Influence of parasite encoded inhibitors of serine peptidases in early infection of macrophages with Leishmania major

Ecotin is a potent inhibitor of family S1A serine peptidases, enzymes lacking in the protozoan parasite Leishmania major . Nevertheless, L. major has three ecotin-like genes, termed inhibitor of serine peptidase (ISP). ISP1 is expressed in vector-borne procyclic and metacyclic promastigotes, whereas ISP2 is also expressed in the mammalian amastigote stage. Recombinant ISP2 inhibited neutrophil elastase, trypsin and chymotrypsin with K _is between 7.7 and 83 nM. L. major ISP2–ISP3 double null mutants (Δ isp 2/3) were created. These grew normally as promastigotes, but were internalized by macrophages more efficiently than wild-type parasites due to the upregulation of phagocytosis by a mechanism dependent on serine peptidase activity. Δ isp 2/3 promastigotes transformed to amastigotes, but failed to divide for 48 h. Intracellular multiplication of Δ isp 2/3 was similar to wild-type parasites when serine peptidase inhibitors were present, suggesting that defective intracellular growth results from the lack of serine peptidase inhibition during promastigote uptake. Δ isp 2/3 mutants were more infective than wild-type parasites to BALB/c mice at the early stages of infection, but became equivalent as the infection progressed. These data support the hypothesis that ISPs of L. major target host serine peptidases and influence the early stages of infection of the mammalian host.     

Leishmania inhibitor of serine peptidase 2 prevents TLR4 activation by neutrophil elastase promoting parasite survival in murine macrophages

Leishmania major is a protozoan parasite that causes skin ulcerations in cutaneous leishmaniasis. In the mammalian host, the parasite resides in professional phagocytes and has evolved to avoid killing by macrophages. We identified L. major genes encoding inhibitors of serine peptidases (ISPs), which are orthologs of bacterial ecotins, and found that ISP2 inhibits trypsin-fold S1A family peptidases. In this study, we show that L. major mutants deficient in ISP2 and ISP3 (Δisp2/3) trigger higher phagocytosis by macrophages through a combined action of the complement type 3 receptor, TLR4, and unregulated activity of neutrophil elastase (NE), leading to parasite killing. Whereas all three components are required to mediate enhanced parasite uptake, only TLR4 and NE are necessary to promote parasite killing postinfection. We found that the production of superoxide by macrophages in the absence of ISP2 is the main mechanism controlling the intracellular infection. Furthermore, we show that NE modulates macrophage infection in vivo, and that the lack of ISP leads to reduced parasite burdens at later stages of the infection. Our findings support the hypothesis that ISPs function to prevent the activation of TLR4 by NE during the Leishmania-macrophage interaction to promote parasite survival and growth.     

Role of the inhibitor of serine peptidase 2 (ISP2) of Trypanosoma brucei rhodesiense in parasite virulence and modulation of the inflammatory responses of the host

Trypanosoma brucei rhodesiense is one of the causative agents of Human African Trypanosomiasis (HAT), known as sleeping sickness. The parasite invades the central nervous system and causes severe encephalitis that is fatal if left untreated. We have previously identified ecotin-like inhibitors of serine peptidases, named ISPs, in trypanosomatid parasitic protozoa. Here, we investigated the role of ISP2 in bloodstream form T. b. rhodesiense. We generated gene-deficient mutants lacking ISP2 (Δisp2), which displayed a growth profile in vitro similar to that of wild-type (WT) parasites. C57BL/6 mice infected with Δisp2 displayed lower blood parasitemia, a delayed hind leg pathological phenotype and survived longer. The immune response was examined at two time-points that corresponded with two peaks of parasitemia. At 4 days, the spleens of Δisp2-infected mice had a greater percentage of NOS2⁺ myeloid cells, IFN-γ⁺-NK cells and increased TNF-α compared to those infected with WT and parasites re-expressing ISP2 (Δisp2:ISP2). By 13 days the increased NOS2⁺ population was sustained in Δisp2-infected mice, along with increased percentages of monocyte-derived dendritic cells, as well as CD19⁺ B lymphocytes, and CD8⁺ and CD4⁺ T lymphocytes. Taken together, these findings indicate that ISP2 contributes to T. b. rhodesiense virulence in mice and attenuates the inflammatory response during early infection.     

Hemoglobin receptor in Leishmania is a hexokinase located in the flagellar pocket

Hb endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket. To understand the nature of the Hb receptor (HbR), we have purified the 46-kDa protein to homogeneity from Leishmania promastigote membrane. Purified HbR specifically binds Hb. The gene for HbR was cloned, and sequence analysis of the full-length HbR gene indicates the presence of hexokinase (HK) signature sequences, ATP-binding domain, and PTS-II motif. Four lines of evidence indicate that HbR in Leishmania is a hexokinase: 1) the recombinant HbR binds Hb, and the Hb-binding domain resides in the N terminus of the protein; 2) recombinant proteins and cell lysate prepared from HbR-overexpressing Leishmania promastigotes show enhanced HK activity in comparison with untransfected cells; 3) immunolocalization studies using antibodies against the N-terminal fragment (Ld-HbR-DeltaC) of Ld-HbR indicate that this protein is located in the flagellar pocket of Leishmania; and 4) binding and uptake of (125)I-Hb by Leishmania is significantly inhibited by anti-Ld-HbR-DeltaC antibody and Ld-HbR-DeltaC, respectively. Taken together, these results indicate that HK present in the flagellar pocket of Leishmania is involved in Hb endocytosis.     

Calcineurin is required for Leishmania major stress response pathways and for virulence in the mammalian host

Leishmania parasites must adapt to elevated temperatures and other environmental stresses during infection of their mammalian hosts. How these environmental cues are sensed is poorly understood. In this study we show that calcium uptake is required for parasite thermotolerance at 34-37°C. To identify potential downstream targets of calcium influx, a Leishmania major mutant lacking the essential regulatory subunit (CnB) of the Ca(2+) /calmodulin-dependent serine/threonine-specific phosphatase, calcineurin, was generated. The Δcnb mutant grew as well as wild-type parasites at 27°C and differentiated normally to infective metacyclic promastigotes. However, Δcnb parasites lost viability when exposed to increased temperature (34°C) and were hypersensitive to endoplasmic reticulum and membrane stress, induced by tunicamycin and inhibitors of sterol and sphingolipid biosynthesis respectively. Δcnb promastigotes were internalized by macrophages, but their differentiation to the heat adapted amastigote stage was delayed and the resulting parasites failed to proliferate. Strikingly, the Δcnb parasites were completely cleared by susceptible BALB/c mice. Complementation of Δcnb parasites with CnB restored thermotolerance and infectivity in both macrophages and animal models. Our results suggest that Ca(2+) influx and calcineurin signalling are required for both early and long-term adaptive parasite responses to environmental stresses encountered in the mammalian host.     

Leishmania mexicana: comparative fine structure of amastigotes and promastigotes in vitro and in vivo

Amastigotes of Leishmania mexicana pifanoi were cultivated by serial transfers in cell-free medium UM-54 at 33 and 35 C. Electron microscopy was used to analyze the structural relationships among promastigotes, axenically cultured amastigotes, and amastigotes in footpads of infected hamsters. These studies revealed very close structural similarities between culture and hamster derived amastigotes. However, both of these amastigotes differed from the promastigotes in the following aspects. The flagellum of promastigotes contained a paraxial rod originating at the axosome level within the flagellar pocket, whereas the flagellum of amastigotes lacks this structure. The flagellar pocket of promastigotes was usually small whereas amastigotes had a distended reservoir. Subpellicular microtubules of promastigotes terminated at the posterior end, whereas those of amastigotes ended subterminally. Membrane bounded vesicles were present only in amastigotes. These results along with the biologic and antigenic comparisons indicate that amastigotes obtained from axenic cultures are related very closely to amastigotes from infected hamster footpads and that their relationship to promastigotes is far more distant.     

Interacting protein kinases involved in the regulation of flagellar length

A striking difference of the life stages of the protozoan parasite Leishmania is a long flagellum in the insect stage promastigotes and a rudimentary organelle in the mammalian amastigotes. LmxMKK, a mitogen-activated protein (MAP) kinase kinase from Leishmania mexicana, is required for growth of a full-length flagellum. We identified LmxMPK3, a MAP kinase homologue, with a similar expression pattern as LmxMKK being not detectable in amastigotes, up-regulated during the differentiation to promastigotes, constantly expressed in promastigotes, and shut down during the differentiation to amastigotes. LmxMPK3 null mutants resemble the LmxMKK knockouts with flagella reduced to one-fifth of the wild-type length, stumpy cell bodies, and vesicles and membrane fragments in the flagellar pocket. A constitutively activated recombinant LmxMKK activates LmxMPK3 in vitro. Moreover, LmxMKK is likely to be directly involved in the phosphorylation of LmxMPK3 in vivo. Finally, LmxMPK3 is able to phosphorylate LmxMKK, indicating a possible feedback regulation. This is the first time that two interacting components of a signaling cascade have been described in the genus Leishmania. Moreover, we set the stage for the analysis of reversible phosphorylation in flagellar morphogenesis.     

Extracellular cultivation and morphological characterization of amastigote-like forms of Leishmania panamensis and L. braziliensis

Two strains of the Leishmania braziliensis complex have been adapted to grow extracellularly at elevated temperature as amastigote-like forms in a cell-free medium. These parasites can be serially cultivated and maintained at 32 degrees C for L. panamensis (WR442; L. braziliensis panamensis) and at 28 degrees C for L. braziliensis (M5052; L. braziliensis braziliensis). Several observations are presented that the forms adapted at elevated temperature are amastigote-like. Morphologically, the amastigote-like organisms appear rounded to ovoid and are immotile and smaller than promastigotes; the flagellum of the amastigote-like forms does not extend beyond the flagellar pocket. In comparison, the promastigotes are very elongated, with a nucleus at mid-cell length and a very long flagellum. By electron microscopy, the short flagellum of the amastigote-like form is within a distended flagellar pocket; the 9 + 2 axonemal configuration is present but the paraxial rod is not observed. By contrast, the flagellum of the promastigote has a paraxial rod which extends from the axosome level. In addition, these amastigote-like forms of Leishmania are able to infect, to survive and to divide within the macrophage cell line J774.     

Pathogenomics analysis of Leishmania spp.: flagellar gene families of putative virulence factors

The trypanosomatid flagellar apparatus contains conventional and unique features, whose roles in infectivity are still enigmatic. Although the flagellum and the flagellar pocket are critical organelles responsible for all vesicular trafficking between the cytoplasm and cell surface, still very little is known about their roles in pathogenesis and how molecules get to and from the flagellar pocket. The ongoing analysis of the genome sequences and proteome profiles of Leishmania major and L infantum, Trypanosoma cruzi, T. brucei, and T. gambiensi ( www.genedb.org ), coupled with our own work on L. chagasi (as part of the Brazilian Northeast Genome Program- www.progene.ufpe.br ), prompted us to scrutinize flagellar genes and proteins of Leishmania spp. promastigotes that could be virulence factors in leishmaniasis. We have identified some overlooked parasite factors such as the MNUDC-1 (a protein involved in nuclear development and genomic fusion) and SQS (an enzyme of sterol biosynthesis), among the described flagellar gene families. A database concerning the results of this work, as well as of other studies of Leishmania and its organelles, is available at http://nugen.lcc.uece.br/LPGate . It will serve as a convenient bioinformatics resource on genomics and pathology of the etiological agents of leishmaniasis.     

I. Extracellular Cultivation and Morphological Characterization of Amastigote-Like Forms of Leishmania panamensis and L. braziliensis

. Two strains of the Leishmania braziliensis complex have been adapted to grow extracellularly at elevated temperature as amastigote-like forms in a cell-free medium. These parasites can be serially cultivated and maintained at 32°C for L. panamensis (WR442; L. braziliensis panamensis ) and at 28°C for L. braziliensis (M5052; L. braziliensis braziliensis ). Several observations are presented that the forms adapted at elevated temperature are amastigote-like. Morphologically, the amastigote-like organisms appear rounded to ovoid and are immotile and smaller than promastigotes; the flagellum of the amastigote-like forms does not extend beyond the flagellar pocket. In comparison, the promastigotes are very elongated, with a nucleus at mid-cell length and a very long flagellum. By electron microscopy, the short flagellum of the amastigote-like form is within a distended flagellar pocket; the 9 + 2 axonemal configuration is present but the paraxial rod is not observed. By contrast, the flagellum of the promastigote has a paraxial rod which extends from the axosome level. In addition, these amastigote-like forms of Leishmania are able to infect, to survive and to divide within the macrophage cell line J774.     

Biochemical characterization of Leishmania major aquaglyceroporin LmAQP1: possible role in volume regulation and osmotaxis

The Leishmania major aquaglyceroporin, LmAQP1, is responsible for the transport of trivalent metalloids, arsenite and antimonite. We have earlier shown that downregulation of LmAQP1 provides resistance to trivalent antimony compounds whereas increased expression of LmAQP1 in drug-resistant parasites can reverse the resistance. In this paper we describe the biochemical characterization of LmAQP1. Expression of LmAQP1 in Xenopus oocytes rendered them permeable to water, glycerol, methylglyoxal, dihydroxyacetone and sugar alcohols. The transport property of LmAQP1 was severely affected when a critical Arg230, located inside the pore of the channel, was altered to either alanine or lysine. Immunofluorescence and immuno-electron microscopy revealed LmAQP1 to be localized to the flagellum of Leishmania promastigotes and in the flagellar pocket membrane and contractile vacuole/spongiome complex of amastigotes. This is the first report of an aquaglyceroporin being localized to the flagellum of any microbe. Leishmania promastigotes and amastigotes expressing LmAQP1 could regulate their volume in response to hypoosmotic stress. Additionally, Leishmania promastigotes overexpressing LmAQP1 were found to migrate faster towards an osmotic gradient. These results taken together suggest that Leishmania LmAQP1 has multiple physiological roles, being involved in solute transport, volume regulation and osmotaxis.     

Leishmania mexicana: distribution of intramembranous particles and filipin sterol complexes in amastigotes and promastigotes

The density and distribution of intramembranous particles was analyzed in freeze fracture replicas of the plasma membrane of amastigotes, and infective as well as noninfective promastigotes of Leishmania mexicana amazonensis. The density of intramembranous particles on both protoplasmic and extracellular faces was higher in infective than in noninfective promastigotes and it was lower in amastigotes than in promastigotes. Amastigotes purified immediately after tissue homogenization were surrounded by a membrane which corresponded to the membrane which lined the endocytic vacuoles where the parasites were located within the tissue macrophages. Aggregation of the particles was seen in the flagellar membrane at the point of emergence of the flagellum from the flagellar pocket. Differences in the organization of the particles were seen in the membrane which lined the flagellar pocket of amastigotes and promastigotes. The polyene antibiotic, filipin, was used as a probe for the detection of sterols in the plasma membrane of L. m. amazonensis. The effect of filipin in the parasite's structure was analyzed by scanning electron microscopy and by transmission electron microscopy of thin sections and freeze fracture replicas. Filipin sterol complexes were distributed throughout the membrane which lined the cell body, the flagellar pocket, and the flagellum. No filipin sterol complexes were seen in the cell body-flagellar adhesion zone. The density of filipin sterol complexes was lower in the membrane lining the flagellum than in that lining the cell body of promastigotes.     

Introductory Remarks: Symposium on Differentiation of Trypanosomatids

ABSTRACT. Leishmania differentiation in the gut of phlebotomine sand flies was evaluated based on five light and electron microscopic studies of natural (Leishmania panamensis/Lutzomyia gomezi, Leishmania chagasi/Lutzomyia longipalpis) and unnatural (Leishmania mexicana/Lutzomyia abonnenci, Leishmania panamensis/Phlebotomus papatasi, Leishmania major/Lutzomyia longipalpis) life cycles. In the bloodmeal, transformation of amastigotes into stumpy promastigotes occurred before or during division. Further division in pairs or rosettes resulted in the development of spatulate and/or elongate nectomonad (free-swimming) promastigotes. Elongate, short, and metacyclic nectomonad promastigotes, and nectomonad paramastigotes were present in the midgut lumen. Dividing short promastigotes predominated in the cardia, and appeared to generate metacyclic forms which were observed in three life cycles. Haptomonad (attached) forms of Leishmania panamensis in the hindgut were primarily spatulate promastigotes (natural host) or pear-shaped promastigotes (unnatural host); paramastigotes and dividing forms were rare. At the stomodeal valve, short haptomonad promastigotes predominated in unnatural hosts, while both short and pear-shaped haptomonads were abundant, along with paramastigotes in natural hosts. Haptomonad paramastigotes and pear-shaped promastigotes colonized the esophagus, while paramastigotes predominated in the pharynx. Metacyclics were free-swimming in the lumen of the foregut.     

Differential targeting of two glucose transporters from Leishmania enriettii is mediated by an NH2-terminal domain

Leishmania are parasitic protozoa with two major stages in their life cycle: flagellated promastigotes that live in the gut of the insect vector and nonflagellated amastigotes that live inside the lysosomes of the vertebrate host macrophages. The Pro-1 glucose transporter of L. enriettii exists as two isoforms, iso-1 and iso-2, which are both expressed primarily in the promastigote stage of the life cycle. These two isoforms constitute modular structures: they differ exclusively and extensively in their NH2-terminal hydrophilic domains, but the remainder of each isoform sequence is identical to that of the other. We have localized these glucose transporters within promastigotes by two approaches. In the first method, we have raised a polyclonal antibody against the COOH-terminal hydrophilic domain shared by both iso-1 and iso-2, and we have used this antibody to detect the transporters by confocal immunofluorescence microscopy and immunoelectron microscopy. The staining observed with this antibody occurs primarily on the plasma membrane and the membrane of the flagellar pocket, but there is also light staining on the flagellum. We have also localized each isoform separately by introducing an epitope tag into each protein sequence. These experiments demonstrate that iso- 1, the minor isoform, resides primarily on the flagellar membrane, while iso-2, the major isoform, is located on the plasma membrane and the flagellar pocket. Hence, each isoform is differentially sorted, and the structural information for targeting each transporter isoform to its correct membrane address resides within the NH2-terminal hydrophilic domain.     

Implantation serine proteinase 1 exhibits mixed substrate specificity that silences signaling via proteinase-activated receptors

Implantation S1 family serine proteinases (ISPs) are tryptases involved in embryo hatching and uterine implantation in the mouse. The two different ISP proteins (ISP1 and ISP2) have been detected in both pre- and post-implantation embryo tissue. To date, native ISP obtained from uterus and blastocyst tissues has been isolated only as an active hetero-dimer that exhibits trypsin-like substrate specificity. We hypothesised that in isolation, ISP1 might have a unique substrate specificity that could relate to its role when expressed alone in individual tissues. Thus, we isolated recombinant ISP1 expressed in Pichia pastoris and evaluated its substrate specificity. Using several chromogenic substrates and serine proteinase inhibitors, we demonstrate that ISP1 exhibits trypsin-like substrate specificity, having a preference for lysine over arginine at the P1 position. Phage display peptide mimetics revealed an expanded but mixed substrate specificity of ISP1, including chymotryptic and elastase activity. Based upon targets observed using phage display, we hypothesised that ISP1 might signal to cells by cleaving and activating proteinase-activated receptors (PARs) and therefore assessed PARs 1, 2 and 4 as potential ISP1 targets. We observed that ISP1 silenced enzyme-triggered PAR signaling by receptor-disarming. This PAR-disarming action of ISP1 may be important for embryo development and implantation.     

Characterization of secretory leukocyte protease inhibitor as an inhibitor of implantation serine proteinases

We have recently identified and characterized two implantation serine proteinase genes, ISP1 and ISP2, which give rise to a dimeric proteinase, ISP that facilitates embryo invasion during peri-implantation period. As many proteinases have cognate serpins that regulate their proteolytic activity, we have been investigating anti-tryptases, expressed during this window of implantation. Here, we report the differential expression of secretory leukocyte protease inhibitor (SLPI) in uterine endometrium around the implantation period. The co-localization of SLPI and ISP suggests the possibility that SLPI is an ISP serpin and that expression of SLPI may lead to a reduction in ISP activity. The expression of SLPI is down regulated during the window of embryo-uterine receptivity. Our results are consistent with a model suggesting that the drop in SLPI expression may help to refine the opening of the window of implantation, by allowing the proteolytic activity of embryo invasive serine proteinases such as the ISPs.     

Leishmania-host-cell interaction: complexities and alternative views

Leishmania are protozoan parasites that infect various mammalian species, including humans. It is generally thought that random attachment of the flagellated promastigotes to mononuclear phagocytes initiates their uptake via circumferential pseudopods. Intracellularly, the promastigotes become located in phagolysosomes in which they transform to and survive as 'aflagellated' amastigotes that hide their shortened flagellum within the flagellar pocket. Unrestricted replication of these amastigotes is assumed to cause the eventual burst of the host cell, thereby releasing the infectious parasites. Here, Mike Rittig and Christian Bogdan review a large body of literature containing potentially important but poorly appreciated findings, which together with recent results, argue for Leishmania-host-cell interactions that are much more complex than generally thought.     

Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry.     

Kharon1 Null Mutants of Leishmania mexicana Are Avirulent in Mice and Exhibit a Cytokinesis Defect within Macrophages

In a variety of eukaryotes, flagella play important roles both in motility and as sensory organelles that monitor the extracellular environment. In the parasitic protozoan Leishmania mexicana, one glucose transporter isoform, LmxGT1, is targeted selectively to the flagellar membrane where it appears to play a role in glucose sensing. Trafficking of LmxGT1 to the flagellar membrane is dependent upon interaction with the KHARON1 protein that is located at the base of the flagellar axoneme. Remarkably, while Δ kharon1 null mutants are viable as insect stage promastigotes, they are unable to survive as amastigotes inside host macrophages. Although Δ kharon1 promastigotes enter macrophages and transform into amastigotes, these intracellular parasites are unable to execute cytokinesis and form multinucleate cells before dying. Notably, extracellular axenic amastigotes of Δ kharon1 mutants replicate and divide normally, indicating a defect in the mutants that is only exhibited in the intra-macrophage environment. Although the flagella of Δ kharon1 amastigotes adhere to the phagolysomal membrane of host macrophages, the morphology of the mutant flagella is often distorted. Additionally, these null mutants are completely avirulent following injection into BALB/c mice, underscoring the critical role of the KHARON1 protein for viability of intracellular amastigotes and disease in the animal model of leishmaniasis.