To successfully establish infection in the mammalian host, Leishmania parasites must impair the microbicidal repertoire of neutrophils and macrophages. One of the strategies for neutrophil-mediated killing is the release of a lattice composed of DNA associated with histones and granular and cytoplasmic proteins named neutrophil extracellular traps (NETs). NETs can ensnare and kill microorganisms, preventing parasitic infection (Brinkmann et al., 2004; Guimarães-Costa et al., 2012). For several microorganisms, the expression of secreted or membrane-bound nucleases has been reported as a strategy to escape the toxic effects promoted by NETs (Sumby et al., 2005; Berends et al., 2010; Seper et al., 2013; Thammavongsa et al., 2013; Afonso et al., 2021). The bifunctional enzyme 3’-nucleotidase/nuclease (3’-NT/NU), a unique class I nuclease present in several Leishmania species, has been considered an important factor for parasite escape from NET-mediated death through the hydrolysis of extracellular DNA (Guimarães-Costa et al., 2014; Freitas-Mesquita et al., 2019).

3’-NT/NU was first described in Leishmania donovani parasites (Gottlieb and Dwyer, 1983). Acid phosphatase, 5’-nucleotidase, and 3’-nucleotidase activities were observed in the surface membrane fraction isolated from L. donovani promastigotes. Based on biochemical properties, primarily differential sensitivity to inhibitors, these activities were shown to correspond to different enzymes (Gottlieb and Dwyer, 1983). Posterior studies performed with several Leishmania species have shown that 3’-NT/NU presents stage-specific expression. While procyclic and metacyclic promastigotes express the enzyme, no expression is observed in amastigotes (Sopwith et al., 2002; Lakhal-Naouar et al., 2008).

Due to similarities in biochemical parameters and structure, the enzyme 3’-NT/NU was classified as a member of the class I nuclease family (Neubert and Gottlieb, 1990). Sequence analyses of the gene encoding 3’-NT/NU in L. donovani (Debrabant et al., 1995; Debrabant et al., 2000), Leishmania mexicana (Sopwith et al., 2002), Leishmania major (Lakhal-Naouar et al., 2008), and Leishmania amazonensis (Paletta-Silva et al., 2011) have confirmed the presence of the five highly conserved regions associated with the class I nuclease family. 3’-NT/NU is a unique class I nuclease characterized as a cell surface membrane-anchored protein (Debrabant et al., 1995). In accordance with its previously observed ectoactivity, analysis of functional domains has shown the presence of an N-terminal signal peptide that targets the enzyme to the endoplasmic reticulum and a C-terminal transmembrane domain that anchors the enzyme to the parasite surface (Debrabant et al., 2000; Yamage et al., 2000).

In terms of biochemical analyses, the 3’-nucleotidase activity of 3’-NT/NU has been vastly studied, with reports in several species, including L. donovani (Gottlieb and Dwyer, 1983), L. mexicana (Hassan and Coombs, 1987), L. major (Lakhal-Naouar et al., 2008), Leishmania chagasi (Vieira et al., 2011), and L. amazonensis (Paletta-Silva et al., 2011). However, few studies regarding the nuclease activity of 3’-NT/NU, which is referred to as ecto-nuclease activity, are available. The first study to perform full biochemical characterization of ectonuclease activity in Leishmania parasites was recently carried out with L. amazonensis (Freitas-Mesquita et al., 2019). The biochemical parameters were determined by evaluating the hydrolysis of extracellular nucleic acids using the purified recombinant enzyme and living promastigotes. This activity was shown to be more efficient at alkaline pH values, as previously observed for the ecto-3’-nucleotidase activities of L. donovani (Gottlieb and Dwyer, 1983), L. mexicana (Hassan and Coombs, 1987), and L. amazonensis (Paletta-Silva et al., 2011). RNA, DNA, and different polyribonucleotides were efficiently hydrolyzed by L. amazonensis, which showed a preference for RNA, Poly-U, and Poly-A, which is consistent with the substrate specificity previously determined in L. donovani (Campbell et al., 1991).

Trypanosomatids do not express the enzymes responsible for de novo synthesis of purines; therefore, they are strictly dependent on host sources. As nucleotides and nucleic acids cannot be transported across the plasma membrane, their sequential hydrolysis to nucleosides constitute an important step in the purine acquisition process (Hammond and Gutteridge, 1984; Gottlieb, 1989). Through its nucleotidase activity, 3’-NT/NU can generate extracellular nucleosides through dephosphorylation of 3’-monophosphorylated nucleotides. Moreover, hydrolysis of extracellular nucleic acids generates 5’-monophosphorylated nucleotides that can be converted to nucleosides by the action of ecto 5’-nucleotidase, another ectoenzyme present in the plasma membranes of several trypanosomatids (Gottlieb, 1989), as summarized in Figure 1A.

Studies with L. donovani and L. amazonensis have shown that the expression and activity of 3’-NT/NU are stimulated when parasites face purine deprivation during growth, corroborating their involvement in the purine salvage pathway (Gottlieb, 1985; Freitas-Mesquita et al., 2019). The starvation of inorganic phosphate (P_i), which is one of the products of the reactions catalyzed by 3’-NT/NU, can also positively modulate ecto-3’-nucleotidase activity, as observed in L. donovani (Sacci et al., 1990) and L. chagasi (Vieira et al., 2011).

The role of 3’-NT/NU in the significant generation of extracellular adenosine, which is promoted by 3’-AMP hydrolysis or even by the sequential hydrolysis of nucleic acids to nucleosides, is also important from an immunological perspective. A adenosine can interact with purinergic receptors, triggering the release of anti-inflammatory cytokines and impairing the production of proinflammatory cytokines, thus favoring the establishment of parasite infection, as shown in Figure 1A (Paletta-Silva and Meyer-Fernandes, 2012; Freitas-Mesquita and Meyer-Fernandes, 2014; Freitas-Mesquita and Meyer-Fernandes, 2017).

Parasite and macrophage in vitro interaction assays have shown that the addition of 3’-AMP to the coculture medium promotes an increase in association indices, as observed in L. chagasi and L. amazonensis. The positive modulation promoted by 3’-AMP was equivalent to that observed with the same concentration of adenosine (Paletta-Silva et al., 2011; Vieira et al., 2011). Moreover, when 3’-AMP was added together with 3’-NT/NU inhibitors, such as tetrathiomolybdate (TTM) and guanosine 5’-monophosphate (5’-GMP), the stimulatory effect was completely reverted (Paletta-Silva et al., 2011; Freitas-Mesquita et al., 2016). Taken together, these results confirm that the modulation exerted by 3’-AMP is related to its conversion to adenosine by the action of 3’-NT/NU.

Although ectonuclease activity may indirectly contribute to adenosine generation, its major biological role seems to be related to parasite escape from NETosis (Figure 1A). An increase in the survival rate upon interaction with neutrophils was correlated with deprivation of P_i and purines during the growth of L. infantum (Guimarães-Costa et al., 2014) and L. amazonensis (Freitas-Mesquita et al., 2019), respectively. As previously reported, 3’-NT/NU activity is sensitive to P_i and purine contents in culture medium and is increased when parasites are starved of these nutrients (Gottlieb, 1985; Sacci et al., 1990; Vieira et al., 2011). Furthermore, pretreatment of L. infantum parasites with TTM and 5’GMP, two 3’-NT/NU inhibitors, resulted in a decrease in NET degradation (Guimarães-Costa et al., 2014). These correlations strongly suggested the participation of 3’-NU/NU in NET hydrolysis, which was confirmed by the results obtained with the purified recombinant protein. The addition of r3’-NT/NU increased parasite survival after coculture with neutrophils and after incubation with NET-enriched supernatant. The effect promoted by r3’-NT/NU was similar to that obtained by DNase, which is known to be able to destroy NETs (Freitas-Mesquita et al., 2019).

Several different drugs are available for the treatment of leishmaniasis, but pentavalent antimony-containing compounds remain to be used as the standard treatment, mainly in Latin America (Herwaldt and Berman, 1992; Berman, 1997; Ashutosh et al., 2007). However, an increase in therapeutic failure has been noted in the past few years due to the emergence of resistant parasites (Croft and Olliaro, 2011). In this context, the development of a vaccine against leishmaniasis is an extremely important aspect for the control of this disease, which affects millions of people worldwide. Although no effective vaccines against human leishmaniasis are currently available, hundreds of potential candidates are being studied (Singh and Sundar, 2012), including a class I nuclease selectively expressed by amastigotes of different Leishmania species (Kar et al., 2000; Campbell et al., 2003; Fakhraee et al., 2016).

Previous studies have shown that inoculation of living promastigotes is effective for preventing Old World cutaneous leishmaniasis (CL) (Greenblatt, 1980; Modabber, 1995). However, vaccination with virulent parasites is currently considered ethically unacceptable due to adverse reactions in susceptible individuals (Stober et al., 2006). First-generation vaccines have shown low efficacy since they are based on the use of killed parasites that do not mimic natural infection and are less immunogenic (Dunning, 2009; Fakhraee et al., 2016). On the other hand, second-generation vaccines induce protection using parasite antigens that may be obtained from native fractions purified from parasites (Rachamim and Jaffe, 1993; Borja-Cabrera et al., 2009; Moreno et al., 2014) or from recombinant bacteria or viruses carrying Leishmania antigen genes (De Oliveira et al., 2000; Smooker et al., 2004; Palatnik-de-Sousa et al., 2008; Miura et al., 2015). As amastigotes represent the parasite stage responsible for the pathology associated with leishmaniasis, amastigote-specific antigens are of particular interest for the development of an efficient vaccine (Kar et al., 2000; Farajnia et al., 2004). One of the first studies to investigate stage-specific purified antigens reported that three antigens selectively expressed amastigotes (P2, P4, and P8) to confer partial to complete protection against infection with Leishmania pifanoi and L. amazonensis in BALB/c mice (Soong et al., 1995).

To obtain more information on this potential prophylactic target, the gene encoding the P4 antigen of L. pifanoi was cloned and sequenced (Kar et al., 2000). Comparative analyses using DNA-derived protein sequences have revealed significant levels of identity with the 3’-NT/NU of L. donovani (33.7%) and the P1 zinc-dependent nuclease of Penicillium citrinum (20.8%), suggesting that the P4 antigen possesses nuclease activity (Kar et al., 2000). This biological property was further confirmed by biochemical assays. Using different substrates to measure the enzymatic activity of affinity-purified L. pifanoi P4, the protein was observed to display endo- and exonuclease activity and can hydrolyze single-stranded DNA and RNA. 3’-monophosphorylated nucleotides are also substrates for P4 nuclease, revealing the presence of phosphomonoesterase activity (Kar et al., 2000).

Despite the level of homology and the similarities in substrate specificity, P4 nuclease differs from 3’-NT/NU in several aspects (Gottlieb and Dwyer, 1981; Gottlieb and Dwyer, 1983; Debrabant et al., 1995; Kar et al., 2000). While 3’-NT/NU is an external surface membrane protein, P4 nuclease appears to have a perinuclear location. Immunofluorescence analyses showed that P4 protein colocalizes with a binding protein (BiP), a marker of the endoplasmic reticulum (Bangs et al., 1996). Notably, nuclease activities associated with the endoplasmic reticulum are involved in mRNA stability (Bandyopadhyay et al., 1990; Ross, 1996). In this context, P4 nuclease can be speculated to play a role in gene regulation and expression. Due to its endonuclease activity, the P4 protein may also be involved in nucleotide excision and repair. This property may be crucial for parasite survival in the mammalian host once inside the phagolysosome of a macrophage. Leishmania parasites are constantly subjected to the oxidative burst that promotes DNA damage (Kar et al., 2000). These possible biological roles are schematized in Figure 1B.

In addition to localization, the most remarkable difference between 3’-NT/NU and P4 nuclease is their differential expression during the parasite lifecycle. The expression of 3’-NT/NU and P4 nuclease is virtually restricted to the promastigote and amastigote stages, respectively. Northern blot analyses using RNA purified from L. pifanoi and L. amazonensis have confirmed previous reports that P4 protein is exclusively expressed by amastigotes (Soong et al., 1995; Kar et al., 2000)

Southern blot analyses have shown that homologs of the P4 gene are present in other Leishmania species, including L. amazonensis, L. braziliensis, L. major and L. donovani (Kar et al., 2000). A novel class I nuclease was posteriorly identified in L. major (LmaC1N), presenting high similarity (87%) to the P4 nuclease of L. pifanoi (Farajnia et al., 2004). The gene encoding LmaC1N was cloned using primers specific for conserved regions of class I nucleases of trypanosomatids, and deduced sequence analyses confirmed the existence of all five conserved regions (Farajnia et al., 2004). Similar to P4 nuclease and in contrast to 3’-NT/NU, LmaC1N was selectively expressed in amastigotes rather than promastigotes, as observed by RT–PCR and Western blotting assays (Farajnia et al., 2004).

Posteriorly, a homolog of P4 nuclease was identified in L. infantum, the causative agent of visceral leishmaniasis. The P4 nuclease gene was cloned, sequenced, and heterologously expressed for further characterization (Farajnia et al., 2011). Comparative sequence analyses have shown high homology to the P4 nucleases of L. donovani, L. major and L. pifanoi. The alignment results confirmed the existence of the five conserved domains of class I nucleases (Farajnia et al., 2011). Western blot analyses have shown that the P4 nuclease of L. infantum is expressed in both promastigotes and amastigotes. However, in accordance with previous studies involving this enzyme, its expression is significantly higher in amastigote parasites (Farajnia et al., 2011).

The high conservation among several Leishmania species, as well as the extensive expression found in the amastigote stage, renders P4 nuclease an excellent target for the development of a pan-Leishmania vaccine (Kar et al., 2000; Coler and Reed, 2005; Farajnia et al., 2011; Fakhraee et al., 2016). The first observations in this area showed that intraperitoneal injections of P4 antigen administered with Corynebacterium parvum as an adjuvant provided significant protection to BALB/c mice challenged with L. pifanoi promastigotes (Soong et al., 1995). The immunized mice developed smaller or no lesions, showing a significant parasite burden reduction after two weeks of infection. An increase in the levels of interferon gamma (IFN-γ) production was observed when immunized mice were stimulated with parasite antigens, suggesting that the resistance induced by P4 antigen is associated with a T helper 1 (Th1) cell-mediated immune response (Soong et al., 1995). Posteriorly, CD4⁺ T cells of P4-vaccinated mice were observed to produce not only IFN-γ but also macrophage migration inhibitory factor (MIF) and tumor necrosis factor/lymphotoxin (TNF/LT), leading to intracellular parasite destruction in vitro (Kar 2005).

A few years later, a DNA-based vaccine was tested using the L. amazonensis gene encoding the P4 nuclease associated with adjuvant constructs encoding murine interleukin-12 (IL-12) and L. amazonensis heat-shock protein 70 (HSP70) (Campbell et al., 2003). Both IL-12 and HSP70 have been reported to elicit Th1-type responses (Mattner et al., 1996; Wang et al., 2002). P4/IL-12-immunized BALB/c mice developed potent immune protection against L. amazonensis but remained susceptible to L. major infection. On the other hand, the P4/HSP70 vaccine only delayed the emergence of lesions in L. amazonensis-infected mice but was highly efficient against L. major, leading to a self-healing phenotype in infected mice (Campbell et al., 2003). To develop a DNA vaccine that promotes cross-protection against different Leishmania species, determining the optimal combination of several parasite genes and an appropriate adjuvant is crucial. Based on these results, P4 and HSP70 appear to be promising candidates for the development of a DNA-based vaccine for both New and Old World Leishmania species (Campbell et al., 2003).

Other studies in this field were performed based on LmaC1N protein, the P4 nuclease homolog from L. major. To evaluate LmaC1N as a potential human vaccine candidate, cellular immune responses to recombinant LmaC1N (rLmaC1N) were examined in individuals who had recovered from Old World cutaneous leishmaniasis (Farajnia et al., 2005). In addition to being recognized in 90% of the individuals tested, rLmaC1N was shown to elicit strong Th1-like responses characterized by high levels of IFN-γ, low levels of IL-10, and minimal IL-5 production (Farajnia et al., 2005). Later, the first report using animal models showed that the use of liposome-polycation-DNA (LPD) as an immunoadjuvant renders rLmaC1N an appropriate candidate for developing a suitable vaccine against leishmaniasis (Fakhraee et al., 2016). BALB/c mice vaccinated with rLmaC1N plus LPD nanoparticles showed delayed emergence of skin lesions as well as a delay in the spread of L. major from the inoculation site to the spleen. The statistically significant advantageous effects observed for rLmaC1N’s association with LPD nanoparticles compared to the effects obtained without coadministration of this adjuvant demonstrate the importance of selecting an efficient antigen delivery system for better protection (Fakhraee et al., 2016).

Beyond the membrane-bound 3’-NT/NU and the intracellular P4 nuclease, a secretory class I nuclease has also been identified and shown to be conserved in different geographic isolates of L. donovani and L. infantum (Joshi and Dwyer, 2007; Joshi et al., 2012). First, L. donovani promastigotes were observed to constitutively synthesize and release this nuclease, named LdNuc^s, into their growth medium. Then, this activity was identified in axenic amastigotes as well as in vivo-derived amastigotes isolated from infected hamster spleen tissue (Joshi and Dwyer, 2007). Analyses of the LdNuc^s-derived protein have confirmed the presence of the five conserved domains of class I nucleases (Joshi and Dwyer, 2007). Zymogram gels of cell lysates and culture supernatants showed marked differences between 3’-NT/NU and LdNuc^s activities. While 3’-NT/NU is ~43 kDa and insensitive to dithiothreitol (DTT) inhibition, LdNuc^s is a 35-kDa, DTT-sensitive nuclease. As previously described, 3’-NT/NU is a membrane-bound enzyme; therefore, its activity was detected only in cell lysates. On the other hand, LdNuc^s activity was detected in both cell lysates and concentrated cell-free culture supernatants (Joshi and Dwyer, 2007).

Coupled immunoprecipitation-enzymatic assays using epitope-tagged recombinant enzyme were performed to analyze other biochemical properties of LdNuc^s activity. The enzyme was capable of hydrolyzing RNA, single- and double-stranded DNA and a variety of synthetic polynucleotides. LdNuc^s appears to have a broad pH tolerance since nucleic acid hydrolysis occurred under both acidic (pH 5.0) and alkaline conditions (pH 8.5), suggesting that LdNuc^s may be completely functional within the different microenvironments faced by parasites during their lifecycle (Joshi and Dwyer, 2007).

Sera collected from infected visceral leishmaniasis patients from different geographic locations were able to recognize LdNuc^s, which clearly indicates that the enzyme is synthesized and expressed by in vivo amastigotes during the course of human infection (Joshi et al., 2012). The biological role played by LdNuc^s remains to be completely elucidated; however, the enzyme was postulated to participate in the purine salvage pathway. As a secreted protein, LdNuc^s can function far away from the parasite in the mobilization of host-derived nucleic acids. Together with 3’-NT/NU and other enzymes involved in purine salvage, LdNuc^s may contribute to the availability of extracellular nucleosides and nucleobases that can be uptaken by the parasite (Joshi and Dwyer, 2007) (Figure 1B).