Is the domestic dog (Canis
Fund (NTD) MR/P027989/1, Sir J. C. Bose Fellowship, India, familiaris) a reservoir host of American cutaneous
and Council of Scientific and Industrial Research, India. leishmaniasis? A critical review of the current
evidence. Am J Trop Med Hyg. 1999;61:530–541.
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Data availability statement America. Parasit Vectors. 2009;2(Suppl 1):S1.
18. Chan A, Ayala JM, Alvarez F, et al. The role of
All the relevant data (tables and figures) of this review are Leishmania GP63 in the modulation of innate inflam­
available. matory response to Leishmania major infection. PLoS
One. 2021;16:e0262158.
19. Saraiva EM, Pinto-da-silva LH, Wanderley JL, et al.
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