Three genes encode distinct AP33 proteins involved in Trichomonas vaginalis cytoadherence

Adherence to host cells is essential for the initiation and maintenance of infection by mucosal pathogens. The protozoan Trichomonas vaginalis colonizes the human urogenital tract via four surface proteins (AP65, AP51, AP33 and AP23). To characterize AP33 further, six cDNA clones were examined. Restriction mapping indicated that the six clones represented three similar genes. Southern analysis confirmed the existence of three single-copy AP33 genes and suggested a semi-conservative genomic arrangement between T. vaginalis isolates. Analysis of full-length sequences determined that each contained a 930 bp open reading frame encoding a protein of approximately 33 000 Da. Sequence comparisons revealed a high degree of identity at both the DNA and the protein levels. N-terminal protein sequencing established the presence of leader peptides. Each of the three full-length recombinant proteins had a predicted pI of approximately 10, which was verified experimentally for the T. vaginalis AP33 adhesin. A database search revealed that AP33 had significant identity to the succinyl-CoA synthetase α-subunit of several different organisms and virtually 100% identity to the reported T. vaginalis subunit. Unlike commercially purchased enzyme, the recombinant proteins retained adhesive properties equal to the natural T. vaginalis AP33. The characteristics of the AP33 protein are similar to those of the other adhesins and emphasize a complex host–parasite relationship.     

The mechanisms and molecules involved in cytoadherence and pathogenesis of Trichomonas vaginalis

All urogenital mucosal pathogens must overcome host factors and responses in order to colonize the mucosa and establish infection. These include the extensive mucus layer, nutrient-limiting conditions antibody responses and the constant fluid flow of the vagina. In this article, John Alderete, Michael Lehker and Rossana Arroyo review the recent work describing the specificity by which the protozoan. Trichomonas vaginalis, adheres to the vaginal epithelial cells via four surface proteins. They discuss three distinct signals that may be received by the parasite upon recognition and binding to these cells, illustrating the type of adaptive responses evolved in the establishment and maintenance of infection.     

Cloning and characterization of three genes encoding Qb-SNARE proteins in rice

Qb-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and function as important components of the vesicle trafficking machinery in eukaryotic cells. Here, we report three novel plant SNARE (NPSN) genes isolated from rice and named OsNPSN11, OsNPSN12 and OsNPSN13. They have about 70% nucleotide identity over their entire coding regions and similar genomic organization with ten exons and nine introns in each gene. Multiple alignment of deduced amino acid sequences indicate that the OsNPSNs proteins are homologous to AtNPSNs from Arabidopsis, containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region. Semi-quantitative RT-PCR assays showed that the OsNPSNs were ubiquitously and differentially expressed in roots, culms, leaves, immature spikes and flowering spikes. The expression of OsNPSNs was significantly activated in rice seedlings treated with H₂O₂, but down-regulated under NaCl and PEG6000 stresses. Transient expression method in onion epidermal cells revealed that OsNPSNs were located in the plasma membrane. Transformed yeast cells with OsNPSNs had better growth rates than empty-vector transformants when cultured on either solid or liquid selective media containing various concentrations of H₂O₂, but more sensitive to NaCl and mannitol stresses. The 35S:OsNPSN11 transgenic tobacco also showed more tolerance to H₂O₂ and sensitivity to NaCl and mannitol than non-transgenic tobacco. These results indicate that OsNPSNs may be involved in different aspects of the signal transduction in plant and yeast responses to abiotic stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning and expression of two genes encoding auxin-binding proteins from tobacco

Two genes encoding the auxin-binding protein (ABP1) of tobacco (Nicotiana tabacum L.), both of which possess the characteristics of a luminal protein of the endoplasmic reticulum (ER), were isolated and sequenced. These genes were composed of at least five exons and four introns. The two coding exons showed 95% sequence homology and coded for two precursor proteins of 187 amino acid residues with molecular masses of 21 256 and 21 453 Da. The deduced amino acid sequences were 93% identical and both possessed an amino-terminal signal peptide, a hydrophilic mature protein region with two potential N-glycosylation sites and a carboxyl-terminal sorting signal, KDEL, for the ER. Restriction mapping of the cDNAs encoding tobacco ABP1, previously purified by amplification of tobacco cDNA libraries by polymerase chain reaction (PCR) using specific primers common to both genes, indicated that both genes were expressed, although one was expressed at a higher level than the other. Genomic Southern blot hybridization showed no other homologous genes except for these two in the tobacco genome. The apparent molecular mass of the mature form of tobacco ABP1 was revealed to be 25 kDa by SDS polyacrylamide gel electrophoresis using affinity-purified anti (tobacco ABP1) antibodies raised against a fusion protein with maltose-binding protein. Expression of the recombinant ABP1 gene in transgenic tobacco resulted in accumulation of the 25 kDa protein. A single point mutation of an amino acid residue at either of the two potential N-glycosylation sites resulted in a decrease in the apparent molecular mass and produced a 22 kDa protein. Mutations at both sites resulted in the formation of a 19.3 kDa protein, suggesting that tobacco ABP1 is glycosylated at two asparagine residues.     

Signalling of Trichomonas vaginalis for amoeboid transformation and adhesin synthesis follows cytoadherence

The cytoadherence of Trichomonas vaginalis, the sexually transmitted flagellated protozoan, to vaginal epithelial cells (VECs) is the key to infection. Electron microscopy revealed that in vitro-grown parasites having typical globular shape transformed rapidly after contact with VECs into thin, flat, amoeboid cells, maximizing the area of adhesion to the surface of VECs. Amoebic trichomonads formed filopodia and pseudopodia, which interdigitated at distinct sites on the plasma membrane of target cells. In contrast, the amoeboid transformation did not occur for T. vaginalis interacting with He La cells, the previously used in vitro host model cell. Initial parasitism of VECs by a single organism was followed by establishment of a monolayer of trichomonads on the host cell. Finally, parasites adhering to either VECs or HeLa cells were induced to synthesize greater amounts of the four previously described adhesins. Therefore, distinct signals after contact with either epithelial cell type leads to the morphological transformation and/or induction of adhesin synthesis by T. vaginalis.     

Identification of antigenic proteins in Trichomonas vaginalis

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage λ Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, α-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.     

Cloning and characterization of two Lactobacillus casei genes encoding a cystathionine lyase

Volatile sulfur compounds are key flavor compounds in several cheese types. To better understand the metabolism of sulfur-containing amino acids, which certainly plays a key role in the release of volatile sulfur compounds, we searched the genome database of Lactobacillus casei ATCC 334 for genes encoding putative homologs of enzymes known to degrade cysteine, cystathionine, and methionine. The search revealed that L. casei possesses two genes that putatively encode a cystathionine beta-lyase (CBL; EC 4.4.1.8). The enzyme has been implicated in the degradation of not only cystathionine but also cysteine and methionine. Recombinant CBL proteins catalyzed the degradation of L-cystathionine, O-succinyl-L-homoserine, L-cysteine, L-serine, and L-methionine to form alpha-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.     

Cloning and characterization of two Serratia marcescens genes involved in core lipopolysaccharide biosynthesis.

Bacteriocin 28b from Serratia marcescens binds to Escherichia coli outer membrane proteins OmpA and OmpF and to lipopolysaccharide (LPS) core (J. Enfedaque, S. Ferrer, J. F. Guasch, J. Tomás, and M. Requé, Can. J. Microbiol. 42:19-26, 1996). A cosmid-based genomic library of S. marcescens was introduced into E. coli NM554, and clones were screened for bacteriocin 28b resistance phenotype. One clone conferring resistance to bacteriocin 28b and showing an altered LPS core mobility in polyacrylamide gel electrophoresis was found. Southern blot experiments using DNA fragments containing E. coli rfa genes as probes suggested that the recombinant cosmid contained S. marcescens genes involved in LPS core biosynthesis. Subcloning, isolation of subclones and Tn5tac1 insertion mutants, and sequencing allowed identification of two apparently cotranscribed genes. The deduced amino acid sequence from the upstream gene showed 80% amino acid identity to the KdtA protein from E. coli, suggesting that this gene codes for the 3-deoxy-manno-octulosonic acid transferase of S. marcescens. The downstream gene (kdtX) codes for a protein showing 20% amino acid identity to the Haemophilus influenzae kdtB gene product. The S. marcescens KdtX protein is unrelated to the KdtB protein of E. coli K-12. Expression of the kdtX gene from S. marcescens in E. coli confers resistance to bacteriocin 28b.     

Sequence analysis of genes encoding ribosomal proteins of amitochondriate protists: L1 of Trichomonas vaginalis and L29 of Giardia lamblia

Two genes encoding the ribosomal proteins were cloned and sequenced from amitochondriate protists, L1 (L10a in mammalian nomenclature) from Trichomonas vaginalis and L29 (L35 in mammalian nomenclature) from Giardia lamblia. The deduced amino acid sequences were analyzed by sequence alignments and phylogenetic reconstructions. Both the T. vaginalis L1 and the G. lamblia L29 displayed eukaryotic sequence features, when compared with all the homologs from the three primary kingdoms.     

Tubulins in Trichomonas vaginalis: molecular characterization of alpha-tubulin genes, posttranslational modifications, and homology modeling of the tubulin dimer

We have isolated and analysed an alpha-tubulin-encoding gene (atub1) in an early-diverging eukaryote, Trichomonas vaginalis. The complete atub1 open reading frame included 1.356 bp encoding a polypeptide of 452 amino-acyl residues. A second alpha-tubulin gene (atub2) was amplified by PCR using primers derived from consensus alpha-tubulin amino acid sequences. Both T. vaginalis alpha-tubulin sequences showed high identity to those described in other parabasalids (94.4%-97.3%), and exhibited a high degree of similarity to sequences from Metazoa (such as pig brain) and diplomonads (such as Giardia). Despite large evolutionary distances previously observed between trichomonads and mammals, the three-dimensional model of the T. vaginalis tubulin dimer was very similar to that of pig brain. Possible correlations between alpha-tubulin sequences and posttranslational modifications (PTMs) were examined. Our observations corroborated previous data obtained in T. vaginalis using specific anti-PTMs antibodies. As described in the related species Tritrichomonas mobilensis, microtubules are likely acetylated, non-tyrosinated, glutamylated, and non-glycylated in T. vaginalis. Evolutionary considerations concerning the time of appearance of these tubulin PTMs are also discussed since trichomonads are potentially one of the earliest diverging eukaryotic lineages.     

Prediction and analysis of paralogous proteins in Trichomonas vaginalis genome

Trichomonas vaginalis causes trichomoniasis, second most sexually transmitted disease. The genome sequence draft of T. vaginalis was published by The Institute of Genomic Research reveals an abnormally large genome size of 160 Mb. It was speculated that a significant portion of the proteome contains paralogous proteins. The present study was aimed at identification and analysis of the paralogous proteins. The all against all search approach is used to identify the paralogous proteins. The dataset of proteins was retrieved from TIGR and TrichDB FTP server. The BLAST-P program performed all against all database searches against the protein database of Trichomonas vaginalis available at NCBI genome database. In the present study about 50,000 proteins were searched where 2,700 proteins were found to be paralogous under the rigid selection criteria. The Pfam database search has identified significant number of paralogous proteins which were further categorized among different 1496 paralogous protein in pfam families, 1027 paralogous protein contains domain, 60 proteins were having different repeats and 1092 paralogous protein sequences of clans. Such identification and functional annotation of paralogous proteins will also help in removing paralogous proteins from possible drug targets in future. Presence of huge number of paralogous proteins across wide range of gene families and domains may be one of the possible mechanisms involved in the T. vaginalis genome expansion and evolution.     

Centrin protein and genes in Trichomonas vaginalis and close relatives

Anti-centrin monoclonal antibodies 20H5 and 11B2 produced against Clamydomononas centrin decorated the group of basal bodies as well as very closely attached structures in all trichomonads studied and in the devescovinids Foaina and Devescovina. Moreover, these antibodies decorated the undulating membrane in Trichomonas vaginalis, Trichomitus batrachorum, and Tritrichomonas foetus, and the cresta in Foaina. Centrin was not demonstrated in the dividing spindle and paradesmosis. Immunogold labeling, both in pre- and post-embedding, confirmed that centrin is associated with the basal body cylinder and is a component of the nine anchoring arms between the terminal plate of flagellar bases and the plasma-membrane. Centrin is also associated with the hook-shaped fibers attached to basal bodies (F1, F3), the X-fiber, and along sigmoid fibers (F2) at the pelta-axostyle junction, which is the microtubule organizing center for pelta-axostyle microtubules. There was no labeling on the striated costa and parabasal fibers nor on microtubular pelta-axostyle, but the fibrous structure inside the undulating membrane was labeled in T. vaginalis. Two proteins of 22-20 kDa corresponding to the centrin molecular mass were recognized by immunoblotting using these antibodies in the three trichomonad species examined. By screening a T. vaginalis cDNA library with 20H5 antibody, two genes encoding identical protein sequences were found. The sequence comprises the 4 typical EF-hand Ca++-binding domains present in every known centrin. Trichomonad centrin is closer to the green algal cluster (70% identity) than to the yeast Cdc31 cluster (55% identity) or the Alveolata cluster (46% identity).     

Cloning and characterization of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation.

Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.