Acylation-dependent protein export in Leishmania

The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.     

Phenotypic changes associated with deletion and overexpression of a stage-regulated gene family in Leishmania

The LmcDNA16 locus of Leishmania major contains three highly related genes HASPA1 , HASPA2 and HASPB , encoding hydrophilic, acylated surface proteins and a tandem pair of unrelated sequences, SHERP1 and SHERP2 , coding for a small, hydrophilic protein that localizes to the endoplasmic reticulum and outer mitochondrial membrane. Differential regulation of these genes results in expression of a subset of the HASP proteins and SHERP only in infective stage parasites. To assess the contribution of these molecules to parasite virulence, the diploid LmcDNA16 gene locus has been removed by targeted gene deletion. Homozygous null mutants have precise deletions of both alleles and exhibit no HASP or SHERP expression. They are at least as virulent as wild-type parasites in macrophage invasion and intracellular survival assays, both in vitro and in vivo . Conversely, null mutants engineered to overexpress the entire LmcDNA16 gene locus are unable to survive within the intramacrophage environment despite their differentiation into infective metacyclic parasites. Both null and overexpressing null parasites show increased sensitivity to complement-mediated lysis, suggesting perturbation of their surface architecture. Avirulence in overexpressing parasites correlates with selective depletion of a specific lipid species, decreased expression of the major surface glycoprotein GP63, but no significant downregulation of the glycoconjugate lipophosphoglycan.     

The stage‐regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector,Phlebotomus papatasi

The stage‐regulated HASPB and SHERP proteins of Leishmania major are predominantly expressed in cultured metacyclic parasites that are competent for macrophage uptake and survival. The role of these proteins in parasite development in the sand fly vector has not been explored, however. Here, we confirm that expression of HASPB is detected only in vector metacyclic stages, correlating with the expression of metacyclic‐specific lipophosphoglycan and providing the first definitive protein marker for this infective sand fly stage. Similarly, SHERP is expressed in vector metacyclics but is also detected at low levels in the preceding short promastigote stage. Using genetically modified parasites lacking or complemented for the LmcDNA16 locus on chromosome 23 that contains the HASP and SHERP genes, we further show that the presence of this locus is essential for parasite differentiation to the metacyclic stage in Phlebotomus papatasi. While wild‐type and complemented parasites transform normally in late‐stage infections, generating metacyclic promastigotes and colonizing the sand fly stomodeal valve, null parasites accumulate at the earlier elongated nectomonad stage of development within the abdominal and thoracic midgut of the sand fly. Complementation with HASPB or SHERP alone suggests that HASPB is the dominant effector molecule in this process.     

LFR1 ferric iron reductase of Leishmania amazonensis is essential for the generation of infective parasite forms

The protozoan parasite Leishmania is the causative agent of serious human infections worldwide. The parasites alternate between insect and vertebrate hosts and cause disease by invading macrophages, where they replicate. Parasites lacking the ferrous iron transporter LIT1 cannot grow intracellularly, indicating that a plasma membrane-associated mechanism for iron uptake is essential for the establishment of infections. Here, we identify and functionally characterize a second member of the Leishmania iron acquisition pathway, the ferric iron reductase LFR1. The LFR1 gene is up-regulated under iron deprivation and accounts for all the detectable ferric reductase activity exposed on the surface of Leishmania amazonensis. LFR1 null mutants grow normally as promastigote insect stages but are defective in differentiation into the vertebrate infective forms, metacyclic promastigotes and amastigotes. LFR1 overexpression partially restores the abnormal morphology of infective stages but markedly reduces parasite viability, precluding its ability to rescue LFR1 null replication in macrophages. However, LFR1 overexpression is not toxic for amastigotes lacking the ferrous iron transporter LIT1 and rescues their growth defect. In addition, the intracellular growth of both LFR1 and LIT1 null parasites is rescued in macrophages loaded with exogenous iron. This indicates that the Fe(3+) reductase LFR1 functions upstream of LIT1 and suggests that LFR1 overexpression results in excessive Fe(2+) production, which impairs parasite viability after intracellular transport by LIT1.     

The overexpression of a new ABC transporter in Leishmania is related to phospholipid trafficking and reduced infectivity

This paper reports the characterization of a new ABC transporter (LtrABC1.1), related to the human ABCA subfamily, in the protozoan parasite Leishmania tropica. LtrABC1.1 is a tandem duplicated gene flanked by inverted repeats. LtrABC1.1 is expressed mainly in the flagellar pocket of the parasite. Drug resistance studies in Leishmania overexpressing LtrABC1.1 showed the transporter not to confer resistance to a range of unrelated drugs. LtrABC1.1 appears to be involved in lipid movements across the plasma membrane of the parasite since overexpression reduces the accumulation of fluorescent phospholipid analogues. The activity of this protein may also affect membrane movement processes since secreted acid phosphatase (SAP) activity was significantly lower in promastigotes overexpressing LtrABC1.1. In vitro infection experiments with macrophages indicated LtrABC1.1-transfected parasites to be significantly less infective. Together, these results suggest that this new ABC transporter could play a role in lipid movements across the plasma membrane, and that its activity might influence vesicle trafficking. This is the first ABCA-like transporter described in unicellular eukaryotes.     

A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation.

At the end of their growth in the sand fly, Leishmania parasites differentiate into the infective metacyclic promastigote stage, which is transmitted to the mammalian host. Thus, in experimental studies of parasite infectivity toward animals or macrophages, the use of purified metacyclics is generally preferred. While metacyclics of several Leishmania species can be efficiently purified with the aid of lectins or monoclonal antibodies, which differentially exploit stage-specific differences in the structure of the abundant surface glycolipid lipophosphoglycan (LPG), such reagents are unavailable for most species and they are unsuitable for studies involving LPG-deficient mutants. Here we describe a simple density gradient centrifugation method, which allows the rapid purification of infective metacyclic parasites from both wild-type and LPG-deficient Leishmania major. The purified metacyclic promastigotes are authentic, as judged by criteria such as their morphology, expression of the metacyclic-specific gene SHERP, and ability to invade and replicate within macrophages in vitro. Preliminary studies suggest that this method is applicable to other Leishmania species including L. donovani.     

Sphingolipids are essential for differentiation but not growth in Leishmania

Sphingolipids (SLs) play critical roles in eukaryotic cells in the formation of lipid rafts, membrane trafficking, and signal transduction. Here we created a SL null mutant in the protozoan parasite Leishmania major through targeted deletion of the key de novo biosynthetic enzyme serine palmitoyltransferase subunit 2 (SPT2). Although SLs are typically essential, spt2- Leishmania were viable, yet were completely deficient in de novo sphingolipid synthesis, and lacked inositol phosphorylceramides and other SLs. Remarkably, spt2- parasites maintained 'lipid rafts' as defined by Triton X-100 detergent resistant membrane formation. Upon entry to stationary phase spt2- failed to differentiate to infective metacyclic parasites and died instead. Death occurred not by apoptosis or changes in metacyclic gene expression, but from catastrophic problems leading to accumulation of small vesicles characteristic of the multivesicular body/multivesicular tubule network. Stage specificity may reflect changes in membrane structure as well as elevated demands in vesicular trafficking required for parasite remodeling during differentiation. We suggest that SL-deficient Leishmania provide a useful biological setting for tests of essential SL enzymes in other organisms where SL perturbation is lethal.     

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.     

Leishmania-specific surface antigens show sub-genus sequence variation and immune recognition

Background

A family of hydrophilic acylated surface (HASP) proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic) and intracellular (amastigote) stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia) have lost HASP genes from their genomes.

Methods/Principal Findings

We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia) species, L. (V.) braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L.) mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o) HASPs) are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family.

Conclusions/Significance

These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are surface-exposed on amastigotes (although L. (L.) major parasites also express HASPB on the metacyclic plasma membrane). The central repetitive domains of the HASPs are highly variant in their amino acid sequences, both within and between species, consistent with a role in immune recognition in the host.     

Differences in human macrophage receptor usage, lysosomal fusion kinetics and survival between logarithmic and metacyclic Leishmania infantum chagasi promastigotes

The obligate intracellular protozoan, Leishmania infantum chagasi (Lic) undergoes receptor-mediated phagocytosis by macrophages followed by a transient delay in phagolysosome maturation. We found differences in the pathway through which virulent Lic metacyclic promastigotes or avirulent logarithmic promastigotes are phagocytosed by human monocyte-derived macrophages (MDMs). Both logarithmic and metacyclic promastigotes entered MDMs through a compartment lined by the third complement receptor (CR3). In contrast, many logarithmic promastigotes entered through vacuoles lined by mannose receptors (MR) whereas most metacyclic promastigotes did not ( P  < 0.005). CR3-positive vacuoles containing metacyclic promastigotes stained for caveolin-1 protein, suggesting CR3 localizes in caveolae during phagocytosis. Following entry, the kinetics of phagolysosomal maturation and intracellular survival also differed. Vacuoles containing metacyclic parasites did not accumulate lysosome-associated membrane protein-1 (LAMP-1) at early times after phagocytosis, whereas vacuoles with logarithmic promastigotes did. MDMs phagocytosed greater numbers of logarithmic than metacyclic promastigotes, yet metacyclics ultimately replicated intracellularly with greater efficiency. These data suggest that virulent metacyclic Leishmania promastigotes fail to ligate macrophage MR, and enter through a path that ultimately enhances intracellular survival. The relatively quiescent entry of virulent Leishmania spp. into macrophages may be accounted for by the ability of metacyclic promastigotes to selectively bypass deleterious entry pathways.     

Abortive infection of Lutzomyia longipalpis insect vectors by aflagellated LdARL-3A-Q70L overexpressing Leishmania amazonensis parasites

Leishmania donovani ADP-ribosylation factor-like protein 3A (LdARL-3A) is a small G protein isolated from the protozoan parasite L. donovani with no defined physiological function. Previously [Cuvillier, A., Redon, F., Antoine, J.-C., Chardin, P., DeVos, T., and Merlin, G. (2000) J Cell Sci 113: 2065-2074] we have shown that overexpression in L. amazonensis promastigotes of the mutated protein LdARL-3A-Q70L, which remains constitutively associated with GTP, leads to the disappearance of the flagellum but does not impair cell viability or growth. Here we report that parasites overexpressing LdARL-3A-Q70L can invade in vitro cultivated macrophages to the same extent as control cells, demonstrating that the flagellum is not necessary for attachment to or engulfment into macrophages. These infections are productive because amastigotes differentiate and multiply. However, aflagellated LdARL-3A-Q70L-overexpressing Leishmania promastigotes could not survive in experimentally infected Lutzomyia longipalpis insect vectors, in contrast to untransfected or native LdARL-3A-overexpressing cells. Overexpression of the native and mutated proteins did not modify in vitro procyclic to metacyclic lipophosphoglycan maturation or differentiation from procyclic to metacyclic promastigotes, nevertheless there is a block in transmission of Leishmania. Better understanding of LdARL-3A pathways, notably those regarding flagellum biogenesis, may lead to the future development of Leishmania-specific drugs, which may stop parasite transmission in nature without affecting other species.     

Leishmania salvage and remodelling of host sphingolipids in amastigote survival and acidocalcisome biogenesis

Sphingolipids (SLs) play essential roles in most eukaryotes, but in the trypanosomatid protozoan Leishmania major their functions differ significantly. Previously we showed that null mutants defective in de novo sphingoid base synthesis (spt2-) lacked SLs but grew well and retained lipid rafts while replicating as promastigotes in vitro. However, they experienced catastrophic defects in membrane trafficking on entry into stationary phase, and failed to differentiate to the infective metacyclic form. Here we showed this mutant retained the ability to enter macrophages silently and inhibit activation, although as expected most parasites were destroyed. However, in mouse infections, after a delay rapidly progressive lesions appeared, and purified amastigotes were fully virulent to macrophages and mice. Mass spectrometry of spt2- amastigote lipids revealed the presence of high levels of parasite-specific inositol phosphorylceramides (IPCs) not synthesized by the mammalian hosts. Inhibitor studies showed that salvage occurs at the level of complex SLs, suggesting that parasites carry out 'headgroup' remodelling. Additionally, we describe a new defect of the spt2- promastigotes involving 'empty' acidocalcisomes (ACs), which may point to the origin of this organelle from the lysosome-related organelle/multivesicular body biogenesis pathway. However, ACs in spt2- amastigotes appeared quantitatively and morphologically normal. Thus salvage of SLs and other molecules by intracellular amastigotes play key roles in AC biogenesis and parasite survival in the host.     

Overexpression of the Leishmania amazonensis Ca2+-ATPase gene lmaa1 enhances virulence

A gene for a Ca2+-transporting ATPase (lmaa1) from the trypanosomatid parasite Leishmania (mexicana) amazonensis was overexpressed in two clones of L. amazonensis differing in their virulence. RNA and protein expression of the gene was increased in transfectants, as was the infectivity of transfectants versus parental types in both mouse and in vitro macrophage infection experiments. The virulence of the almost avirulent clone was enhanced such that it was more virulent than the parental 'virulent' clone. Growth of the parasites in culture as promastigotes, after isolation from mouse lesions, indicated that transfection led to improved survival of promastigotes during the stationary phase of culture. As it is in this culture phase that infective metacyclic forms develop, the key role of the Lmaa1 protein may be in metacyclogenesis. The protein may be important in the synthesis and trafficking of new proteins through the secretory pathway, as we demonstrate, using a green fluorescent protein hybrid and by immunofluorescence, that the Lmaa1 protein is located in the endoplasmic reticulum in promastigotes and amastigotes of L. amazonensis.     

Fibronectin shedding by Leishmania may influence the parasite-macrophage interaction.

Fibronectin (FN) is a large extracellular matrix protein involved in the endocytosis of several types of particles by different phagocytes. Here we investigated the role of FN in the entry and destruction of Leishmania amazonensis promastigotes (flagellated form) by murine resident peritoneal macrophages. We also studied the lateral mobility of this protein on the surface of the parasite cells using a immunogold technique. We compared the effects of addition and depletion of FN on infective and non-infective populations of Leishmania promastigotes. The invasion by the latter but not by the former, was increased by FN, and the uptake of these cells was more sensitive to FN depletion from the culture medium. We also observed enhanced killing of intracellular infective promastigotes upon FN addition to the macrophage cultures. Immunocytochemical localization of FN on the surface of the flagellates revealed that the parasite cells released bound FN by membrane shedding in a constitutive fashion. Therefore we conclude that FN removal by shedding may be part of a physiological mechanism by which the parasites evade intracellular destruction by host cells.     

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 cell surface prohibitin: role in host–parasite interaction

Proteins selectively upregulated in infective parasitic forms could be critical for disease pathogenesis. A mammalian prohibitin orthologue is upregulated in infective metacyclic promastigotes of Leishmania donovani, a parasite that causes visceral leishmaniasis. Leishmania donovani prohibitin shares 41% similarity with mammalian prohibitin and 95–100% within the genus. Prohibitin is concentrated at the surface of the flagellar and the aflagellar pole, the aflagellar pole being a region through which host–parasite interactions occur. Prohibitin is attached to the membrane through a GPI anchor. Overexpression of wild‐type prohibitin increases protein surface density resulting in parasites with higher infectivity. However, parasites overexpressing a mutant prohibitin with an amino acid substitution at the GPI anchor site to prevent surface expression through GPI‐link show lesser surface expression and lower infective abilities. Furthermore, the presence of anti‐prohibitin antibodies during macrophage–Leishmania interaction in vitro reduces infection. The cognate binding partner for Leishmania prohibitin on the host cell appears to be macrophage surface HSP70, siRNA mediated downregulation of which abrogates the capability of the macrophage to bind to parasites. Leishmania prohibitin is able to generate a strong humoral response in visceral leishmaniasis patients. The above observations suggest that prohibitin plays an important role in events leading to Leishmania–host interaction.     

The dendritic cell receptor DC-SIGN discriminates among species and life cycle forms of Leishmania

Infection of dendritic cells by the human protozoal parasite Leishmania is part of its survival strategy. The dendritic cell receptors for Leishmania have not been established and might differ in their interactions among Leishmania species and infective stages. We present evidence that the surface C-type lectin DC-SIGN (CD 209) is a receptor for promastigote and amastigote infective stages from both visceral (Leishmania infantum) and New World cutaneous (Leishmania pifanoi) Leishmania species, but not for Leishmania major metacyclic promastigotes, an Old World species causing cutaneous leishmaniasis. Leishmania binding to DC-SIGN was found to be independent of lipophosphoglycan, the major glycoconjugate of the promastigote plasma membrane. Our findings emphasize the relevance of DC-SIGN in Leishmania-dendritic cell interactions, an essential link between innate and Leishmania-specific adaptive immune responses, and suggest that DC-SIGN might be a therapeutic target for both visceral and cutaneous leishmaniasis     

The overexpression of a new ABC transporter in Leishmania is related to phospholipid trafficking and reduced infectivity

This paper reports the characterization of a new ABC transporter (LtrABC1.1), related to the human ABCA subfamily, in the protozoan parasite Leishmania tropica. LtrABC1.1 is a tandem duplicated gene flanked by inverted repeats. LtrABC1.1 is expressed mainly in the flagellar pocket of the parasite. Drug resistance studies in Leishmania overexpressing LtrABC1.1 showed the transporter not to confer resistance to a range of unrelated drugs. LtrABC1.1 appears to be involved in lipid movements across the plasma membrane of the parasite since overexpression reduces the accumulation of fluorescent phospholipid analogues. The activity of this protein may also affect membrane movement processes since secreted acid phosphatase (SAP) activity was significantly lower in promastigotes overexpressing LtrABC1.1. In vitro infection experiments with macrophages indicated LtrABC1.1-transfected parasites to be significantly less infective. Together, these results suggest that this new ABC transporter could play a role in lipid movements across the plasma membrane, and that its activity might influence vesicle trafficking. This is the first ABCA-like transporter described in unicellular eukaryotes.     

Expression of calreticulin P-domain results in impairment of secretory pathway in Leishmania donovani and reduced parasite survival in macrophages

The secretory proteins of Leishmania are thought to be involved in the parasite survival inside the insect vector or mammalian host. It is clear from studies in higher eukaryotes that proper folding in the endoplasmic reticulum and targeting out of the endoplasmic reticulum is critical for the function of secretory proteins. The endoplasmic reticulum chaperones such as calreticulin play an important role in the quality control of secretory proteins. However, very little is known about the secretory pathway of trypanosomatid parasites such as Leishmania. In the present study, we show that overexpression of the P-domain of Leishmania donovani calreticulin in transfected L. donovani resulted in a significant reduction in the secretion of the parasite secretory acid phosphatases. This effect is associated with an intracellular accumulation of active enzyme in these transfected parasites. In addition, parasites expressing the P-domain calreticulin showed a significant decrease in survival inside human macrophages. This study suggests that altering the function of an endoplasmic reticulum chaperone such as calreticulin in Leishmania may affect the targeting of proteins that are associated with the virulence of the parasite during their trafficking through the parasite secretory pathway.     

Cell surface nanoanatomy of Leishmania major as revealed by fracture-flip. A surface meshwork of 44 nm fusiform filaments identifies infective developmental stage promastigotes

Fracture-flip (Anderson-Forsman and Pinto da Silva, J. Cell Sci. 90, 531-541; 1988) was used to reveal the nanoanatomy of the surface of Leishmania major promastigotes. Over the cell surface of infective metacyclic promastigotes we identify a meshwork of 44 nm long, fusiform filaments. These filaments are not seen in noninfective stages of the parasite. Replica-staining immunocytochemistry with monoclonal antibody against infective metacyclic lipophosphoglycan shows a uniform distribution of protein A-colloidal gold complexes over the cell surface. Thin sections show that acquisition of the high molecular weight lipophosphoglycan is reflected in a thicker glycocalyx. Conventional freeze-fracture shows that in infective metacyclic promastigotes there is a reversal of the partition of intramembrane particles--an additional morphological marker for the infective developmental stage. We hypothesize that the fusiform filaments represent metacyclic developmental lipophosphoglycan.