Overview of Leishmaniasis with Special Emphasis on Kala-azar in South Asia

Abstract

Leishmaniasis is a complex disease caused by Leishmania infection, producing variable clinical symptoms, e.g., cutaneous, mucocutaneous, and visceral leishmaniases [1–3]. Cutaneous leishmaniasis (CL) caused, for example, by Leishmania major/L. tropica is marked by the appearance of skin lesion in various forms, which are often innocuous and self-healing, while mucocutaneous leishmaniasis (MCL) caused, for example, by L. braziliensis is a protracted disease, resulting sometimes in facial disfigurement of the ear, mouth, and nose. Neither CL nor MCL is life-threatening per se. Only in non-healing case has death of these patients been reported due to secondary infections or other causes, e.g., suicide as a result of unbearable psychological stress. Visceral leishmaniasis (VL) caused by L. donovani/L. infantum is far more severe. It is often fatal, if untreated, resulting from systemic and progressive infection of macrophages by Leishmania in the reticuloendothelial systems or lymphoid organs, chiefly the spleen, liver, and bone marrow. Disorders of hematological and hepatosplenic functions are thus the clinical manifestations of VL, including hepatosplenomegaly, fever, anemia, leucopenia, hypergammaglobulinemia, and cachexia. The development of all leishmaniases follows a chronic course lasting for months and sometimes years.

For collaborator details please see the list provided at the end.

Appendix

Box 1: Malnutrition, Autophagy, and Susceptibility to Kala-azar

• Contributed by: Syamal Roy

• National Institute of Pharmaceutical Education & Research

• Jadavpur, Kolkata, India

There is an interesting relationship between nutritional deficiency and aggravation of kala-azar. Kala-azar patients in Bihar are malnourished. Starvation induces autophagy. When autophagy is triggered in macrophages in vitro either by pharmacological mediators or by starvation, infection of these cells with SAG-resistant Leishmania donovani (LD) results in its exuberant intracellular replication (1). Interestingly, this was not seen when these macrophages were infected with SAG-sensitive parasites (1). The autophagic cells after infection undergo apoptosis, which then may favor parasites to egress and accelerate cell-to-cell transmission and dissemination (1), as shown in the cases of a wide variety of bacteria and apicomplexan parasites (2). In our earlier work, GP63 was shown to cleave dicer that inhibits maturation of miR 122, which constitutes ~80% of the hepatic microRNAs and is important for lipid metabolism (3). This is known to cause hypocholesterolemia, as generally noted to be severe among kala-azar patients (4). The cholesterol level in some patients is lowered to one-tenth of the normal level. It is well known that cholesterol is important in maintaining the conformation of membrane proteins like acetylcholine receptor and serotonin receptor (5), MHC-II protein (6, 7), and also for the lateral mobility of membrane protein (8). Leishmania infection of antigen-presenting cells, like macrophages and dendritic cells, has been shown to significantly alter the kinetic parameters of peptide-MHC-II stability (K _on and K _off kinetics), resulting in immune dysfunction (9). This is perceived as part of the mechanisms (10), coupled with decrease in membrane cholesterol (11, 12) by which intracellular LD manipulates host metabolic pathways and contributes to the aggravated pathogenesis. Thus, autophagy pathway may contribute to aggressive infection in the mammalian host by the antimony resistant LD as compared to the sensitive ones. Metabolic dysfunction induced by the LD infection may contribute to the establishment of the infection in the mammalian host.

References

• 1. Mukherjee B, Mukhopadhyay R, Sundar S, Dujardin J-C, Roy S. Probing the strategies of antimony resistant Leishmania donovani to induce aggressive infection. Session “Molecular basis of virulence & Tropism”, 6th World Congress of Leishmaniasis, May 16–20 Toledo, Spain. 2017.

• 2. Friedrich N, Hagedorn M, Soldati-Favre D, Soldati T. Prison break: pathogens’ strategies to egress from host cells. Microbiol Mol Biol Rev. 2012;76(4):707–20.

• 3. Ghosh J, Bose M, Roy S, Bhattacharyya SN. Cell Host Microb. 2013;13:277–88.

• 4. Ghosh J, Lal CS, Pandey K, Das VNR, Das P, Roy Choudhuri K, Roy S. Ann Trop Med Hyg. 2011;105:267–71.

• 5. Gimpl G. Cholesterol protein interaction: methods and cholesterol reporter molecules. Subcell Biochem. 2010;51:1–45.

• 6. Roy K, Ghosh M, Pal TK, Chakrabarti S, Roy S. J Lipid Res. 2013;54:3106–15.

• 7. Roy K, Mandloi S, Chakrabarti S, Roy S. PLoS Neg Trop Dis. 2016;10: e0004710.

• 8. Ghosh M, Roy K, Das Mukherjee D, Chakrabarti G, Roy Choudhury K, Roy S. PLoS Negl Trop Dis. 2014;4(8):e3367.

• 9. Roy K, Naskar K, Ghosh M, Roy S. J Immunol. 2014;192:5873–80.

• 10. Chakraborty D, Banerjee S, Sen A, Banerjee KK, Das P, Roy S. J Immunol. 2005;175:3214–24.

• 11. Sen S, Roy K, Mukherjee S, Mukhopadhyay R, Roy S. PLoS Pathog. 2011;7:e1002229.

• 12. Benerjee S, Ghosh J, Sen S, Guha R, Dhar R, Ghosh M, Datta S, Raychaudhury B, Naskar K, Holder AK, Las CS, Pandey K, Das VNR, Das P, Roy S. Infect Immun. 2009;77:2330–42.

Box 2: Leishmania Acquires Heme from Host Hemoglobin

Contributed by: Amitabha Mukhopadhyay

National Institute of Immunology, New Delhi, India

A rational approach to search for a novel drug/vaccine target against intracellular pathogens is the exploitation of biochemical differences between the parasite and its mammalian host. Leishmania is auxotroph for heme, as the parasites lack complete heme biosynthetic pathway (1). Heme is a critical prosthetic group required by the parasites for several metabolic pathways. Thus, heme acquisition process in Leishmania could be a potential target (2). However, how parasites acquire heme is not well depicted. Interestingly, it has been shown that Leishmania expresses a high-affinity receptor for hemoglobin (Hb) in the flagellar pocket of the parasites (3). Hemoglobin first binds to this high-affinity receptor (HbR) and endocytosed via a clathrin-mediated process (4). Subsequently, Hb is internalized into early endosomal compartment in the parasite via Rab5-regulated process (5, 6). Finally, internalized Hb is targeted to the parasite lysosomes by Rab7-dependent process where it is degraded to generate intracellular heme, which parasites use for their survival (7). Interestingly, it has been shown that HbR is a surface-localized hexokinase, a glycolytic protein (8). Thus, HbR regulates two major functions in parasite: (a) it acts as Hb receptor on cell surface to acquire heme and (b) it also regulates glycolysis. Moreover, it has been shown that blocking the Hb uptake by anti-receptor antibody or inhibiting the targeting of internalized Hb to the lysosomes is detrimental for the parasites, rendering them unable to acquire heme from Hb degradation. In addition, it has been shown that newly synthesized HbR exit the endoplasmic reticulum (ER) via COPII-regulated process and targeted to the cell surface by Rab1-independent unconventional secretory pathway (9, 10). Interestingly, knocking down of these regulatory proteins by specific siRNA inhibits parasites’ growth. These results unequivocally prove that parasites acquire heme from Hb.

As HbR is found to regulate two major functions in parasite, therefore HbR could be a potential new target. Consequently, HbR is evaluated as potential vaccine candidate against visceral leishmaniasis. It has been shown that vaccination of mice and hamsters with HbR-DNA constructs inhibits more than 99% splenic and hepatic parasite burden in comparison to infected and vector control animals. It has been shown that impaired T-cell response and inhibition of IL-2 production are associated with VL. Interestingly, it has been shown that HbR vaccination can reverse the impaired T-cell response with higher production of IL-2 and induce Th1 protective response (11). These results demonstrate that HbR-DNA immunization offers major advantages over other vaccine candidates against VL because it is functionally important in the parasite life cycle, conserved across various Leishmania species, and naturally immunogenic in VL patients.

References

• 1. Sah JF, Ito H, Kolli BK, Peterson DA, Sassa S, Chang KP. Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy. J Biol Chem. 2002;277:14902–9.

• 2. Kelly JX, Ignatushchenko MV, Bouwer HG, Peyton DH, Hinrichs DJ, Winter RW, Riscoe M. Antileishmanial drug development: exploitation of parasite heme dependency. Mol Biochem Parasitol. 2003;126:43–9.

• 3. Sengupta S, Tripathi J, Tandon R, Raje M, Roy RP, Basu SK, Mukhopadhyay A. Hemoglobin endocytosis in Leishmania is mediated through a 46 kD protein located in the flagelar pocket. J Biol Chem. 1999;274:2758–65.

• 4. Agarwal S, Rastogi R, Gupta D, Patel N, Raje M, Mukhopadhyay A. Clathrin-mediated hemoglobin endocytosis is essential for survival of Leishmania. BBA Mol Cell Res. 2013;1833:1065–77.

• 5. Singh SB, Tandon R, Krishnamurthy G, Vikram R, Sharma N, Basu SK, Mukhopadhyay A. Rab5 mediated endosome-endosome fusion regulates hemoglobin endocytosis in Leishmania donovani. EMBO J. 2003;22:5712–22.

• 6. Rastogi R, Kapoor JKV, Langsley G, Mukhopadhyay A. Rab5 isoforms specifically regulate different modes of endocytosis in Leishmania. J Biol Chem. 2016;291:14732–46.

• 7. Patel N, Singh SB, Basu SK, Mukhopadhyay A. Leishmania requires Rab7-mediated degradation of endocytosed hemoglobin for their growth. Proc Natl Acad Sci USA.2008;105:3980–5.

• 8. Krishnamurthy G, Vikram R, Singh SB, Patel N, Agarwal S, Mukhopadhyay G, Basu SK, Mukhopadhyay A. Hemoglobin receptor in leishmania is a hexokinase located in the flagellar pocket. J Biol Chem. 2005;280:5884–91.

• 9. Bahl S, Parashar S, Malhotra H, Raje M, Mukhopadhyay A. Functional characterization of monomeric GTPase Rab1 in the secretory pathway of Leishmania. J Biol Chem. 2015;290:29993–30005.

• 10. Parashar S, Mukhopadhyay A. GTPase Sar1 regulates the trafficking and secretion of the virulence factor gp63 in Leishmania. J Biol Chem. 2017. doi:https://doi.org/10.1074/jbc.M117.784033 (in press).

• 11. Guha R, Gupta D, Rastogi R, Vikram R, Krishnamurthy G, Bimal S, Roy S, Mukhopadhyay A. Vaccination with Leishmania hemoglobin-receptor-encoding-DNA protects against visceral Leishmaniasis. Science Transl Med. 2013;5:202ra121.

Box 3: Leishmania Survive in Phagolysosomes (Misnomer)

• Contributed by: Amitabha Mukhopadhyay

• National Institute of Immunology, New Delhi, India

Ref: Verma JK, Rastogi A, Mukhopadhyay A. Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494. Plos Pathog. 2017;13:e1006459.

Several intracellular pathogens block the phagosome maturation to lysosomes in the host cells for their survival. Rab GTPases are the central regulators of membrane trafficking pathways; therefore, most of the intracellular pathogens modulate the function of host endocytic Rab GTPase specially the Rab5 to inhibit their lysosomal targeting. In contrast, Leishmania are thought to reside in phagolysosomal compartment in mouse macrophages as the Leishmania-containing parasitophorous vacuole (PV) recruits lysosomal markers such as Lamp1, Lamp2, and cathepsin D. However, how parasites survive in such a detrimental compartment in a cell is not well demonstrated. Recently, we have shown that Leishmania donovani specifically upregulates the expression of Rab5a by inhibiting the synthesis of miR-494 in human macrophages which negatively regulates the expression of Rab5. Leishmania downregulates the expression of miR-494 by degrading c-Jun via their metalloprotease gp63. Subsequently, L. donovani recruits and retains these overexpressed Rab5a along with early endosome-associated antigen (EEA1) on PV to reside in early endosomes. Recruitment of Rab5a on Leishmania-containing PV promotes fusion with early endosomes to inhibit transport to the lysosomes. Finally, we have found that the parasite also modulates the early endosome by recruiting Lamp1 and inactive pro-cathepsin D on PV via the overexpression of Rab5a in human macrophages. Thus, Leishmania resides in early endosomes not in phagolysosomes as thought earlier. But PV also recruits lysosomal enzymes in immature and inactive form in human macrophages which help the parasites to survive in human macrophages.

Interestingly, overexpression of Rab5 by downregulating the synthesis of miR-494 happens only in human and hamster macrophages, but not in mouse macrophages as miR-494 target site is absent in the 3’-UTR of mouse Rab5a. Thus, our results unequivocally prove that Leishmania resides in modified early endosomes in human macrophages but also resolve the controversy why it was thought that Leishmania resides in phagolysosomal compartment using mainly mouse macrophages. Thus, these results also indicate why among the two animal models of leishmaniasis, hamster model mimics human infection, whereas Leishmania infection is self-healing in mouse.

Commentary on “Leishmania Survive in Phagolysosomes: Misnomer” by KP Chang

Since the 1970s, Leishmania have been recognized as a phagolysosomal parasite of the macrophages—its exclusive host cells in susceptible animals. This conclusion was drawn by a number of early investigators from their work on Leishmania infection of macrophages in vitro and in vivo in animal models. In infected macrophages, Leishmania-containing vacuoles (PVs) and phagolysosomes are congruent in their physical and chemical properties, as shown by multiple experimental approaches, i.e., (a) particulate or fluorescent tags of the secondary lysosomes emerge in the endosomes, which contain Leishmania, e.g., L. donovani, in human peripheral blood monocyte-derived macrophages (1); (b) acidity of the PVs, as measured under living conditions of L. donovani-infected macrophages based on pH-dependent changes in the fluorescence intensity of FITC-dextran (2); (c) cytochemical localization of lysosomal enzyme activities in the PV, e.g., alkaline phosphatase and myeloperoxidase reaction products deposited in the PV of L. donovani-infected human primary phagocytes-monocytes, neutrophils, and eosinophils (3); and (d) co-localization of L. donovani with phagolysosomes in the liver from infected animals after subcellular fractionation (Andre Trouet; 4). Together, all these lines of evidence indicate that L. donovani does reside in phagolysosomes shortly after in vitro infection of macrophages from human and other mammalian hosts and after in vivo infection of animals to a steady state.

Inconsistent with the previous conclusion are more recent work based chiefly on the “Rab cascade model” to explain the regulation of directional and orderly trafficking of vacuoles/vesicles for the transport of their cargoes along the mammalian endocytic and secretory pathways. There are dozens of Rabs or GTPase isoforms and other membrane proteins, which tether to the cytoplasmic side of the vacuoles. Some RabGTPases are thought to be the master regulators, which order the events of membrane trafficking and regulate the localization of the subsequent Rabs, thereby determining indirectly the identity of vacuoles/endosomes and their functional status. These and other membrane-associated proteins are regulated by a network of signal pathways and indirectly by microRNAs. The readout of these and related vacuolar membrane molecules is based invariably on immunofluorescent microscopy of fixed cell samples and Western blot analysis in conjunction with the use of inhibitors and cutting edge, albeit globally affecting genetic approaches: specific gene knockdown/knock-in, transcriptome/miRNA analysis, etc. This powerful combination of cellular and molecular tools allows one to scrutinize the PV membrane proteins and, more importantly, to manipulate them for predicting the intracellular location and fate of intracellular pathogens. The burgeoning literature in this field includes excellent work on the PV-associated membrane molecules after endocytosis of Leishmania by macrophages. Interested readers are referred to the publications in this area of investigation for details. It suffices to briefly mention a couple of examples: Albert Descoteaux and his colleagues have long reported inhibition/modulation of phagosome maturation by Leishmania lipophosphoglycans (LPG) and Zn-metalloprotease (gp63) in macrophages after infection in vitro with metacyclic promastigotes of L. major (5, 6); Peter Kima and his colleagues described the association of ER markers with Leishmania-containing endosomes, thereby considering them as chimeric (7, 8). The most recent paper described above by Amitaba Mukhopadhyay and his colleagues presents an excellent piece of work to further advance our understanding on the molecular events of the PV membrane proteins during the early infection of human macrophages in vitro by L. donovani. Key points of relevance are recapitulated very briefly as follows: the parasite-secreted gp63 apparently downregulates c-Jun in the pathway necessary for the expression of miR494, which regulates Rab5a negatively. The resulting upregulation of Rab5a promotes its sequestration to the PV, thereby keeping them as early endosomes and preventing its replacement with Rab7 necessary for their maturation into late endosomes and phagolysosomes. Extensive data of excellent quality are presented in support of the interpretations based on the “Rab cascade model” and the novel discovery of miR494 with regulatory role specific to THP-1- and HPBM-derived human macrophages.

The foregoing paragraph provides a glimpse of the current conceptual basis and technical approaches to dissect early Leishmania-macrophage membrane interactions in vitro. New discoveries as described warrant further investigation in greater details to bridge the gap of their discordance with the previous findings and to advance the field. Some recommendations are given below for consideration:

Foremost is perhaps to examine the PV in the infected macrophages ex vivo derived from lesion aspirates of patients’ spleen, bone marrow, or skin and, if known, reservoir animals. Examination of such samples for the vacuolar membrane marker proteins and the vacuolar contents will shed light on the properties of well-established PV in clinical infection with direct relevance to the diseases. Clinical correlation of laboratory discoveries has become increasingly mandatory for acceptance by examining archived disease tissues for verification. Such clinical materials are readily available from kala-azar patients for investigation in the endemic countries, such as India. It would be highly desirable to directly examine, in the natural setting, the very early infection of human macrophages by sand fly-delivered promastigotes. This is difficult, if not impossible, to accomplish. The closest simulation of such natural infection is to develop an in vitro organ system, which mimics human skin, e.g., 3D printed skin with draining vasculatures for examining macrophages and other phagocytes in vitro for endocytosis of Leishmania delivered by infected vectors. Given that such an experimental model is not available, the next best to consider is perhaps to obtain in vivo infected macrophages for ex vivo study of their PV, e.g., inoculate mammalian peritoneal cavity or artificially produced skin blister/pouch with infective promastigotes plus sand fly saliva. In vivo infected macrophages are then withdrawn from these sites periodically for ex vivo examinations of their PV in a time course. While still artificial, this experimental approach is perhaps closer to reality than the methodology in use, i.e., exposure of glass- or plastic-adhered macrophages to in vitro grown promastigotes alone in culture medium. The merit of this in vitro system is its simplicity for use to study endocytosis of inert particles, from which “Rab cascade model” is derived as a plausible explanation for phagosome maturation and its regulation as discussed. In that sense, by using the similar in vitro system, the work under discussion contributes significantly to this model by the discovery of miR494 for its novel role in regulating Rab5a. Intervention of this and other regulatory molecules by gp63 and LPG is a very acceptable scenario, considering that both are released, as they are downregulated during promastigote-to-amastigote differentiation after Leishmania infection of macrophages. Leishmania differentiation, akin to cellular development, is expected to follow an orderly program of molecular reorganization. There are known changes of the surface architectures and secretory molecules, in addition to gp63 and LPG, released by Leishmania from early to late stages of this differentiation. All these programed events are expected to work in tandem, contributing to the remodeling the PV for its maturation, i.e., creation of a microenvironment conducive to the replication of amastigotes. At least in one in vitro model, intracellular Leishmania differentiation appears to take a week or longer to complete based on the switch in tubulin biosynthesis as the molecular marker (9). Thus, a large gap appears to emerge in the experimental approaches to assess the molecular events and in the time frame of the observations between previous and more current studies, i.e., the week-long maturation of the PV for parasite replication versus a couple of days or less for phagosome maturation. In addition, information collected after short-term infection, e.g., 48 h does not foretell events beyond this time frame, including phagosome-lysosome fusion, as reported previously. Further investigation to bridge the gaps entails the consideration of all experimental approaches using in vitro models, which enable Leishmania not only to complete their differentiation but also to replicate as amastigotes, i.e., long-term infection of human macrophages (1) and macrophage cell lines (10, Fig. 4), and those from experimentally well-infected animals for PV in a steady-state infection.

Recent work, including the latest paper under discussion, has significant bearing on our quest for understanding the Leishmania mechanisms of intracellular parasitism. Our renewed attention in that direction is necessitated by the state-of-the-art approach, as it represents progresses in the science of cell biology research. Whether or not the discussion provided is viewed as pertinent, it brings up a significant issue. That is, a close and proactive collaboration among leishmaniacs in different fields will be necessary for advances toward the resolution of the issue at hand in the context of leishmaniasis.

References

• 1. Berman JD, Dwyer DM, Wyler DJ. Multiplication of Leishmania in human macrophages in vitro. Infect Immun. 1979;26:375–9.

• 2. Chang KP. Endocytosis of Leishmania-infected macrophages. Fluorometry of pinocytic rate, lysosome-phagosome fusion and intralysosomal pH, p. 231-234. In: Van den Bossche H, editor. The host invader interplay. Amsterdam: Elsevier/NorthHolland, Biomedical Press; 1980.

• 3. Chang KP. Leishmanicidal mechanisms of human polymorphonuclear phagocytes. Am J Trop Med Hyg. 1981;30:322–33.

• 4. Trouet A. Isolation of modified liver lysosomes. Methods Enzymol. 1974;31:323–9. PMID: 4370710; Tulkens P, Trouet A. The concept of drug-carriers in the treatment of parasitic diseases. In: Müller-Ruchholtz W, Müller-Hermelink HK, editors. Function and structure of the immune system. Advances in experimental medicine and biology, Vol. 114. Boston, MA: Springer; 1979.

• 5. Moradin N, Descoteaux A. Leishmania promastigotes: building a safe niche within macrophages. Front Cell Infect Microbiol. 2012;2:121. doi:https://doi.org/10.3389/fcimb.2012.00121. PMID: 23050244.

• 6. Matte C, Descoteaux A. Exploitation of the host cell membrane fusion machinery by Leishmania is part of the infection process. PLoS Pathog. 2016;12:e1005962. doi:https://doi.org/10.1371/journal.ppat.1005962.

• 7. Ndjamen B, Kang BH, Hatsuzawa K, Kima PE. Leishmania parasitophorous vacuoles interact continuously with the host cell’s endoplasmic reticulum; parasitophorous vacuoles are hybrid compartments. Cell Microbiol. 2010;12:1480–94. doi:https://doi.org/10.1111/j.1462-5822.2010.01483.x.

• 8. Canton J, Kima PE. Interactions of pathogen-containing compartments with the secretory pathway. Cell Microbiol. 2012;14:1676–86. doi:https://doi.org/10.1111/cmi.12000.

• 9. Fong D, Chang KP. Tubulin biosynthesis in the developmental cycle of a parasitic protozoan, Leishmania mexicana: changes during differentiation of motile and nonmotile stages. Proc Natl Acad Sci U S A. 1981;78:7624–8.

• 10. Chang KP, Reed SG, McGwire BS, Soong L. Leishmania model for microbial virulence: the relevance of parasite multiplication and pathoantigenicity. Acta Trop. 2003;85:375–90.

Box 4: Programmed Cell Death in the Leishmania Parasite

• Contributed by: Chandrima Shaha

• Professor of Eminence and Former Director, National Institute of Immunology and Vice President (Foreign Affairs) Indian National Science Academy, New Delhi 110067

Programmed cell death (PCD), commonly manifested as apoptosis, plays crucial roles in a multitude of physiological processes starting from embryogenesis to maintenance of the immune system. Initially believed to be the prerogative of multicellular organisms to use PCD for maintaining cellular homeostasis, it was later found to be prevalent in unicellular organisms as well (1). The term PCD and apoptosis have been used interchangeably and describe cell death with typical features of apoptosis. PCD was described in Trypanosoma cruzi and Leishmania amazonensis during the 1990s (2, 3).

Subsequently, with the demonstration of cell death in different Leishmania species, showing a phenotype similar to apoptosis generated a great interest in the field of Leishmania biology. The digenetic life cycle of this parasite provides possibilities of PCD at several points during their life cycle for maintenance of fitness of the colony. The fittest promastigotes residing in the midgut of the female sand fly have to pass onto the pharynx of the fly by removing unfit cells, likely discarded through PCD as necrotic removal would endanger the health of the sand fly. Although the type of death in the gut of the sand fly has not been examined, free-swimming forms of the parasite in culture were shown to undergo PCD under various stress conditions (4–6). Within the vertebrate host cells, the mammalian macrophages, the parasites are removed through the process of PCD to maintain the optimum number, thus creating a niche for favorable growth of the remaining amastigotes, the nonmotile intracellular forms (7). Several features of mammalian apoptosis like chromatin condensation, DNA fragmentation, loss of mitochondrial membrane potential, cell shrinkage, caspase-like activities, phosphatidylserine exposure, and cytochrome c release were demonstrated in the Leishmania parasite in vitro (4, 7). Cell lysates from Leishmania undergoing apoptosis were shown to cleave substrates for caspase-3, although no caspase has been identified in the Leishmania except for a metacaspase (6–8). Interestingly, pretreatment of cells with specific caspase inhibitors reduced the number of cells showing apoptosis-like features, e.g., DNA breakage and cleavage of a PARP-like protein, suggesting existence of proteins with caspase-like activity (4–6).

It was not only during developmental stages of the life cycle that PCD features were shown, exposure to agents that the parasites are normally exposed to, like the reactive oxygen species or drugs, also induced PCD features. Anti-leishmanial drugs like antimony, miltefosine, and amphotericin B were reported to precipitate PCD (9–11). Exposure to reactive oxygen species, heat shock, and staurosporine treatment also precipitates apoptosis of the parasites (4, 5, 12, 13). Like the higher eukaryotic system, the single mitochondrion of Leishmania spp. plays a pivotal role in PCD where imbalances in mitochondrial membrane potential like a fall or increase lead to cell death by apoptosis (5). Calcium appears to be heavily involved in Leishmania PCD. It is increased by exposure to several PCD-inducing agents. Reducing cytosolic calcium by chelating extracellular or intracellular calcium during oxidative stress prevents apoptosis that is preceded by abrogation of a loss of mitochondrial membrane potential (5, 7, 9, 14). Inhibitors of respiratory chain complexes I, II, and III provoke PCD in Leishmania donovani promastigotes. Mitochondrial hyperpolarization resulting from Complex I inhibition is preceded by increased superoxide production. Thenoyltrifluoroacetone and antimycin A, inhibitors of complexes II and III, respectively, dissipate the membrane potential causing PCD (15). Therefore, respiratory chain inhibition is an interesting prospect for drug targeting (16). Exposure of these protozoa to a mixture of reactive oxygen and nitrogen species can cause PCD that is reversible by antioxidants, like glutathione and calcium channel blockers (17). Leishmania spp. react to two related metalloids, arsenic and antimony, leading to cell death accompanied by typical apoptotic features that is preceded by an increase in reactive oxygen species. Mitochondrial dysfunction and a drop in ATP level are observed with a loss of membrane potential. During arsenic treatment, prevention of calcium influx reduces cell death, whereas supplementation of glutathione during antimony treatment saved cell loss (9). Therefore, multiple agents with different mechanisms of action could precipitate apoptosis-like death. Recently, apoptotic death in the Leishmania has been shown after exposure to amphotericin B, and zinc flux causes mitochondrial disruption, resulting from the accumulation of reactive oxygen species (18). Caspase-like activity was detected in Leishmania raising the possibility for the existence of this enzyme, although genome sequence did not reveal any ORF homologous to typical caspases. Caspase-independent death was described in the trypanosomatid parasites where endonuclease G, a mitochondrial enzyme, appears to be responsible for DNA fragmentation during apoptosis (19).

Interestingly, PCD may function beyond the provision of unwanted cell elimination to maintain fitness of the colony; it can be used to drive other functions like the ability to infect. These parasites have been shown to mimic an apoptotic cell phenotype by phosphatidylserine exposure. As a result, a given infective inoculum may consist of both live and apoptotic cells to facilitate a successful infection (20). In the case of Leishmania spp. infection in mice, such apoptotic mimicry in amastigotes has been described (21). Leishmania expresses a variety of defense mechanisms against exogenous stress, preventing them from undergoing apoptosis. For example, ergosterol upsurge during antimony treatment prevents cell death (22). Upregulation of defensive enzymes like tryparedoxin peroxidases of both the mitochondrial and cytosolic origin also prevents cell death induced by reactive oxygen species (17). Therefore, it is evident that PCD of Leishmania parasites may play a significant role in infection (23).

Although many aspects of the PCD have come to light, the molecular mechanism remains to be defined. Elucidation of the molecular events linked to apoptotic death of Leishmania spp. is of great importance because this information has the potential to help define a more comprehensive view of the cell death machinery in terms of evolutionary origin and identify new target molecules for chemotherapeutic drug development and therapeutic intervention.

References

• 1. Arnoult D, Akarid K, Grodet A, Petit PX, Estaquier J, Ameisen JC. On the evolution of programmed cell death: apoptosis of the unicellular eukaryote Leishmania major involves cysteine proteinase activation and mitochondrion permeabilization. Cell Death Differ. 2002;9:65–81.

• 2. Murphy NB, Welburn SC. Programmed cell death in procyclic Trypanosoma brucei rhodesiense is associated with differential expression of mRNAs. Cell Death Differ. 1997;4:365–70.

• 3. Moreira ME, Del Portillo HA, Milder RV, Balanco JM, Barcinski MA. Heat shock induction of apoptosis in promastigotes of the unicellular organism Leishmania (Leishmania) amazonensis. J Cell Physiol. 1996;167:305–13.

• 4. Das M, Mukherjee SB, Shaha C. Hydrogen peroxide induces apoptosis-like death in Leishmania donovani promastigotes. J Cell Sci. 2001;114:2461–9.

• 5. Mukherjee SB, Das M, Sudhandiran G, Shaha C. Increase in cytosolic Ca²⁺ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes. J Biol Chem. 2002;277:24717–27.

• 6. Lee N, Bertholet S, Debrabant A, Muller J, Duncan R, Nakhasi HL. Programmed cell death in the unicellular protozoan parasite Leishmania. Cell Death Differ. 2002;9:53–64.

• 7. Sudhandiran G, Shaha C. Antimonial-induced increase in intracellular Ca²⁺ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes. J Biol Chem. 2003;278:25120–32.

• 8. Casanova M, Gonzalez IJ, Sprissler C, Zalila H, Dacher M, Basmaciyan L, Späth GF, Azas N, Fasel N. Implication of different domains of the Leishmania major metacaspase in cell death and autophagy. Cell Death Dis. 2015;6:e1933.

• 9. Mehta A, Shaha C. Mechanism of metalloid-induced death in Leishmania spp.: role of iron, reactive oxygen species, Ca²⁺, and glutathione. Free Radic Biol Med. 2006;40:1857–68.

• 10. Paris C, Loiseau PM, Bories C, Bréard J. Miltefosine induces apoptosis-like death in Leishmania donovani promastigotes. Antimicrob Agents Chemother. 2004;48:852–9.

• 11. Shadab M, Jha B, Asad M, Deepthi M, Kamran M, Ali N. Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B. PLoS One. 2017;12:e0171306.

• 12. Sen N, Banerjee B, Das BB, Ganguly A, Sen T, Pramanik S, Mukhopadhyay S, Majumder HK. Apoptosis is induced in Leishmanial cells by a novel protein kinase inhibitor withaferin A and is facilitated by apoptotic topoisomerase I-DNA complex. Cell Death Differ. 2007;14:358–67.

• 13. Kumari A, Singh KP, Mandal A, Paswan RK, Sinha P, Das P, Ali V, Bimal S, Lal CS. Intracellular zinc flux causes reactive oxygen species mediated mitochondrial dysfunction leading to cell death in Leishmania donovani. PLoS One. 2017;12:e0178800.

• 14. Dolai S, Pal S, Yadav RK, Adak S. Endoplasmic reticulum stress-induced apoptosis in Leishmania through Ca²⁺-dependent and caspase-independent mechanism. J Biol Chem. 2011;286:13638–46.

• 15. Mehta A, Shaha C. Apoptotic death in Leishmania donovani promastigotes in response to respiratory chain inhibition: complex II inhibition results in increased pentamidine cytotoxicity. J Biol Chem. 2004;279:11798–813.

• 16. Fidalgo LM, Gille L. Mitochondria and trypanosomatids: targets and drugs. Pharm Res. 2011;28:2758–70.

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• 20. Wanderley JL, Pinto da Silva LH, Deolindo P, Soong L, Borges VM, Prates DB, de Souza AP, Barral A, Balanco JM, do Nascimento MT, Saraiva EM, Barcinski MA. Cooperation between apoptotic and viable metacyclics enhances the pathogenesis of Leishmaniasis. PLoS One. 2009;4:e5733.

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• 23. Shaha C. Apoptosis in Leishmania species and its relevance to disease pathogenesis. Indian J Med Res. 2006;123:233–44.

Box 5: One Health for Leishmaniasis

• Contributed by:

• Dr Gautam Kumar Saha

• Senior Program Officer

• Policy Center for Biomedical Researcher

• Translational Health Science and Technology Institute

• Faridabad, Haryana, India

• Email gautamksaha@thsti.res.in, gautamkumarsaha@gmail.com

• Prof N. K. Ganguly, MD

• Visiting Professor of Eminence

• Translational Health Science & Technology Institute

• Former Director General

• Indian Council of Medical Research

• nkganguly@nii.ac.in

One of the major necessities, for more effective Leishmania elimination program in South Asia and for even the rest of the world, can be an integrative approach to introduce One Health programs and education with welfare as well as development programs. Hence a comprehensive policy framework is required for incorporation of One Health program for control and elimination of leishmaniasis. One Health programs are an amalgamation of multidisciplinary-integrated approach that brings about multiple benefits. One Health encompasses unification of animal, human, and environmental health into an interdisciplinary field of health sciences. The synergy between interdisciplinary fields helps in achieving the goals of biomedical research, education, and more effective public health programs as well as environmental protection. The One Health programs are all encompassing, which further aids toward better effective implementation of welfare programs for achieving sustainable development goals and the overall well-being of the community.

One Health program ensures a creation of a platform for information gathering, training of health workers, and educating the masses of an integrated approach for disease control and elimination both in human and animals (1). By utilization of modern information and communication technologies, along with an effective training of health workers, a robust surveillance system can be designed for areas that are endemic for anthroponotic VL (AVL) or zoonotic VL.

Comprehensive One Health approach explores and strengthens the existing programs using a multidimensional road map of all possible scientific streams. First parameter to analyze the effectiveness of control program should be to ascertain the mode of transmission of disease-causing VL parasite. The best method to prevent or curb VL disease is vector control especially in the endemic regions for AVL. Xenomonitoring of vector infection through a real-time dynamic surveillance design is needed to monitor the transmission of the Leishmania parasite vector species in endemic areas. The best example is the infection caused by L. donovani parasite through vector Phlebotomus argentipes in South Asia, predominantly in India, Nepal, and Bangladesh. The information of percentage of vector infected will be essential to ascertain the degree of spread of Leishmania parasite in the VL endemic regions.

Also in the context for vector control program, now there is a definite shift toward using synthetic pyrethroids that are pesticides derived from naturally occurring pyrethrins. The use of dichlorodiphenyltrichloroethane (DDT) is gradually being discontinued in many VL endemic regions of the world including South Asia due to environmental concerns. There has been introduction of pressure pumps for insecticide spraying; effectiveness of the same has to be also ascertained. Here a policy is also needed for proper use of pesticide as well as continuous monitoring for identifying the development of resistance against the pesticides among the vector population. Another important parameter is the reporting for occurrence of any adverse reaction to human population and the environment. The vector, i.e., the sand fly’s ecological role, cannot be ignored and have to be researched thoroughly. The vector control envisages stopping the overpopulation of the vector and preventing transmission of VL infection but definitely not the total eradication of the vector population.

Further the second step is to monitor human reservoirs of VL parasite. In the AVL areas, the asymptomatic human populations, which harbor Leishmania parasites, including the cases of Post-kala-azar dermal leishmaniasis, assume significance. Even an active surveillance at short regular intervals will be helpful in evaluation of the load of parasite circulating in the environment at any given time. The use of dynamic surveillance becomes more important in the areas of zoonotic VL. In addition to the vector and the patients, the animal reservoirs have to be monitored for circulating parasites.

Third important step toward Leishmania control is to monitor the zoonotic reservoirs for VL, including the environmental changes it affects. The environmental changes are a continuous process and it affects inevitably the life cycle of the organisms that occupy its habitat. Thus it is important that monitoring of the leishmanial parasitic spread if any, also among the domesticated cattle, be undertaken (2). Domestication of cattle is a major source of livelihood among the farmers and the rural community in the VL endemic regions like the Indian subcontinent. The cattle shelters in most of the times harbor conducive habitats for several insects like mosquitoes and sand fly that are health hazard. In the Indian subcontinent, the VL disease is found to be anthroponotic in nature till date with L. donovani as the main causative agent of the VL infection and sand fly as vector, but constant monitoring of the VL parasite among the animal population is important still.

In other zoonotic VL endemic areas, for example, L. infantum, animal reservoirs are found in the canid population along with human population. These are mainly found in the Mediterranean regions, the Middle East, Central Asia, China, and the Americas. A thorough surveillance of the disease in both canine and human populations will help prevent disease outbreaks.

In several studies (3–5) to control zoonotic VL, researchers have emphasized One Health programs as required for effective management of transmission of disease. This can be achieved through a combined approach on one hand by obtaining information regularly from human, vectors, and animal reservoirs for the parasite in the endemic area and on the other hand as an integrated approach, by analysis of environmental factors necessary for disease spread. The environmental factors as we know lead to random genetic mutations; this can increase or decrease parasite infectivity and can also give rise to phenotypic changes in the parasite which also needs to be monitored periodically. The advent of the omics technology has opened new tools to monitor genetic and epigenetic changes among the organisms. It is imperative that the genomic and protein profiling of the parasite circulating in the environment have to be carried out periodically. Another important aspect is reporting of adverse reactions for the chemotherapeutic treatment agents. The policy thus would design mandatory protocols for health systems in reporting adverse events in a full proof and robust manner as part of surveillance system. Another addition to the surveillance policy is to have a comprehensive monitoring for the development of resistance against the chemotherapeutic agents. The grassroots public health clinics have involved in the policy framework.

The important step now is how to implement the concept with the given resources. The surveillance system requires adequate tools for diagnosis that has to be rapid, sensitive, easy to conduct, and cost-effective. The diagnosis with rK39 rapid diagnostic dip test is a sensitive proposition in detecting the presence of anti-Leishmania antibodies in the serum of the patients at a field level. Also now new variant of novel rapid rKE16 antigen-based test is being evaluated to be introduced in the VL elimination programs (6). The rapid dip test for VL mentioned here is routinely carried out in blood samples in place of serum due to lack of resources. In rKE39 test carried out with whole blood, the sensitivity is lesser in cases where the antibody produced is below the normal range, mostly among immunocompromised patients (7). The policy in such cases is such that patients will be screened and identified for suspected VL based on symptoms, even if rK39 test or another dip test comes out negative. Patients with symptoms can be referred to public health clinics (PHC). The PHC have to be equipped to carry out definitive test and provide treatment. India and other South Asian countries are slowly progressing toward equipping their PHC in VL endemic areas to be self-sufficient to provide treatment. VL as we know is the disease prevalent among the impoverished and immunocompromised. Thus the other problem is that of coinfection with diseases like pneumonia and TB that can occur in VL patients and that have to be properly diagnosed. PHC can also screen for HIV which is found to be prevalent in VL-endemic areas too. The PHC should be nodal points of training centers for ground-level health workers, so that they can identify symptoms in patients in the community, carry out surveillance and diagnosis in the field, and learn data gathering. The use of mobile net and telephony application tools can be a viable and speedy option for data collation and distribution to the block-, district-, state-, and national-level program managers as required. The One Health program also envisages as stated before that the environment is protected and its degradation is minimized and the community gets access to both proper sanitation and nutrition in a sustainable manner. As a holistic approach in addition to welfare programs, sustainable development goals have to be achieved. The PHC through health workers will also ascertain whether benefits of the other welfare program reach the target community. Further the One Health program will ensure educating the community about VL and other infections and about how to protect and prevent the infection. Policy should envisage that primary school teachers at rural level along with health workers have to be given proper training and incentives to hire them over a long period of time to educate and generate awareness among the masses about VL and other diseases along the need for environmental protection and conservation.

Thus the One Health approach when adopted in full measure will ensure that data are gathered properly, stored securely, and analyzed, which will aid to ascertain the NTD elimination program gaps and drawbacks. This will ensure proper course correction carried out to keep the elimination program for VL on track.

References

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• 2. Bhattarai NR, Van der Auwera G, Rijal S, Picado A, Speybroeck N, Khanal B, De Doncker S, Das ML, Ostyn B, Davies C, Coosemans M, Berkvens D, Boelaert M, Dujardin J-C. Domestic animals and epidemiology of visceral leishmaniasis, Nepal. Emerg Infect Dis. 2010;16(2). www.cdc.gov/eid.

• 3. Lorusso V, et al. Paediatric visceral leishmaniasis in Italy: a ‘One Health’ approach is needed. Parasit Vectors. 2013;6:123.

• 4. Hailu T. One Health approach prospect for integrated control and elimination of visceral leishmaniasis in Ethiopia: a narrative review article. Iran J Parasitol 2016;11(1):1–9.

• 5. Palatnik-de-Sousa CB, Day MJ. One Health: the global challenge of epidemic and endemic leishmaniasis. Parasit Vectors. 2011;4:197. http://www.parasitesandvectors.com/content/4/1/197.

• 6. Vaish M, Sharma S, Chakravarty J, Sundar S. Evaluation of two novel rapid rKE16 antigen-based tests for diagnosis of visceral leishmaniasis in India. Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India. J Clin Microbiol. 2012;50(9):3091–2. jcm.asm.org 3091.

• 7. Matlashewski G, Das VNR, Pandey K, Singh D, Das S, Ghosh AK, et al. Diagnosis of visceral leishmaniasis in Bihar, India: comparison of the rK39 rapid diagnostic test on whole blood versus serum. PLoS Negl Trop Dis. 2013;7(5):e2233. https://doi.org/10.1371/journal.pntd.0002233.