Role of AP-1 in Developmentally Regulated Lysosomal Trafficking in Trypanosoma brucei

African trypanosomes are the causative agents of human trypanosomiasis (sleeping sickness). The pathogenic stage of the parasite has unique adaptations to life in the bloodstream of the mammalian host, including upregulation of endocytic and lysosomal activities. We investigated stage-specific requirements for cytoplasmic adaptor/clathrin machinery in post-Golgi apparatus biosynthetic sorting to the lysosome using RNA interference silencing of the Tbμ1 subunit of adaptor complex 1 (AP-1), in conjunction with immunolocalization, kinetic analyses of reporter transport, and quantitative endocytosis assays. Tbμ1 silencing was lethal in both stages, indicating a critical function(s) for the AP-1 machinery. Transport of soluble and membrane-bound secretory cargoes was Tbμ1 independent in both stages. In procyclic parasites, trafficking of the lysosomal membrane protein, p67, was disrupted, leading to cell surface mislocalization. The lysosomal protease trypanopain was also secreted, suggesting a transmembrane-sorting receptor for this soluble hydrolase. In bloodstream trypanosomes, both p67 and trypanopain trafficking were unaffected by Tbμ1 silencing, suggesting that AP-1 is not necessary for biosynthetic lysosomal trafficking. Endocytosis in bloodstream cells was also unaffected, indicating that AP-1 does not function at the flagellar pocket. These results indicate that post-Golgi apparatus sorting to the lysosome is critically dependent on the AP-1/clathrin machinery in procyclic trypanosomes but that this machinery is not necessary in bloodstream parasites. We propose a simple model for stage-specific default secretory trafficking in trypanosomes that is consistent with the behavior of other soluble and glycosylphosphatidylinositol-anchored cargos and which is influenced by upregulation of endocytosis in bloodstream parasites as an adaptation to life in the mammalian bloodstream.African trypanosomes (Trypanosoma brucei subspecies), the agents of African sleeping sickness, are alone among the kinetoplastid parasites (including Trypanosoma cruzi and Leishmania spp.) in having a pathogenic bloodstream stage that exists and replicates extracellularly in the mammalian host. This places unique constraints on the parasite in terms of dealing with host immune responses and on acquisition of essential nutrients. The parasite has evolved many strategies to deal with these constraints, the best known of which is the process of antigenic variation (9). Another is the lysosome, which impacts the host-pathogen balance in multiple ways. Trypanosomes have a single terminal lysosome that is the final repository of endocytic cargo acquired from the host serum for nutritional purposes (30), as well as for potentially lytic immune complexes removed from the cell surface (4, 8). Both endocytosis and lysosomal hydrolytic activities are differentially regulated through the trypanosome life cycle (11, 30), and there are stage-specific differences in the biosynthetic trafficking of essential lysosomal components (discussed below). The release of lysosomal proteases is a factor in the signature event of human infection, penetration of the central nervous system (36). Finally, lysosomal physiology is critical to the activity of an innate human serum resistance trait, trypanolytic factor, which limits the host range of Trypanosoma species (38).Clearly, given its multiple roles in pathogenesis, biogenesis of the lysosome is critical to the success of trypanosomes as human parasites. As in all eukaryotes, lysosomal biogenesis is a balance between the proper sorting of newly synthesized membranes and proteins and recycling of established membranes and proteins internalized from the cell surface. In each case, protein sorting involves recognition of specific signals in cargo molecules by cellular machinery for inclusion in nascent transport vesicles destined for downstream delivery. Unique sets of cytoplasmic coat complexes at discrete intracellular locations serve the dual purpose of simultaneously mediating vesicle formation and selective cargo loading. The best characterized of these machineries is the clathrin/adaptin system for formation of coated vesicles at the Golgi apparatus and the plasma membrane (10, 41). Adaptor complexes (APs) are cytosolic heterotetramers that interact with specific signals in the cytoplasmic domains of membrane cargo proteins, such as dileucine motifs ([E/D]XXXL[L/I]) and tyrosine motifs (YXXØ, where Ø is a bulky hydrophobic residue). The prototypic AP complexes are AP-1 and AP-2, which function at the trans-Golgi network and plasma membrane, respectively. Both are composed of two large subunits (γ/β1 in AP-1; α/β2 in AP-2) and two smaller subunits (σ1/μ1 in AP-1; σ2/μ2 in AP-2). YXXØ motifs interact with μ adaptins, and dileucine motifs interact with combinations of adaptin subunits in both AP-1 and AP-2 (26, 40, 42). It is the large subunits, particularly β adaptin, that mediate clathrin recruitment (19, 44). Other APs, AP-3 and AP-4, with discrete subunit compositions, also exist. AP-3 functions in trafficking to lysosome-related organelles, such as melanosomes, and AP-4 may be involved in basolateral trafficking in polarized epithelial cells (10). The genome of the African trypanosome, T. brucei, encodes a complete complement of orthologous subunits for AP-1, AP-3, and AP-4 but has no genes for AP-2, the major adaptor complex mediating endocytosis in vertebrate cells (16). This is likely due to evolutionary loss, since the closely related T. cruzi has orthologues of all four APs.Two major lysosomal cargo proteins have been studied in T. brucei, the LAMP (lysosome-associated membrane protein)-like protein p67 and the cathepsin L orthologue trypanopain. p67 is a type I membrane protein with a large glycosylated lumenal domain and a short cytoplasmic domain (1, 27). In procyclic insect stage (PCF) trypanosomes, the cytoplasmic domain is both necessary and sufficient for lysosomal targeting of a heterologous reporter, and its deletion results in mistargeting of p67 to the cell surface (1). The cytoplasmic domain contains two canonical dileucine motifs, mutation of which also results in delivery to the cell surface (47). These findings strongly indicate the existence of cognate cytoplasmic machinery for lysosomal delivery of p67 in PCF trypanosomes. Strikingly, however, the cytoplasmic domain, and its motifs, are totally dispensable for lysosomal targeting in bloodstream stage (BSF) trypanosomes (1). Deletion of the cytoplasmic domain results in minor mislocalization to the cell surface, but p67 is still overwhelmingly delivered to the lysosome. Ongoing lysosomal targeting cannot easily be attributed to misfolding of the lumenal domain, as suggested by others (3), since the normal transport-associated patterns of p67 glycosylation and cleavage prevail in these deletion constructs.Less is known about targeting of soluble trypanopain. In mammalian cells, soluble hydrolases are targeted to the lysosome by the addition of mannose-6-phosphate (M6P) moieties in the Golgi apparatus, which serve as ligands for recognition and lysosomal targeting by downstream M6P receptors (28). Soluble hydrolases can also be sorted by receptors that recognize polypeptide motifs, such as sortilins in mammalian cells (12) and Vps10 in yeast (13, 32). These receptors have lumenal cargo recognition domains and cytoplasmic domains containing signals for late endosomal targeting and recycling. M6P-modified N-linked glycans are not found in trypanosomes, and genes encoding the necessary enzymatic activities are absent from the genome (16), ruling out this possibility for trypanopain sorting. However, the T. cruzi orthologue, cruzipain, has been shown to rely on peptide motifs in the N-terminal prodomain for targeting (24), raising the possibility of a sortilin/Vps10p-like sorting receptor. Although there are no obvious orthologues of these proteins in the T. brucei genome, overexpression of trypanopain in PCF trypanosomes leads to secretion, an observation that is consistent with saturation of a specific sorting receptor (S. S. Sutterwala and J. D. Bangs, unpublished observations).Having previously studied the innate signals involved in p67 targeting (1, 47), we now turned our attention to the cognate machinery for post-Golgi apparatus sorting. Specifically, we investigate the role of trypanosomal AP-1 in stage-specific biosynthetic trafficking to the lysosome using RNA interference (RNAi)-mediated silencing of the Tbμ1 (geneDB no. Tb927.7.3180 [www.genedb.org]) subunit as our primary strategy. Our results demonstrate that AP-1 and clathrin are critical for lysosomal targeting of p67 and trypanopain in PCF trypanosomes but that they are essentially dispensable in BSF parasites. These data, in conjunction with the behavior of p67-targeting mutants (1) and other trypanosomal secretory reporters, lead us to propose a simple model for stage-specific default trafficking in African trypanosomes. Although in some respects our results are similar to those of a recent publication using RNAi silencing of the Tbγ1 subunit of AP-1 (3), they differ in key aspects, leading us to significantly different conclusions.     

Heterologous Expression Studies of Saccharomyces cerevisiae Reveal Two Distinct Trypanosomatid CaaX Protease Activities and Identify Their Potential Targets

The CaaX tetrapeptide motif typically directs three sequential posttranslational modifications, namely, isoprenylation, proteolysis, and carboxyl methylation. In all eukaryotic systems evaluated to date, two CaaX proteases (Rce1 and Ste24/Afc1) have been identified. Although the Trypanosoma brucei genome also encodes two putative CaaX proteases, the lack of detectable T. brucei Ste24 activity in trypanosome cell extracts has suggested that CaaX proteolytic activity within this organism is solely attributed to T. brucei Rce1 (J. R. Gillespie et al., Mol. Biochem. Parasitol. 153:115-124. 2007). In this study, we demonstrate that both T. brucei Rce1 and T. brucei Ste24 are enzymatically active when heterologously expressed in yeast. Using a-factor and GTPase reporters, we demonstrate that T. brucei Rce1 and T. brucei Ste24 possess partially overlapping specificities much like, but not identical to, their fungal and human counterparts. Of interest, a CaaX motif found on a trypanosomal Hsp40 protein was not cleaved by either T. brucei CaaX protease when examined in the context of the yeast a-factor reporter but was cleaved by both in the context of the Hsp40 protein itself when evaluated using an in vitro radiolabeling assay. We further demonstrate that T. brucei Rce1 is sensitive to small molecules previously identified as inhibitors of the yeast and human CaaX proteases and that a subset of these compounds disrupt T. brucei Rce1-dependent localization of our GTPase reporter in yeast. Together, our results suggest the conserved presence of two CaaX proteases in trypanosomatids, identify an Hsp40 protein as a substrate of both T. brucei CaaX proteases, support the potential use of small molecule CaaX protease inhibitors as tools for cell biological studies on the trafficking of CaaX proteins, and provide evidence that protein context influences T. brucei CaaX protease specificity.Certain isoprenylated proteins are synthesized as precursors having a highly degenerate C-terminal tetrapeptide CaaX motif (C, cysteine; a, aliphatic amino acid; X, one of several amino acids). This motif typically directs three posttranslational modifications that include covalent attachment of an isoprenoid lipid to the cysteine residue, followed by endoproteolytic removal of the terminal three residues (i.e., aaX), and lastly, carboxyl methyl esterification of the farnesylated cysteine (49, 50). Relevant examples of proteins subject to the above modifications, also referred to as CaaX proteins, include the Ras and Ras-related GTPases, Gγ subunits, prelamin A, members of the Hsp40 family of chaperones, and fungal mating pheromones.Isoprenylation of CaaX proteins is performed by either the farnesyltransferase (FTase) or the geranylgeranyl transferase I (GGTase I). The particular isoprenoid attached, C15 farnesyl or C20 geranylgeranyl, respectively, depends in part on the sequence of the CaaX motif (8, 26, 31). Proteolysis of isoprenylated intermediates is carried out by the otherwise unrelated Rce1p (Ras converting enzyme 1) and Ste24p (sterile mutant 24) enzymes, collectively referred to as CaaX proteases, which are integral membrane proteins residing within the endoplasmic reticulum (3, 40, 45). Studies to elucidate the specificities of the CaaX proteases have often involved reporters designed from biological substrates (e.g., Ras GTPases) (2, 3, 16, 21, 22, 24, 34). Although these studies suggest that isoprenylated CaaX tetrapeptides alone are sufficient for recognition as a substrate, insufficient evidence exists to assert whether this sequence contains all of the necessary information for substrate specificity. Reporters are typically cleaved by either Rce1p or Ste24p. The Saccharomyces cerevisiae a-factor mating pheromone is a rather unusual biological reporter since it is cleaved by both yeast CaaX proteases. Orthologs of the CaaX proteases from humans, worms, and plants can also cleave a-factor when heterologously expressed in yeast, thereby making a-factor a convenient reporter for comparative analyses of CaaX protease activities (3, 5, 6, 36). Where evaluated using the a-factor reporter, Rce1p and Ste24p display partially overlapping target specificity, and this is an expected property of CaaX proteases in all eukaryotic systems (5, 6, 36, 47). Unlike the isoprenylation and proteolysis steps, carboxyl methyl esterification exclusively relies on a single enzyme, the isoprenylcysteine carboxyl methyltransferase (ICMT) (23, 50). A farnesylated cysteine appears to be the sole recognition determinant of the endoplasmic reticulum-localized ICMT (10, 23, 38).Disruption of the posttranslational modifications associated with CaaX proteins is often perceived as an anticancer strategy because of the prominent role of CaaX proteins in cellular transformation (i.e., the Ras GTPases) (49). To date, the most advanced drug discovery efforts have focused on farnesyltransferase inhibitors (FTIs) (9, 53). Inhibitors of the CaaX proteases and ICMT are also being developed (1, 11, 28, 37, 39, 48). Disrupting CaaX protein modifications has therapeutic application to other diseases as well. The relief of prelamin A toxicity by FTIs is a well-documented example (51). Accumulation of the farnesylated but unproteolysed precursor of lamin A results in a progeroid phenotype in individuals lacking ZmpSte24 proteolytic activity. The treatment of parasitic disease is another area under investigation (13). A number of FTIs have been developed that inhibit protozoan FTases, and in vivo testing is a continued effort (15, 32). Although research is less advanced with respect to CaaX protease and ICMT inhibitors, RNA interference experiments on the bloodstream form of Trypanosoma brucei indicate that CaaX processing enzymes are required for viability and proliferation of the parasite (20).In the present study, we evaluated the enzymatic properties of the trypanosomal CaaX proteases. We establish through the use of a variety of in vivo and in vitro assays that T. brucei Rce1 and T. brucei Ste24 are active when heterologously expressed in S. cerevisiae and have partially overlapping substrate specificities. The assays rely on various reporters, specifically the yeast a-factor mating pheromone, a K-Ras4B-based fluorogenic peptide, a green fluorescent protein (GFP)-GTPase fusion, and a T. brucei Hsp40 protein. All but the GTPase reporter could be effectively cleaved by both T. brucei CaaX proteases. We also demonstrate that the trypanosomal CaaX proteases can be targeted for inhibition by small molecules both in vitro and when heterologously expressed in yeast, suggesting that the trypanosomal CaaX proteases may be attractive drug targets for pharmacological inhibition.     

Evidence for Mucin-Like Glycoproteins That Tether Sporozoites of Cryptosporidium parvum to the Inner Surface of the Oocyst Wall

Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wall that surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomifera agglutinin (MPA), which binds GalNAc, and the C. parvum wall contains at least two unique proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) identified by monoclonal antibodies. C. parvum sporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40 and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appear to attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from the sporozoites, some of these fibrillar tethers appear to collapse into globules on the inner surface of oocyst walls. The most abundant proteins of purified oocyst walls, which are missing the tethers and outer veil, were COWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. In contrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls and sporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser protease inhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] to CpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 and gp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linked GalNAc on glycoproteins were localized to the globules. These results suggest that mucin-like glycoproteins, which are associated with the sporozoite surface, may contribute to fibrils and/or globules that tether sporozoites to the inner surface of oocyst walls.Cryptosporidium parvum and the related species Cryptosporidium hominis are apicomplexan parasites, which are spread by the fecal-oral route in contaminated water and cause diarrhea, particularly in immunocompromised hosts (1, 12, 39, 47). The infectious and diagnostic form of C. parvum is the oocyst, which has a single, multilayered, spherical wall that surrounds four sporozoites, the invasive forms (14, 27, 31). The outermost layer of the C. parvum oocyst wall is most often absent from electron micrographs, as it is labile to bleach used to remove contaminating bacteria from C. parvum oocysts (27). We will refer to this layer as the outer veil, which is the term used for a structure with an identical appearance on the surface of the oocyst wall of another apicomplexan parasite, Toxoplasma gondii (10). At the center of the C. parvum oocyst wall is a protease-resistant and rigid bilayer that contains GalNAc (5, 23, 43). When excysting sporozoites break through the oocyst wall, the broken edges of this bilayer curl in, while the overall shape of the oocyst wall remains spherical.The inner, moderately electron-dense layer of the C. parvum oocyst wall is where the Cryptosporidium oocyst wall proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) have been localized with monoclonal antibodies (4, 20, 28, 32). COWPs, which have homologues in Toxoplasma, are a family of nine proteins that contain polymorphic Cys-rich and His-rich repeats (37, 46). Finally, on the inner surface of C. parvum oocyst walls are knob-like structures, which cross-react with an anti-oocyst monoclonal antibody (11).Like other apicomplexa (e.g., Toxoplasma and Plasmodium), sporozoites of C. parvum are slender, move by gliding motility, and release adhesins from apical organelles when they invade host epithelial cells (1, 8, 12, 39). Unlike other apicomplexa, C. parvum parasites are missing a chloroplast-derived organelle called the apicoplast (1, 47, 49). C. parvum sporozoites have on their surface unique mucin-like glycoproteins, which contain Ser- and Thr-rich repeats that are polymorphic and may be modified by O-linked GalNAc (4-7, 21, 25, 26, 30, 32, 34, 35, 43, 45). These C. parvum mucins, which are highly immunogenic and are potentially important vaccine candidates, include gp900 and gp40/gp15 (4, 6, 7, 25, 26). gp40/gp15 is cleaved by furin-like proteases into two peptides (gp40 and gp15), each of which is antigenic (42). gp900, gp40, and gp15 are shed from the surface of the C. parvum sporozoites during gliding motility (4, 7, 35).The studies presented here began with electron microscopic observations of C. parvum oocysts stained with ruthenium red (23), which revealed novel fibrils or tethers that extend radially from the inner surface of the oocyst wall to the outer surface of sporozoites. We hypothesized that at least some of these fibrillar tethers might be the antigenic mucins, which are abundant on the surface of C. parvum sporozoites. To test this hypothesis, we used mass spectroscopy to identify oocyst wall proteins and sporozoite glycoproteins and used deconvolving and immunoelectron microscopy (immuno-EM) with lectins and anti-C. parvum antibodies to directly label the tethers.     

Trypanosoma brucei UDP-Glucose:Glycoprotein Glucosyltransferase Has Unusual Substrate Specificity and Protects the Parasite from Stress

In this paper, we describe the range of N-linked glycan structures produced by wild-type and glucosidase II null mutant bloodstream form Trypanosoma brucei parasites and the creation and characterization of a bloodstream form Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase null mutant. These analyses highlight peculiarities of the Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase, including an unusually wide substrate specificity, ranging from Man₅GlcNAc₂ to Man₉GlcNAc₂ glycans, and an unusually high efficiency in vivo, quantitatively glucosylating the Asn263 N-glycan of variant surface glycoprotein (VSG) 221 and 75% of all non-VSG N glycosylation sites. We also show that although Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase is not essential for parasite growth at 37°C, it is essential for parasite growth and survival at 40°C. The null mutant was also shown to be hypersensitive to the effects of the N glycosylation inhibitor tunicamycin. Further analysis of bloodstream form Trypanosoma brucei under normal conditions and stress conditions suggests that it does not have a classical unfolded protein response triggered by sensing unfolded proteins in the endoplasmic reticulum. Rather, judging by its uniform Grp78/BiP levels, it appears to have an unregulated and constitutively active endoplasmic reticulum protein folding system. We suggest that the latter may be particularly appropriate for this organism, which has an extremely high flux of glycoproteins through its secretory pathway.Trypanosoma brucei is a protozoan parasite with two main proliferative stages in its life cycle: the procyclic form that grows in the tsetse fly midgut, and the bloodstream form that causes African sleeping sickness in humans and nagana in cattle. The bloodstream form is covered in a densely packed layer of 5 × 10⁶ glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) dimers. This coat protects the parasites from the alternative pathway of complement-mediated lysis, shields other cell surface proteins from the host immune system, and by the process of antigenic variation allows these parasites to persist for long periods in the host bloodstream (16, 54). The trypanosome genome contains several hundreds of silent VSG genes, most of which are pseudogenes in subtelomeric arrays (40). T. brucei evades host-acquired immunity through differential activation of these genes, which encode immunologically distinct GPI-anchored glycoproteins with one to three N glycosylation sites (27, 43).Protein N glycosylation is the most common covalent protein modification in eukaryotic cells (25). N-glycans contribute to “quality control” in the endoplasmic reticulum (ER) through a series of oligosaccharide-processing and lectin-binding reactions that contribute to protein folding and the targeting of misfolded glycoproteins for degradation (24, 47, 58, 65). As nascent protein chains enter the ER lumen, they are modified covalently in most eukaryotes by the addition of the Glc₃Man₉GlcNAc₂ core glycan via the action of oligosaccharyltransferase (OST). After deglucosylation by α-glucosidases I (GI) and II (GII), misfolded glycoproteins can be reglucosylated in the ER by the UDP-Glc:glycoprotein glucosyltransferase (UGGT), recreating the same monoglucosylated trimming intermediate generated by GII (9, 64, 66). UGGT behaves as a sensor of glycoprotein conformation and is a key constituent of ER quality control (50, 61). Calnexin and calreticulin are ER-resident lectin-like quality control chaperones that recognize the monoglucosylated glycans on glycoproteins and help them to fold properly through their close association with the oxidoreductase ERp57 (49). On reaching the proper tertiary structure, the glycoproteins are still substrates of GII but no longer of UGGT. Properly folded molecules, thus liberated from the lectins, are then free to continue their transit to the Golgi apparatus (64). When exposure to the folding machinery in the ER is not sufficient to promote a native conformation, proteins are eventually degraded by ER-associated degradation (49, 64).Most eukaryotes under conditions of stress, such as heat shock, undergo an unfolded protein response (UPR) that is triggered by sensing unfolded proteins in the ER. The UPR typically leads to increased expression of ER quality control components, such as calnexin and calreticulin and the ER chaperone Gpr78/BiP, as well inhibition of protein synthesis and cell cycle arrest (53, 57, 60).In contrast to the situation in most other eukaryotes, none of the trypanosomatid dolichol-linked oligosaccharides are capped with glucose residues, as these parasites do not synthesize the sugar donor dolichol-phosphate-glucose for these reactions (41, 59). The mature dolichol-phosphate-oligosaccharide species used for transfer to protein vary according to trypanosomatid species (17, 51, 52, 56). Therefore, in these organisms, monoglucosylated glycans are exclusively formed through UGGT-dependent glucosylation (12). Furthermore, trypanosomatids lack calnexin, which binds and participates in the refolding of glucosylated proteins, and it has been suggested that differences in the N-glycan precursor have profound effects on N-glycan-dependent quality control of glycoprotein folding and ER-associated degradation (4). These protozoa do not present a conventional OST complex and express only the catalytic stt3 protein subunit that, at least for the Trypanosoma cruzi and Leishmania major enzymes, shows little specificity toward the structure of the dolichol-phosphate-oligosaccharide donor (4, 11, 26, 31, 32, 45). In the case of T. brucei, while the insect-dwelling procyclic form makes and transfers Man₉GlcNAc₂-phosphate-dolichol (1), previous work from our group showed that the bloodstream form of the parasite transfers both Man₉GlcNAc₂ and Man₅GlcNAc₂ to VSG in a site-specific manner (29). Regarding ER folding and quality control, although in vitro assays have shown that T. cruzi and higher eukaryotic UGGTs exclusively glucosylate high-mannose glycans in misfolded glycoproteins (66), in T. brucei the UGGT and GII enzymes use Man₅GlcNAc₂ and Glc₁Man₅GlcNAc₂, respectively, as their substrates in the processing of VSG variant 221 (VSG221) (29). However, it could not be concluded from that study whether this apparent preference for atypical biantennary Man₅GlcNAc₂ and Glc₁Man₅GlcNAc₂ structures reflected the substrate specificity of the enzymes or the location of the glycosylation site in the VSG polypeptide chain (30).In this work, we further analyze the specificity and function of the UGGT/GII quality control system of T. brucei by analyzing the non-VSG N-glycans of our α-GII null mutant and creating and characterizing a T. brucei UGGT null mutant.     

Isolation and Characterization of a Novel Lysine Racemase from a Soil Metagenomic Library

A lysine racemase (lyr) gene was isolated from a soil metagenome by functional complementation for the first time by using Escherichia coli BCRC 51734 cells as the host and d-lysine as the selection agent. The lyr gene consisted of a 1,182-bp nucleotide sequence encoding a protein of 393 amino acids with a molecular mass of about 42.7 kDa. The enzyme exhibited higher specific activity toward lysine in the l-lysine-to-d-lysine direction than in the reverse reaction.Amino acids are the building blocks of proteins and play an important role in the regulation of the metabolism of living organisms. Among two enantiomers of naturally occurring amino acids, l-amino acids are predominant in living organisms, while d-amino acids are found in both free and bound states in various organisms like bacteria (36), yeasts (35), plants (47), insects (11), mammals (17), bivalves (39), and fish (28). The d-amino acids are mostly endogenous and produced by racemization from their counterparts by the action of a racemase. Thus, the amino acid racemases are involved in d-amino acid metabolism (29, 46). Since the discovery of alanine racemase in 1951 (42), several racemases toward amino acids, such as those for glutamate, threonine, serine, aspartate, methionine, proline, arginine, and phenylalanine, have been reported in bacteria, archaea, and eukaryotes, including mammals (1, 2, 15, 30, 31, 44). They are classified into two groups: pyridoxal 5′-phosphate (PLP)-dependent and PLP-independent enzymes (9, 36).Lysine racemase (Lyr, EC 5.1.1.5) was first reported in Proteus vulgaris ATCC 4669 (19) and proposed to be involved in the lysine degradation of bacterial cells (5, 19). Catabolism of lysine occurs via two parallel pathways. In one of the pathways, δ-aminovalerate is the key metabolite, whereas in the other l-lysine is racemized to d-lysine, and l-pipecolate and α-aminoadipate (AMA) are the key metabolites (5). d-Lysine catabolism proceeds through a series of cyclized intermediates which are necessary to regenerate an α-amino acid and comprise the following metabolites (AMA pathway): d-lysine→α-keto-ɛ-amino caproate→Δ¹-piperideine-2-carboxylate→pipecolate→Δ¹-piperideine-6-carboxylate→α-amino-δ-formylcaproate→α-AMA→α-ketoadipate (6, 7, 12, 27). The final product is converted to α-ketoglutarate via a series of coenzyme A derivatives and subsequently participates as an intermediate in the Krebs cycle. This pathway suggests that the biological function of d-lysine in the bacteria is that of a carbon or nitrogen source. Racemization of added l-lysine to d-lysine by whole cells of Proteus spp. and Escherichia spp. (19) and by the cell extract of Pseudomonas putida ATCC 15070 (5, 20) has been found. However, the enzyme has not been purified to homogeneity, and thus, its molecular and catalytic characteristics, including its gene structure, have not been elucidated. In this study, we explored a metagenomic library constructed from a garden soil to isolate a novel Lyr enzyme. After expression in Escherichia coli, the purified enzyme was characterized in terms of optimal pH and temperature, thermal stability, and racemization activity.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei

A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB³H₄ labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB³H₄ labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.The tsetse fly-transmitted protozoan parasite Trypanosoma brucei causes human sleeping sickness and the cattle disease Nagana in sub-Saharan Africa. The organism undergoes a complex life cycle between the mammalian host and the insect, tsetse, vector. The bloodstream form of the parasite expresses a dense monolayer of glycosylphosphatidylinositol (GPI)- anchored variant surface glycoprotein dimers and avoids specific immune responses through antigenic variation (32, 47). Following ingestion in a blood meal, the parasites differentiate into procyclic-form parasites that colonize the tsetse midgut. The procyclic trypanosomes express a radically different cell surface coat that includes about 3 × 10⁶ procyclin glycoproteins (28, 36, 37) and about 1 × 10⁶ poly-N-acetyllactosamine-containing free GPIs (19, 29, 39, 55). The procyclins are polyanionic, rod-like (38, 50) proteins encoded by procyclin genes. In T. brucei strain 427, used in this study, the parasites contain (per diploid genome) two copies of the GPEET1 gene, encoding 6 Gly-Pro-Glu-Glu-Thr repeats; one copy each of the EP1-1 and EP1-2 genes, encoding EP1 procyclins with 30 and 25 Glu-Pro repeats, respectively; two copies of the EP2-1 gene, encoding EP2 procyclin with 25 Glu-Pro repeats; and two copies of the EP3-1 gene, encoding EP3 procyclin with 22 Glu-Pro repeats (1). The EP1 and EP3 procyclins contain a single N-glycosylation site, occupied exclusively by a conventional Man₅GlcNAc₂ oligosaccharide, at the N-terminal side of the Glu-Pro repeat domain (1, 50). Whereas neither EP2 nor GPEET procyclin is N-glycosylated, GPEET1 procyclin is phosphorylated on six out of seven Thr residues (25). In culture, the procyclin expression profile depends on the carbon source (56) and metabolic state of the cells (27), and in the tsetse fly, there appears to be a program of procyclin expression such that GPEET procyclin is expressed early, giving way to EP1 and EP3 procyclin expression (2, 54). GPEET and EP procyclins contain similar GPI membrane anchors. These are based on the ubiquitous ethanolamine-P-6Manα1-2Manα1-6Manα1-4GlcNα1-6PI core (where, in this case, the PI lipid is a 2-O-acyl-myo-inositol-1-P-sn-2-lyso-1-O-acylglycerol structure [50]), but they also contain the largest and most complex known GPI side chains. These side chains are large poly-disperse-branched poly-N-acetyllactosamine structures (with an average of about 8 to 12 repeats, depending on the preparation) that can terminate with α2- and α3-linked sialic acid residues (9, 50). Sialic acid is transferred from serum sialoglycoconjugates to terminal β-galactosidase residues by the action of a cell surface GPI-anchored trans-sialidase enzyme (7, 26, 34). The trans-sialylation of surface components plays a role in the successful colonization of the tsetse fly (29). In vivo, the N termini of the procyclins are removed by tsetse fly gut proteases (2), though the role of this event is unclear (20) and it is thought that the underlying (protease-resistant) anionic repeat units and associated GPI anchor side chains might protect the parasite from the approach of tsetse fly gut hydrolases (2).The cell surface architecture of procyclic trypanosomes has been manipulated by the gene knockout of the procyclin genes themselves (55, 57), by galactose starvation (39), and by the knockout or knockdown of genes encoding enzymes of the GPI biosynthetic pathway, i.e., TbGPI10, TbGPI8, and TbGPI12 (11, 19, 29, 30). The procyclin TbGPI10 and TbGPI8 knockouts all resulted in parasites devoid of GPI-anchored procyclins, but this was compensated for by an upregulation in free GPI expression. However, the TbGPI12 null mutants that cannot synthesize GPI structures beyond GlcNAc-PI, revealed the presence of previously unidentified non-GPI-anchored surface coat components. In this paper, we characterize the fate of non-GPI-anchored procyclin protein and characterize the non-GPI-anchored surface coat components.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).