Whole-Exome Sequencing Indicated New Candidate Genes Associated with Unilateral Cryptorchidism in Pigs.

INTRODUCTION: Cryptorchidism is a hereditary anomaly characterized by the incomplete descent of one or both testicles to the scrotum. One of the challenges of this anomaly is that the retained testicle maintains its endocrine function. As a consequence, cryptorchid animals produce hormone-tainted meat in comparison to castrated animals and are likely to be more aggressive. Cryptorchidism can lead to reduced animal welfare outcomes and cause economic losses. Identifying genetic markers for cryptorchidism is an essential step toward mitigating these negative outcomes and may facilitate genome manipulation to reduce the occurrence of cryptorchidism. Attempts to identify such markers have used genome-wide association studies. Using whole-exome sequencing, we aimed to identify single nucleotide polymorphisms (SNPs) in the coding regions of cryptorchid pigs and to characterize functional pathways concerning these SNPs. METHODS: DNA was extracted and sequenced from 5 healthy and 5 cryptorchid animals from the Landrace breed, using the Illumina HiSeq 2500 platform. Data were pre-processed using the SeqyClean tool and further mapped against the swine reference genome (Sus scrofa 11.1) using BWA software. GATK was used to identify polymorphisms (SNPs and InDels), which were annotated using the VEP tool. Network prediction and gene ontology enrichment analysis were conducted using the Cytoscape platform, and STRING software was used for visualization. RESULTS: A total of 63 SNPs were identified across the genes PIGB, CCPG1, COMMD9, LDLRAD3, TRIM44, MYLPF, SEPTIN, ZNF48, TIA1, FAIM2, KRT18, FBP1, FBP2, CTSL, DAPK1, DHX8, GPR179, DEPDC1B, ENSSSCG00000049573, ENSSSCG00000016384, ENSSSCG00000022657, ENSSSCG00000038825, and ENSSSCG00000001229. Using pathway enrichment analyses and network prospection, we have identified the following significant adjusted p value threshold of 0.001 involved with the biological function pathways of estrogen signaling, cytoskeleton organization, and the pentose phosphate pathway. CONCLUSION: Our data suggest the involvement of new SNPs and genes in developing cryptorchidism in pigs. However, further studies are needed to validate our results in a larger cohort population. Variations in the GPR179 gene, with implications at the protein level, may be associated with the appearance of this anomaly in the swine. Finally, we are showing that the estrogen signaling pathway may be involved in the pathophysiological mechanisms of this congenital anomaly as previously reported in GWAS.

Mannosyltransferase (GPI-14) overexpression protects promastigote and amastigote forms of Leishmania braziliensis against trivalent antimony.

BACKGROUND: Glycosylphosphatidylinositol is a surface molecule important for host-parasite interactions. Mannosyltransferase (GPI-14) is an essential enzyme for adding mannose on the glycosylphosphatidyl group. This study attempted to overexpress the GPI-14 gene in Leishmania braziliensis to investigate its role in the antimony-resistance phenotype of this parasite. RESULTS: GPI-14 mRNA levels determined by quantitative real-time PCR (qRT-PCR) showed an increased expression in clones transfected with GPI-14 compared to its respective wild-type line. In order to investigate the expression profile of the surface carbohydrates of these clones, the intensity of the fluorescence emitted by the parasites after concanavalin-A (a lectin that binds to the terminal regions of α-D-mannosyl and α-D-glucosyl residues) treatment was analyzed. The results showed that the clones transfected with GPI-14 express 2.8-fold more mannose and glucose residues than those of the wild-type parental line, indicating effective GPI-14 overexpression. Antimony susceptibility tests using promastigotes showed that clones overexpressing the GPI-14 enzyme are 2.4- and 10.5-fold more resistant to potassium antimonyl tartrate (SbIII) than the parental non-transfected line. Infection analysis using THP-1 macrophages showed that amastigotes from both GPI-14 overexpressing clones were 3-fold more resistant to SbIII than the wild-type line. CONCLUSIONS: Our results suggest the involvement of the GPI-14 enzyme in the SbIII-resistance phenotype of L. braziliensis.

Cell Autoaggregation, Biofilm Formation, and Plant Attachment in a Sinorhizobium meliloti lpsB Mutant.

Bacterial surface molecules are crucial for the establishment of a successful rhizobia-legume symbiosis, and, in most bacteria, are also critical for adherence properties, surface colonization, and as a barrier for defense. Rhizobial mutants defective in the production of exopolysaccharides (EPSs), lipopolysaccharides (LPSs), or capsular polysaccharides are usually affected in symbiosis with their plant hosts. In the present study, we evaluated the role of the combined effects of LPS and EPS II in cell-to-cell and cell-to-surface interactions in Sinorhizobium meliloti by studying planktonic cell autoaggregation, biofilm formation, and symbiosis with the host plant Medicago sativa. The lpsB mutant, which has a defective core portion of LPS, exhibited a reduction in biofilm formation on abiotic surfaces as well as altered biofilm architecture compared with the wild-type Rm8530 strain. Atomic force microscopy and confocal laser microscopy revealed an increase in polar cell-to-cell interactions in the lpsB mutant, which might account for the biofilm deficiency. However, a certain level of biofilm development was observed in the lpsB strain compared with the EPS II-defective mutant strains. Autoaggregation experiments carried out with LPS and EPS mutant strains showed that both polysaccharides have an impact on the cell-to-cell adhesive interactions of planktonic bacteria. Although the lpsB mutation and the loss of EPS II production strongly stimulated early attachment to alfalfa roots, the number of nodules induced in M. sativa was not increased. Taken together, this work demonstrates that S. meliloti interactions with biotic and abiotic surfaces depend on the interplay between LPS and EPS II.

Functional characterization of the Sporothrix schenckii Ktr4 and Ktr5, mannosyltransferases involved in the N-linked glycosylation pathway.

Sporothrix schenckii is one of the causative agents of the deep-seated mycosis sporotrichosis, a fungal infection with worldwide distribution. Fungus-specific molecules and biosynthetic pathways are potential targets for the development of new antifungal drugs. The MNT1/KRE2 gene family is a group of genes that encode fungus-specific Golgi-resident mannosyltransferases that participate in the synthesis of O-linked and N-linked glycans. While this family is composed of five and nine members in Candida albicans and Saccharomyces cerevisiae, respectively, the S. schenckii genome contains only three putative members. MNT1 has been previously characterized as an enzyme that participates in the synthesis of both N-linked and O-linked glycans. Here, we aimed to establish the functional role of the two remaining family members, KTR4 and KTR5, in the protein glycosylation pathways by using heterologous complementation in C. albicans mutants lacking genes of the MNT1/KRE2 family. The two S. schenckii genes restored defects in the elaboration of N-linked glycans, but no complementation of mutants that synthesize truncated O-linked glycans was observed. Therefore, our results suggest that MNT1 is the sole member with a role in O-linked glycan elaboration, whereas the three family members have redundant activity in the S. schenckii N-linked glycan synthesis.

Saccharomyces cerevisiae KTR4, KTR5 and KTR7 encode mannosyltransferases differentially involved in the N- and O-linked glycosylation pathways.

Saccharomyces cerevisiae is a model to understand basic aspects of protein glycosylation pathways. Although these metabolic routes have been thoroughly studied, there are still knowledge gaps; among them, the role of the MNT1/KRE2 gene family. This family is composed of nine members, with only six functionally characterized. The enzymes Ktr1, Ktr3, and Mnt1/Kre2 have overlapping activities in both O-linked and N-linked glycan synthesis; while Ktr2 and Yur1 participate exclusively in the elongation of the N-linked glycan outer chain. KTR6 encodes for a phosphomannosyltransferase that synthesizes the cell wall phosphomannan. Here, we aimed to establish the functional role of KTR4, KTR5 and KTR7 in the protein glycosylation pathways, by using heterologous complementation in Candida albicans null mutants lacking members of the MNT1/KRE2 gene family. The three S. cerevisiae genes restored defects in the C. albicans N-linked glycosylation pathway. KTR5 and KTR7 partially complemented a C. albicans null mutant with defects in the synthesis of O-linked glycans, and only KTR4 fully elongated the O-linked glycans like wild-type cells. Therefore, our results suggest that the three genes have a redundant activity in the S. cerevisiae N-linked glycosylation pathway, but KTR4 plays a major role in O-linked glycan synthesis.

Evaluation of Swallowing in Infants with Congenital Heart Defect

Introduction Surgical repair of congenital heart disease in the first years of life compromises the coordination of the suction, breathing, and swallowing functions. Objective To describe the alterations in swallowing found in infants with congenital heart defect during their hospitalization. Methods Prospective, cross-sectional study in a reference hospital for heart disease. The sample consisted of 19 postsurgical patients who underwent an evaluation of swallowing. The infants included were younger than 7 months and had a diagnosis of congenital heart defect and suspected swallowing difficulties. Results Of the 19 infants with congenital heart defect, the median age was 3.2 months. A significant association was found between suction rhythm and dysphagia (p = 0.036) and between oral-motor oral feeding readiness and dysphagia (p = 0.014). Conclusions The data suggest that dysphagia often occurs after surgery in infants with congenital heart defect. Infants with congenital heart defect had very similar behavior to preterm infants in terms of oral feeding readiness. .

Isolation of Sporothrix schenckii MNT1 and the biochemical and functional characterization of the encoded α1,2-mannosyltransferase activity.

Sporothrix (Sp.) schenckii is a pathogenic fungus that infects humans and animals, and is responsible for the disease named sporotrichosis. The cell wall of this fungus has glycoproteins with a high content of mannose and rhamnose units, which are synthesized by endoplasmic reticulum- and Golgi-localized glycosyltransferases. Little is known about the enzymic machinery involved in the synthesis of these oligosaccharides in Sp. schenckii, or the genes encoding these activities. This is in part because of the lack of an available genome sequence for this organism. Using a partial genomic DNA library we identified SsMNT1, whose predicted product has significant similarity to proteins encoded by members of the Saccharomyces (Sa.) cerevisiae KRE2/MNT1 gene family. In order to biochemically characterize the putative enzyme, SsMNT1 was heterologously expressed in the methylotrophic yeast Pichia pastoris. Recombinant SsMnt1 showed Mn(2+)-dependent mannosyltransferase activity and the ability to recognize as acceptors α-methyl mannoside, mannose, Man(5)GlcNAc(2) oligosaccharide and a variety of mannobiosides. The characterization of the enzymic products generated by SsMnt1 revealed that the enzyme is an α1,2-mannosyltransferase that adds up to two mannose residues to the acceptor molecule. Functional complementation studies were performed in Sa. cerevisiae and Candida albicans mutants lacking members of the KRE2/MNT1 gene family, demonstrating that SsMnt1 is involved in both the N- and O-linked glycosylation pathways, but not in phosphomannan elaboration.

Expression, purification and biochemical characterization of GumI, a monotopic membrane GDP-mannose:glycolipid 4-{beta}-D-mannosyltransferase from Xanthomonas campestris pv. campestris.

We describe the first biochemical characterization of the gumI gene product, an essential protein for xanthan polysaccharide synthesis. Cellular fractionation experiments reveal the presence of a protein associated with the membrane fraction, even in the absence of the other proteins responsible for the synthesis of glycolipid intermediates and the proteins involved in the polymerization and transport of the xanthan chains. By alkaline buffer extraction and detergent phase partitioning, GumI was categorized as a monotopic membrane protein. GumI was overexpressed in Escherichia coli, solubilized and purified in an active and stable form using a simple and reproducible two-step procedure. The purified recombinant GumI is a nonprocessive ß-mannosyltransferase that uses GDP-Man as a donor substrate and glucuronic acid-ß-1,2-mannose-α-1,3-glucose-ß-1,4-glucose-PP-polyisoprenyl as an acceptor. We also established the optimal biochemical conditions for GumI enzymatic activity. Sequence analysis revealed the presence of a conserved domain for glycosyltransferases (GTs) of the GT-B superfamily and homologous proteins in several prokaryote organisms. On the basis of this biochemical characterization, GumI may represent the founding member of a new GT family in the Carbohydrate-Active EnZymes classification.

A metabolic pathway leading to mannosylfructose biosynthesis in Agrobacterium tumefaciens uncovers a family of mannosyltransferases.

A metabolic pathway for biosynthesis of the nonreducing disaccharide mannosylfructose (beta-fructofuranosyl-alpha-mannopyranoside), an important osmolyte in Agrobacterium tumefaciens, was discovered. We have identified and functionally characterized two ORFs that correspond to genes (named mfpsA and mfppA) encoding the rare enzymes mannosylfructose-phosphate synthase and mannosylfructose-phosphate phosphatase, an associated phosphohydrolase. The mfpsA and mfppA genes are arranged in an operon structure, whose transcription is up-regulated by NaCl, resulting in the accumulation of mannosylfructose in the cells. Not only is the biosynthesis of mannosylfructose mechanistically similar to that of sucrose, but the corresponding genes for the biosynthesis of both disaccharides are also phylogenetic close relatives. Importantly, a protein phylogeny analysis indicated that mannosylfructose-phosphate synthase defines a unique group of mannosyltransferases.

Biosynthesis of glycoproteins in the human pathogenic fungus Sporothrix schenckii: synthesis of dolichol phosphate mannose and mannoproteins by membrane-bound and solubilized mannosyl transferases.

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg2+ and Mn2+. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A-Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26-112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.

Biosynthesis of glycoproteins in the pathogenic fungus Candida albicans: activation of dolichol phosphate mannose synthase by cAMP-mediated protein phosphorylation.

Following incubation with ATP and a cAMP-dependent protein kinase under optimal conditions of lipid acceptor, phospholipid and metal ion requirements, the transfer activity of partially purified dolichol phosphate mannose synthase (DPMS) increased about 60% and this activation correlated with a 50% increase in V(max) with no alteration in the apparent K(m) for GDP-Manose. Phosphorylation with [gamma-(32)P]ATP resulted in the labeling of several polypeptides, one of which exhibited the molecular weight of the enzyme (30 kDa) and was also recognized using a specific anti-DPMS monoclonal antibody. This and the fact that the phosphate label could be removed by an alkaline phosphatase indicate that Candida DPMS may be regulated by phosphorylation-dephosphorylation, a mechanism that has been proposed for the enzyme in other organisms.

Insulin up-regulates a Glc3Man9GlcNAc2-PP-Dol pool in capillary endothelial cells not essential for angiogenesis.

Endothelial cells line blood vessels, and their proliferation during neovascularization ( i.e., angiogenesis) is essential for a normal growth and development as well as for tumor progression and metastasis. Mechanistic details indicated that down-regulation of Glc(3)Man(9)GlcNAc(2)-PP-Dol level reduced angiogenesis and induced apoptosis in capillary endothelial cells (Martínez JA, Torres-Negrón I, Amigó LA, Banerjee DK, Cellular and Molec Biochem 45, 137-152 (1999)). Unlike in any other insulin-responsive cells, insulin reduced capillary endothelial cell proliferation by increasing the cell doubling time. But, when analyzed, the rate of lipid-linked oligosaccharide-PP-Dol (LLO) synthesis as well as its turnover ( i.e., t(1/2)) were increased in insulin treated cells. No major differences in their molecular size were observed. This corroborated with an enhanced glycosylation of Factor VIIIC, an N-linked glycoprotein (essential cofactor of the blood coagulation cascade) and a marker for the capillary endothelial cell. Increased LLO synthesis was independent of elevating either Dol-P level or Man-P-Dol synthase gene (dpm) transcription. Insulin however, enhanced 2-deoxy-glucose transport across the endothelial cell plasma membrane and caused increased secretion of Factor VIIIC, thus, supporting the existence of additional LLO pool(s), and arguing favorably that growth retardation of capillary endothelial cells by insulin turned a highly proliferative cell into a highly secretory cell.

Partial purification and characterization of a mannosyl transferase involved in O -linked mannosylation of glycoproteins in Candida albicans.

Incubation of a mixed membrane fraction of C. albicans with the nonionic detergents Nonidet P-40 or Lubrol solubilized a fraction that catalyzed the transfer of mannose either from endogenously generated or exogenously added dolichol-P-[14C]Man onto endogenous protein acceptors. The protein mannosyl transferase solubilized with Nonidet P-40 was partially purified by a single step of preparative nondenaturing electrophoresis and some of its properties were investigated. Although transfer activity occurred in the absence of exogenous mannose acceptors and thus depended on acceptor proteins isolated along with the enzyme, addition of the protein fraction obtained after chemical de-mannosylation of glycoproteins synthesized in vitro stimulated mannoprotein labeling in a concentration-dependent manner. Other de-mannosylated glycoproteins, such as yeast invertase or glycoproteins extracted from C. albicans, failed to increase the amount of labeled mannoproteins. Mannosyl transfer activity was not influenced by common metal ions such as Mg(2+), Mn(2+) and Ca(2+), but it was stimulated up to 3-fold by EDTA. Common phosphoglycerides such as phosphatidylglycerol and, to a lower extent, phosphatidylinositol and phosphatidylcholine enhanced transfer activity. Interestingly, coupled transfer activity between dolichol phosphate mannose synthase, i.e., the enzyme responsible for Dol-P-Man synthesis, and protein mannosyl transferase could be reconstituted in vitro from the partially purified transferases, indicating that this process can occur in the absence of cell membranes.

Dolichol phosphate mannose synthase is differentially expressed in male and female worms of Schistosoma mansoni.

The cDNA encoding the Schistosoma mansoni dolichol phosphate mannose synthase was completely sequenced, displaying the highest homology with Cricetulus griseus and Saccharomyces pombe genes. The Schistosome enzyme had a K(m) of 0.127 microM, a value that is within the range of those reported for several other species. Thin-layer chromatography of the radiolabelled schistosome lipid intermediate showed it was identical to dolichol-phosphate (C80-C105). Expression of dolichol phosphate mannose synthase of S. mansoni (SmDPMS) was analysed by Northern blot and quantified by semi-quantitative RT-PCR with cDNA from mature and immature male and female worms. Northern blot analysis revealed a single 1-kb band. Both approaches confirmed a higher level of expression in mature female worms, as compared to immature and male worms.

Regulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in Leishmania mexicana.

The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites.

Identification of essential amino acids in the bacterial alpha -mannosyltransferase aceA.

The alpha-mannosyltransferase AceA from Acetobacter xylinum belongs to the CaZY family 4 of retaining glycosyltransferases. We have identified a series of either highly conserved or invariant residues that are found in all family 4 enzymes as well as other retaining glycosyltransferases. These residues included Glu-287 and Glu-295, which comprise an EX(7)E motif and have been proposed to be involved in catalysis. Alanine replacements of each conserved residue were constructed by site-directed mutagenesis. The mannosyltransferase activity of each mutant was examined by both an in vitro transferase assay using recombinant mutant AceA expressed in Escherichia coli and by an in vivo rescue assay by expressing the mutant AceA in a Xanthomonas campestris gumH(-) strain. We found that only mutants K211A and E287A lost all detectable activity both in vitro and in vivo, whereas E295A retained residual activity in the more sensitive in vivo assay. H127A and S162A each retained reduced but significant activities both in vitro and in vivo. Secondary structure predictions of AceA and subsequent comparison with the crystal structures of the T4 beta-glucosyltransferase and MurG suggest that AceA Lys-211 and Glu-295 are involved in nucleotide sugar donor binding, leaving Glu-287 of the EX(7)E as a potential catalytic residue.

Dolichol phosphate is a rate-limiting factor in mannosyl transferase activity of adult male worms of Schistosoma mansoni.

The formation of mannolipid through catalysis by mannosyl transferase of adult females of Schistosoma mansoni was found to be 2-3-fold higher than male worms. In contrast, mannosyl transferase in immature females generated approximately the same amount of mannolipid as male worms, immature or not. Exogenous dolichol phosphate added to homogenates of male worms produced a stoichiometric increase in mannolipid formation. Saturating amounts of dolichol phosphate generated similar mannosyl transferase activities in male and female homogenates, showing that in S. mansoni, dolichol phosphate is the lipid intermediate in the glycosylation reaction and that this mannolipid is a rate-limiting substrate. Thin layer chromatography revealed that the mannolipid was identical in male and female worms. Adult males incubated with 14C-acetate synthesised several apolar compounds, one of which displayed a Rf identical to that of the mannolipid. When exposed to 14C-acetate treated males in vitro, untreated females were able to incorporate a compound, which partitioned in the same way as the mannolipid. The increased mannosyl transferase-dependent mannolipid formation in adult females may reflect a higher energy demand by these parasites, which is probably associated with oogenesis.

Glycosylphosphatidylinositol biosynthetic enzymes are localized to a stable tubular subcompartment of the endoplasmic reticulum in Leishmania mexicana.

Glycosylphosphatidylinositols (GPI) are essential components in the plasma membrane of the protozoan parasite Leishmania mexicana, both as membrane anchors for the major surface macromolecules and as the sole class of free glycolipids. We provide evidence that L.mexicana dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis, is localized to a distinct tubular subdomain of the endoplasmic reticulum (ER), based on the localization of a green fluorescent protein (GFP)-DPMS chimera and subcellular fractionation experiments. This tubular membrane (termed the DPMS tubule) is also enriched in other enzymes involved in GPI biosynthesis, can be specifically stained with the fluorescent lipid, BODIPY-C5-ceramide, and appears to be connected to specific subpellicular microtubules that underlie the plasma membrane. Perturbation of microtubules and DPMS tubule structure in vivo results in the selective accumulation of GPI anchor precursors, but not free GPIs. The DPMS tubule is closely associated morphologically with the single Golgi apparatus in non-dividing and dividing cells, appears to exclude luminal ER resident proteins and is labeled, together with the Golgi apparatus, with another GFP chimera containing the heterologous human Golgi marker beta1,2-N-acetylglucosaminyltransferase-I. The possibility that the DPMS-tubule is a stable transitional ER is discussed.

Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana.

The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all mannose additions in the biosynthesis of both the anchor and free GPIs, except for a alpha1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol-phosphate-mannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.

Isolation and nucleotide sequence of the GDP-mannose:cellobiosyl-diphosphopolyprenol alpha-mannosyltransferase gene from Acetobacter xylinum.

A genetic locus from Acetobacter xylinum involved in acetan polysaccharide synthesis has been characterized. The chromosomal region was identified by screening a genomic library of A. xylinum in a Xanthomonas campestris mutant defective in xanthan polysaccharide synthesis. The A. xylinum cosmid clone can functionally complement a xanthan-negative mutant. The polymer produced by the recombinant strain was found to be indistinguishable from xanthan. Insertion mutagenesis and subcloning of the cosmid clone combined with complementation studies allowed the identification of a 2.3-kb fragment of A. xylinum chromosomal DNA. The nucleotide sequence of this fragment was analyzed and found to contain an open reading frame (aceA) of 1,182 bp encoding a protein of 43.2 kDa. Results from biochemical and genetic analyses strongly suggest that the aceA gene encodes the GDP-mannose:cellobiosyl-diphosphopolyprenol alpha-mannosyltransferase enzyme, which is responsible for the transfer of an alpha-mannosyl residue from GDP-Man to cellobiosyl-diphosphopolyprenol. A search for similarities with other known mannosyltransferases revealed that all bacterial alpha-mannosyltransferases have a short COOH-terminal amino acid sequence in common.