The therapeutic effect of PTP inhibitor treatments was in line with observations made during in vivo treatment of L. donovani-infected mice with okadaic acid, a STP PP2A inhibitor (see above). The comparative analysis of both inhibitors in macrophages revealed that PP2A synergistically inhibited the activity of the MAPK ERK1/2 together with a phosphatase belonging to the PTP family, the DUSP6, also known as MAP kinase phosphatase (MKP) 3 (60). The inhibition of MAPKs downstream of PKCζ following Leishmania infection led to a decreased iNOS expression (60). In the same study, the role of DUSP1, also termed MKP1, was analyzed. Activation of DUSP1 downstream of PKCε resulted in the inhibition of p38 MAPK (60). A host-damaging role for these two DUSPs during visceral leishmaniasis was also demonstrated in L. donovani-infected BALB/c mice treated with 18β-glycyrrhetinic acid (GRA), a compound derived from the medicinal plant licorice (Glycyrrhizza glabra). Treatment with GRA caused a shift of the immune response from a detrimental Th2 response to a protective Th1 response (61). At the molecular level, the shift was explained by a decreased DUSP1 and DUSP6 expression and the concomitant activation of mitogen- and stress-activated protein kinase 1 (MSK1), which ultimately led to the production of leishmanicidal NO (62).

Findings with L. major-infected macrophages, however, indicated that the activity of DUSP6 is not always detrimental for the outcome of Leishmania infections. In L. major-infected macrophages activated by anti-CD40, siRNA-mediated inhibition of DUSP6 led to ERK activation, enhanced production of IL-10, and reduced expression of IL-12 and iNOS, whereas inhibition of DUSP1 caused p38 activation and increased generation of IL-12 and iNOS. Consequently, in vivo overexpression of DUSP6 or pharmacological inhibition of DUSP1 both ameliorated the course a cutaneous L. major infection (63). This functional dichotomy between DUSP6 and DUSP1 has also been reported in the context of macrophage coinfections with the immunomodulatory bacterium Mycobacterium indicus pranii and L. donovani. The protective effect of this bacterial infection relied on a toll-like receptor (TLR) 4-dependent increase of DUSP6 and simultaneous decrease of DUSP1. Whereas DUSP1 inhibition led to the activation of p38, the production of NO and the release of IL-12, the activation of DUSP6 blocked the ERK1/2 pathway and the expression of IL-10 and arginase-1 (Arg1). Overall, M. indicus pranii conferred protection against visceral leishmaniasis by reciprocal regulation of DUSP1 and DUSP6 and a shift toward a Th1 response (64).

A phenotype similar to the one of DUSP6 was also observed for DUSP4 (syn. MKP2). Mice deficient for the dusp4 gene were more susceptible to a cutaneous infection with L. mexicana than wild type mice. This was due to an enhanced JNK and p38 phosphorylation in macrophages that increased the expression of Arg1 and was associated with a disease-promoting Th2 immune response (65, 66).

In macrophages infected with L. donovani, a PTP activity identified as Src homology region 2 domain-containing phosphatase-1 (SHP-1; also known as protein tyrosine phosphatase non-receptor type 6 [PTPN6]) was rapidly activated and induced hypo-responsiveness to IFN-γ by directly interacting with the kinase JAK2 recruited to the IFN-γ receptor (31, 43, 44). The confirmation that SHP-1 played a key role in the pathogenicity of leishmaniasis came from infection experiments with viable motheaten (me^v) mice that are deficient for this phosphatase (67). Me^v mice were largely protected against L. major infection, most likely because of an increased NO production by their phagocytes (68) and an enhanced secretion of proinflammatory cytokines like TNF, IL-1β, and IL-6 (69). Another group, however, did not observe an impact of the SHP-1 deficiency in me^v mice on the course of L. major-infection and questioned a selective role of SHP-1 in macrophages as SHP-1 is also regulating T and B cell responses (70). It is also important to point out that even in the absence of SHP-1 activity different species of Leishmania (L. major, L. braziliensis, L. mexicana, and L. donovani) caused degradation and reduced nuclear translocation of STAT1 in primary mouse macrophages (71). This finding indicates that at least part of the suppression of the JAK2/STAT1 pathway in Leishmania-infected macrophages and the subsequent reduced release of cytokines and NO in response to IFN-γ is SHP-1 independent.

Infection of SHP-1-deficient macrophages also revealed the importance of this phosphatase for the downregulation of MAPK activity after IFN-γ (31, 44). Control of MAPK by SHP-1 was proposed to be partially responsible for the inhibition of iNOS during IFN-γ stimulation (44, 72). However, involvement of individual MAPK seemed to differ depending on the stimuli used to trigger iNOS expression during Leishmania infection. Although SHP-1 negatively regulated ERK1/2 and JNK after IFN-γ stimulation (72), this phosphatase boosted the activity of ERK1/2 after CD40 stimulation, which promoted the secretion of IL-10 and simultaneously inhibited p38 MAPK activation (73).

During Leishmania infection, SHP-1 did not only alter the macrophage responses to proinflammatory stimuli originating from T cell such as IFN-γ and CD40 ligand. Signaling pathways triggered by pathogen recognition receptors (PRRs), e.g., TLR4, were also disturbed. In mouse bone marrow-derived macrophages, infection with different Leishmania species (L. major, L. mexicana, L. donovani) caused rapid activation of SHP-1, which bound to a kinase tyrosyl-based inhibitory motif (KTIM) of the interleukin 1 receptor-associated kinase 1 (IRAK-1) and thereby blocked the intrinsic kinase activity of IRAK-1. The inactivation of IRAK-1 by SHP-1 was associated with the inability of IRAK-1 to detach from the TLR4 adaptor MyD88 and to attach to the downstream signal transducer TRAF6 so that the LPS-induced macrophage responses (e.g., release of NO and TNF) were severely impaired (74). According to the authors of this study KTIMs are also present in other targets of SHP-1, such as ERK1/2, p38, and JNK, and therefore offer a molecular explanation for the multiple effects of SHP-1 (74). A SHP-1 mediated hypo-responsiveness to LPS was also observed in human macrophages infected by L. donovani (75).

Two recent studies demonstrated that SHP-1 also modulates the signaling downstream of PRRs of the lectin receptor family during leishmaniasis. C-type lectin receptor Mincle expressed by dendritic cells recognized a still unknown ligand present on the surface of L. major. Ligation of the Mincle receptor by L. major impaired the activation of dendritic cells due to recruitment of SHP-1, which converted the canonical immunoreceptor tyrosine-based activation motif (ITAM) of the FcRγ-chain signaling component of the receptor into an inhibitory motif (ITAMi). In accordance with these in vitro findings Mincle-deficient mice developed less severe skin lesion after infection with L. major (76). Likewise, I-type lectin receptors Siglec-1 and -5 expressed by macrophages were described to recognize sialylated ligands on the surface of L. donovani parasites. Engagement of Siglec-1 facilitated the uptake of the parasites by the macrophages, whereas ligation of Siglec-5 led to a reduced production of leishmanicidal ROS and NO accompanied by a shift toward a Th2 response. The latter resulted from the recruitment of SHP-1 to the Siglec-5 receptor promoting here again the deactivation of the immune cell (77).

Together, these data illustrate that phosphatases are critical regulators of the immune response against Leishmania parasites. Parasite-driven activation of PTPs, notably SHP-1, clearly impairs the effector function of macrophages [reviewed in Ref. (78)]. On the other hand, host cells have developed strategies to counteract the evasion mechanisms of Leishmania parasites. As an example, natural resistance-associated macrophage protein 1 (NRAMP-1) inhibited the function of PTPs, either via a direct interaction between PTPs and the metal cation (Fe²⁺) transported by NRAMP-1 or by oxidation of the phosphatases resulting from NRAMP-1/iron-dependent ROS-formation (79). However, in the arm race between host and parasite, Leishmania parasites were shown to again antagonize this ROS-mediated inhibition of host PTPs in macrophages by inducing the expression of the uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS generation. This led to lower ROS production resulting in an enhanced host PTP activity (80).

Leishmania major, the model species used for this review, encodes 58 genes for serine/threonine phosphatases (STP) which are members of the three different groups of enzymes within this family: the PPP group (30 genes), the PPMs (15 genes), and the aspartate-based phosphatases (FCP/SCP; 13 genes) (see Table 1). These enzymes are highly conserved in the different Leishmania species (Table S1 in Supplementary Material).

Within the PPP-group, the eight Leishmania phosphatases that belong to the PP1 subgroup have not yet been biochemically and/or functionally characterized. Surprisingly, five of them are encoded by genes organized in tandem on chromosome 34. This organization suggests a common regulation of the expression of the different isoforms. PP2A phosphatases are also not yet fully characterized, but their increased expression has been linked to resistance against antibiotics such as paromomycin (111). Additional information on their functions in Leishmania parasites could be derived from studies of orthologs in other kinetoplastid. PP1 and PP2A phosphatases of Trypanosoma parasites participate in amastigote transformation (110) and, interestingly, share the highest homology with Leishmania orthologs (Table S1 in Supplementary Material).

Regarding the calcineurin-like phosphatase subgroup (PP2B), a calcium- and calmodulin-dependent phosphatase activity was purified from L. donovani parasites but their respective genes have not yet been sequenced (124). In L. major two genes, LmjF26.2530 and LmjF36.1980, might account for this activity (Table 1) as they encode proteins carrying a calcineurin A catalytic domain and the three canonical regulatory domains: the calcineurin B (CnB) binding domain, the calmodulin (CaM) binding domain and an autoinhibitory domain (possibly missing in LmjF36.1980) (125). Noteworthy, the ortholog found in T. cruzi does not contain the last two domains which likely causes a substantial functional difference (125). Interestingly, Leishmania CnB (LmjF.21.1630), the regulatory subunit of calcineurin A, was shown to control the temperature adaptation of L. major. Promastigotes deficient for CnB failed to differentiate into amastigotes and were completely avirulent in macrophages and during in vivo infection (126).

The PP7-like phosphatase, encoded by the gene LmjF.12.0660, showed a strong homology to protein phosphatases with a E-helix/F-helix (EF)-hand (PPEF) that were identified in sensory cells of higher eukaryotes. However, LmPPEF did not bind calcium because its EF-hand domains appeared degenerated and the functionality of its N-terminal calmodulin binding domain was not ascertained (113). Due to a N-myristoylation modification, LmPPEF appears to be membrane-bound and localized in the vicinity of the kinetoplast and the flagellar pocket (113), an active secretory site, which might explain its detection in the exosome content of promastigotes (83, 92).

PP5 phosphatases showed some similarities with PP7 proteins, but instead of calcium-dependent regulatory domains they carry N-terminal tricopeptide repeat (TPR) domains, which can mediate protein–protein interaction and autoinhibition. The PP5 phosphatase encoded by L. major (LmjF18.0150) has been biochemically characterized. Mutation analysis of the recombinant protein confirmed its phosphatase activity and the autoinhibitory role of the TPR domains (112). Functional data on this protein are scarce but suggested its involvement in stress response as also seen with the T. brucei homolog (110). Expression of the unique PP5 gene in L. infantum correlated with increased resistance to antimony (114).

Regarding the eukaryotic-like PP6 phosphatases or the eukaryotic phosphatases homologous to the bacterial enzyme diadenosine tetraphosphate hydrolase ApaH (ApaH-like phosphatases [ALPHs]), only few data exist on their biochemical or physiological role. In Trypanosoma brucei, ALPH1 was shown to have all features of an mRNA decapping enzyme (127).

The last subgroup of the PPPs comprises kinetoplastid-specific proteins (kPPP) that carry a mutation in their catalytic site. These pseudo-phosphatases (kSTP pseudo) show some similarities to plant and fungal PPPs. They likely add an extra level of regulation to the kinase/phosphatase activities by stably binding to phosphorylated substrates and shielding them against other phosphatase activities (110). These adaptations to numerous substrates combined with the absent catalytic activity might explain the higher divergence observed between kPPP from Leishmania and Trypanosoma (Table S1 in Supplementary Material). Expression of such pseudo-enzyme, encoded by the gene LinJ.34.2610, has been negatively correlated with the resistance of L. infantum to antimony treatment (115).

Regarding the 15 phosphatases of the PPM (or PP2C) group, their biological functions have not yet been unraveled, but their dependency on divalent manganese or magnesium cations has been validated in L. donovani and L. infantum/L. chagasi promastigotes (116, 128). In case of L. infantum/chagasi, a 42 kDa PP2C-like phosphatase named LcPP2C, encoded by the gene LinJ.25.0780, was biochemically characterized in promastigotes and amastigotes (116). Interestingly, the ortholog of this protein was secreted by L. mexicana along with 71 other proteins detected (84, 85). However, whether this enzyme contributes to the modulation of the host cell signaling (e.g., via activation of the host phosphatases SHP-1 and PTP-1B) by the L. mexicana secretome, was not investigated (85). Other PP2C-like proteins have been detected in L. mexicana promastigotes but they still lack functional characterization (129). Moreover, expression of two additional PP2C genes, LinJ.34.2310 and LinJ.34.2320, was recently linked to methotrexate resistance of L. infantum (114).

Altogether, only 6 of the 58 STP genes have been partly characterized despite the suggested crucial role in amastigote transformation (Table 1). More research on this phosphatase family could therefore open new avenues toward the development of alternative therapeutic approaches.

In additions to STPs, 31 putative protein tyrosine phosphatases (PTPs) are encoded in the L. major genome (Table 2). All of them share a common catalytic site surrounded by a cysteine and an arginine (CX₅R). They are well conserved among Leishmania species and are divided into four classes (Table S1 in Supplementary Material). Class I PTPs, which form the largest group, contain enzymes with either proven PTP activity or a structural organization characteristic for PTPs, whereas classes II and III, represented by a single member each, might not convey such activities. Class IV enzymes were not detected in the genome of Leishmania parasites.

Proteins of the class II PTPs are homologous to low-molecular-weight (LMW) PTPs found in human or bacteria. However, the protein encoded by the gene LmjF.01.0200 carries a mutated catalytic cysteine as revealed during our own sequence analyses, suggesting that it is probably inactive and might therefore act, similar to the kPPPs, as substrate trapping molecule. Another degenerated LMW PTP-like enzyme is encoded by the gene LTRL590_050007600.1, whose expression correlated to antimony resistance in L. tropica field isolates (130).

Regarding the class III PTPs, the enzyme named LmACR2 has been initially characterized in L. major as an arsenate (As)/antimony (Sb) reductase despite containing a canonical PTP catalytic site. The antimony reductase activity of LmACR2 converted Sb(V), present in Sb-containing antileishmanial drugs (e.g., sodium stibogluconate), into Sb(III) which is the active microbicidal molecule. Overexpression of LmACR2 in promastigotes supported this observation as parasites became more sensitive to antimonials (120). However, metalloids like Sb are not commonly encountered by Leishmania parasites in nature. Therefore, the evolutionary pressure to develop such activity should have been low. Thus, the concomitant PTP activity of LmACR2 detected in vitro might reflect the true physiological function of this enzyme (121, 122).

Most of the PTPs expressed in Leishmania parasites belong to the class I PTPs that can be further divided into two subgroups of enzymes. The first subgroup consists of classical PTPs homologous to human PTP1B. However, in Leishmania and other kinetoplastid parasites, this group does not contain any receptor-PTP usually found in higher eukaryotes. Among the three non-receptor-PTPs encoded by Leishmania parasites, only the eukaryotic-like LdPTP1 was functionally characterized. The deletion of the gene encoding for this enzyme, LdBPK_365610.1, resulted in complete loss of amastigote transformation during infection of macrophages with L. donovani promastigotes and in a dramatic decrease of the parasite virulence in vivo (117). The molecular mechanism underlying this drastic phenotype has not yet been elucidated. However, the study of the TbPTP1 ortholog in Trypanosoma brucei revealed that this family of phosphatase could control the integration of environmental differentiation signal (131, 132).

The second subgroup of class I PTPs comprises dual specificity phosphatases (DUSP) (Table 2). These phosphatases are characterized by a substrate spectrum that extends beyond phosphotyrosine residue (e.g., dephosphorylation of serine and threonine residues). Some of these DUSPs are even described as lipid phosphatases: (a)

Myotubularins (MTMs) were shown to target phosphoinositides like PI(3)P or PI(3,5)P₂ in mammals, and therefore control endocytosis and membrane trafficking (133). In Leishmania parasites, the MTM group consists of only two genes (instead of the 16 encoded by the human genome making it the largest group of human DUSP). Leishmania MTMs are characterized by their very large size with up to 3,245 amino acid (as in the case of LmjF.12.0320), which is due to long N-terminal extensions. However, their function has not been yet studied in trypanosomatids.

In addition to these phospholipid phosphatases, DUSPs dephosphorylating proteins on tyrosine, serine or threonine residues were also identified in the Leishmania genomes: (a)

Three Leishmania genes encode eukaryotic-like DUSPs (eDUSPs). These include a homolog of the human CDC14 phosphatase that was described to control mitosis by targeting cyclin dependent kinase (136). However, this crucial function has not been yet documented in trypanosomatids. The two genes homologous to phosphatase of the regenerating liver (PRL) will be discussed later in the context of their importance for the virulence of L. major.

Although the large majority of the Leishmania DUSPs have so far not been functionally studied in Leishmania, it is interesting to note that two of them (LmjF.16.0230 and LmjF34.2190) were secreted in exosomes by Leishmania parasites (83, 92) (Table 2). These two DUSPs along with the six STPs also detected in exosomes (i.e., LmPPEF, LcPP2C, two PP1, and two kSTPs) represent a small pool of Leishmania phosphatases that might directly interact with phosphorylation cascades inside host cells during infection.

All MAcPs share the same structural organization starting with a signal peptide at the N-terminus followed by the catalytic domain containing the histidine residues and a transmembrane domain at the C-terminus (Figure 4B). Phylogenetically, MAcPs are organized in four distinct groups (Figure 4A, groups I–IV), all represented by one MAcP gene sequence derived from each of the six analyzed Leishmania genomes. Interestingly, an additional MAcP gene, LinJ.36.6740, could be detected in the genome of L. infantum. Despite being more related to SAcP and being substantially shorter than all other MAcP (only 35 kDa instead of usually around 60 kDa), this phosphatase harbors a signal peptide and a putative transmembrane domain at its end, illustrating the genetic plasticity of Leishmania parasites in which diverging paralogous genes are common. Gene divergence was also seen in the MAcP gene of L. donovani. The MAcP protein of the L. donovani strain MHOM/SD/62/1S-CL2D (148) carried a transmembrane domain, which was found to be replaced by a not-functionally defined C terminal sequence of 91 amino acids in the MAcP gene of the L. donovani strain BPK282A1 (149). Comparing the published sequences, the variation between these Leishmania strains was not related to frameshifts or aberrant stop codons but might result from a more complex evolutionary process.

Numerous studies aimed to characterize the function of MAcPs as they represented the first enzymatic activities identified on the surface of Leishmania promastigotes (150). Having been transported via the classical intracellular secretion pathway, it was an expected finding that the MAcPs were phospho-glycosylated (151). Interestingly, expression of MAcP varied during the different life stages of Leishmania parasites. In L. major, MAcP expression increased during metacyclogenesis of promastigotes and correlated with the translocation of the protein from the cytoplasm to the membrane (152). Increased expression of some of the MAcPs was also detected during metacyclogenesis and differentiation into amastigotes following macrophage infection (153, 154). Most recently, MAcPs were found to be differentially expressed in clinical isolates of the same Leishmania species (155). A MAcP of L. tropica (LTRL590_280034100, annotated as LmjF.28.2650) was described as one of the 30 most diversely expressed genes in a collection of 14 clinical isolates of L. tropica from different geographic areas. This might indicate that the parasite adapts the level of extracellular phosphatase activity depending on the specific infection situation. Functionally, early reports suggested that these ectophosphatases altered the response of neutrophils (156). Partially purified MAcP activity from L. donovani specifically interfered with the production of ROS by neutrophils after stimulation with the tripeptide N-formylmethionine-leucyl-phenylalanine (fMLP), whereas activation by PMA was resistant to MAcP activity (157). Other authors proposed that a phosphoinositide phosphatase activity of MAcP might play a role in this inhibition, because the phagocyte NADPH oxidase (Phox) relies on these signaling molecules for its activation (139). In accordance with this hypothesis, overexpression of LdMAcP by L. donovani promastigotes resulted in an increased survival during in vitro infection of a mouse macrophage cell line (158). However, deletion of the MAcP gene of L. mexicana (LmxM.36.2570 [LmxMBAP]) raised doubts about an essential role of this class of phosphatases for the virulence of L. mexicana parasites; LmxMBAP-deficient parasites were as virulent as wild-type parasites during in vitro infection of macrophages or in vivo infection of susceptible BALB/c mice (159). However, a definitive statement on the function of MAcPs is still not possible because of potential compensatory mechanisms as Leishmania parasites carry a minimum of four MAcP genes.

Secreted acid phosphatases are among the most abundant proteins secreted by Leishmania parasites (137, 160). However, a few Leishmania species, such as L. major, were shown to express very little SAcP as promastigotes (161). This difference could be due to the number of genes coding for truly secreted phosphatases. Our in silico analysis of the three L. major histidine phosphatases devoid of transmembrane regions revealed that only one, LmjF.36.2470, might have a functional signal peptide (Table S2 in Supplementary Material), whereas other Leishmania species such as L. panamensis, L. braziliensis, L. mexicana, and L. donovani can express more than one intact SAcP. The phylogenetic analysis also allowed the classification of SAcPs in two different families that are substantially divergent. One group of phosphatases (Figure 4A, group VI) branched out early from the histidine phosphatase family but is highly conserved across species, while the other group (Figure 4A, group V) is more closely related to MAcPs but is also more diverse. In members of group V, a signal peptide is sometimes absent, the enzymes can be significantly shorter than other orthologs and truncation could occur in the catalytic domain. This is the case for the two putatively non-secreted phosphatases of L. major, LmjF.36.6460, and LmjF.36.6480, indicating that the function of these two genes probably differs from their orthologs in case they are expressed.

For parasites expressing large amounts of SAcP, such as L. donovani, soluble phosphatase activity could be detected all along their differentiation from promastigotes to amastigotes (162). Interestingly, SAcP activity was also found in the cytosol of macrophages after 24 h of infection (11). However, the exact molecular identity of the phosphatase was not determined and this family of enzymes was also not detected inside exosomes that represent the main transit route of Leishmania cargo toward host cell cytosol. Like MAcPs, SAcPs are phospho-glycosylated proteins (163, 164). This modification relies on the same family of enzymes responsible for the LPG biosynthesis as a deletion of lpg2 gene, but not lpg1, abolished the phospho-glycosylation of SAcPs in L. donovani (165). Immunoprecipitation of SAcP suggested that the glycosylation pattern might increase during the parasite differentiation into amastigotes (11, 162). In addition to this posttranslational modification, SAcPs can form filamentous polymers. This quaternary structure has been extensively described for L. mexicana, but, surprisingly, was not seen with SAcPs from L. donovani, which remained mono- or oligomeric and non-filamentous (166). In case of L. mexicana, the filament formation occurred in the flagellar pocket of promastigotes (167) to create homo- or hetero-polymers of two phosphatases, LmSAP1 and LmSAP2. The genes encoding these two SAcPs are tandemly arranged and nearly identical, only differing by the addition of a long stretch of serine-threonine repetitions after the catalytic domain of LmSAP2 (168). This stretch is the site of intense glycosylation but does not interfere with the formation of the filament backbone constructed around the catalytic domains of LmSAPs (169). Remarkably, the published sequences of LmSAPs derived from L. mexicana MNYC/BZ/62/M379 could not be fully retrieved from the L. mexicana MHOM/GT/2001/U1103 strain that was sequenced, illustrating again the high plasticity of Leishmania genomes. An ortholog of the LmSAPs, LmxM.36.6480, has even a higher number of serine-threonine repetitions and seems to lack a functional signal peptide. In general, our search of the different Leishmania genomes showed that all SAcPs share a similar architecture but the size of the serine/threonine rich domain varies. This probably reflects the need to adapt the level of phospho-glycosylation to the different environments required by each parasite species.

Comparative genetic analysis of two different isolates of L. major from patients with contrasting severity of disease indicated that the hypo-virulent strain carried a mutated version of SAcP (170), suggesting a role of SAcP in virulence. Interestingly, a similar study looking at the genetic differences between two strains of L. donovani isolated from patients with cutaneous or visceral leishmaniasis, respectively, did not report a link between Leishmania AcPs and virulence (171). The function of SAcP in the virulence Leishmania has been directly addressed with parasites lacking the genomic locus encoding the two LmSAPs. Deletion of this locus abolished the virulence of L. mexicana promastigotes as they failed to transform into amastigotes during macrophage infection (172). However, this phenotype was independent of the LmSAPs because phenotypic complementation was achieved by adding back the MAPK gene that was identified in the intergenic region between the two LmSAP genes (172).

Together, these results demonstrate that the exact contribution of MAcPs and SAcPs to maintain the life cycle of Leishmania parasites is still unknown. First, the lack of a phenotype in a Leishmania mutant deficient for an AcP does not exclude its functional relevance, because compensation between different genes of each group could occur. Second, MAcP could also compensate for SAcP deficiency and vice versa. Finally, a role for these phosphatases also needs to be considered during sand fly infection by promastigotes. To experimentally address these hypotheses, the nature of the AcP substrate needs to be defined. If, however, these enzymes are primarily responsible for providing sufficient inorganic phosphate to the parasite as it has been proposed by Freitas-Mesquita et al. (140), it will be difficult to dissect the functional role of the different classes of AcPs and/or to overcome compensatory mechanisms.