Cloning and Characterization of Immunological Properties of Haemophilus influenzae Enolase.

Haemophilus influenzae is a common organism of the human upper respiratory tract; this bacterium is responsible of a wide spectrum for respiratory infections and can generate invasive diseases such as meningitis and septicemia. These infections are associated with H. influenzae encapsulated serotype b. However, the incidence of invasive disease caused by nontypeable H. influenzae (NTHi) has increased in the post-H. influenzae serotype b (Hib) vaccine era. Currently, an effective vaccine against NTHi is not available; due to this, it is important to find an antigen capable to confer protection against NTHi infection. In this study, 10 linear B cell epitopes and 13 CTL epitopes and a putative plasminogen-binding motif (252FYNKENGMY260) and the presence of enolase on the surface of different strains of H. influenzae were identified in the enolase sequence of H. influenzae. Both in silico and experimental results showed that recombinant enolase from H. influenzae is immunogenic that could induce a humoral immune response; this was observed mediating the generation of specific polyclonal antibodies anti-rNTHiENO that recognize typeable and nontypeable H. influenzae strains. The immunogenic properties and the superficial localization of enolase in H. influenzae, important characteristics to be considered as a new candidate for the development of a vaccine, were demonstrated.

Combination of neuron-specific enolase measurement and initial neurological examination for the prediction of neurological outcomes after cardiac arrest.

This study aimed to investigate the efficacy of the combination of neuron-specific enolase (NSE) measurement and initial neurological examination in predicting the neurological outcomes of patients with cardiac arrest (CA) by retrospectively analyzing data from the Korean Hypothermia Network prospective registry. NSE levels were recorded at 48 and 72 h after CA. The initial Full Outline of UnResponsiveness (FOUR) and Glasgow Coma Scale (GCS) scores were recorded. These variables were categorized using the scorecard method. The primary endpoint was poor neurological outcomes at 6 months. Of the 475 patients, 171 (36%) had good neurological outcomes at 6 months. The areas under the curve (AUCs) of the categorized NSE levels at 72 h, GCS score, and FOUR score were 0.889, 0.722, and 0.779, respectively. The AUCs of the combinations of categorized NSE levels at 72 h with categorized GCS scores and FOUR score were 0.910 and 0.912, respectively. Each combination was significantly higher than the AUC value of the categorized NSE level at 72 h alone (with GCS: p = 0.015; with FOUR: p = 0.026). Combining NSE measurement and initial neurological examination improved the prediction of neurological outcomes.

Dramatic Changes in Oligomerization Property Caused by Single Residue Deletion in Staphylococcus aureus Enolase.

Studies were conducted to understand the role of C-terminal lysine residues in the catalytic activity, structural stability and oligomeric properties of Staphylococcus aureus enolase. Interestingly, the S. aureus enolase, in solution, shows its presence as a stable dimer as well as the catalytically active fragile octamer. Compared to the hexa-histidine tagged S. aureus enolase (rSaeno), the deletion mutant showed the negligible difference in Km, but approximately 20-25% reduction in maximum reaction velocity (Vmax) and 2% reduction in turnover number were observed. These kinetic parameters indicate that K-434Δ deletion mutation does not drastically compromise the enzyme efficiency. The secondary structure and the octameric conformation of both the rSaeno and the K-434Δ mutant are very much stable between pH ranging from 6 to 9, temperatures ranging from 20 to 40 °C and in the presence of divalent metal ions Mg2+, Zn2+ and Mn2+. Under these conditions, the recombinant enzyme and the mutant are also catalytically very active. Intrinsic tryptophan fluorescence (320-380 nm) and CD spectral (195-260 nm) analysis revealed that the secondary structure and the surface architecture of the proteins are not majorly altered by the mutation. But, a significant correlation was observed between the time-dependent decrease in the catalytic activity and the oligomeric stability of rSaeno and K-434Δ mutant. The C-terminal lysine residues in the inter-dimer groove aid in folding and oligomerization of S. aureus enolase.

Effect of ions and inhibitors on the catalytic activity and structural stability of S. aureus enolase.

The glycolytic enzyme enolase of Staphylococcus aureus is a highly conserved enzyme which binds to human plasminogen thereby aiding the infection process. The cloning, over expression and purification of S. aureus enolase as well as the effect of various metals upon the catalytic activity and structural stability of the enzyme have been reported. The recombinant enzyme (rSaeno) has been purified to homogeneity in abundant amounts (60 mg/L of culture) and the kinetic parameters (Km = 0.23 +/- 0.013 x 10-3 M; Vmax = 90.98 +/- 0.00052 U/mg) and the optimum pH were calculated. This communication further reports that increasing concentrations of Na+ ions inhibit the enzyme while increasing concentrations of K+ ions were stimulatory. In case of divalent cations, it was found that Mg2+ stimulates the activity of rSaeno while the rest of the divalent cations (Zn2+, Mn2+, Fe2+, Cu2+, Ni2+ and Ca2+) lead to a dose-dependent loss in the activity with a total loss of activity in the presence of Hg2+ and Cr2+. The circular dichroism data indicate that other than Hg2+, Ni2+ and to a certain extent Cu2+, none of the other ions destabilized rSaeno. The inhibitory roles of fluorides, as well as neurotoxic compounds upon the catalytic activity of rSaeno, have also been studied. Conformational changes in rSaeno (induced by ions) were studied using partial trypsin digestion.

PtCu nanoprobe-initiated cascade reaction modulated iodide-responsive sensing interface for improved electrochemical immunosensor of neuron-specific enolase.

The method of transferring the immunoreaction from electrode surface to tube can greatly improve the analytical performance of electrochemical immunosensor. In this work, based on PtCu nanoprobe-mediated and iodide-catalyzed cascade reaction, an improved electrochemical immunosensor was elaborately designed for neuron-specific enolase (NSE) detection. PtCu nanoparticles combined with NSE antibody (Ab2) were used as immunoprobes to trigger cascade reaction. With high catalytic activity towards the oxidation of iodide, PtCu nanoprobes catalyzed iodide to iodine in the presence of H2O2, leading to the decrease of iodide. Subsequently, as a bridge between the tube and iodide-responsive sensing interface, the residual iodide in tube was employed to catalyze the transition from thiol substances (RSH) to disulfide substances (RSSR) on electrode surface. On the basis of this property, thiol-modified DNA (DNA-SH) and 6-mercaptohexanol (MCH) reacted with H2O2 and the residual iodide to form disulfide substances and detach from the electrode surface, causing the decrease of interface resistance in different degrees. Then square wave voltammetry (SWV) was applied for current signal of electrochemical sensing interface to achieve the quantitative detection of NSE. Under optimal conditions, this improved biosensor demonstrated excellent selectivity, stability and reproducibility with wide linear range from 0.0001 to 100 ng mL-1 and ultralow detection limit of 52.14 fg mL-1 for NSE, thus holding great promise for sensitive determination of tumor markers.

Cloning and identification of Bartonella α-enolase as a plasminogen-binding protein.

Bartonella infection is distributed worldwide with animal and public health. Recent studies have shown that host cells infection by Bartonella has a series of different infection stages, beginning with encounter and adherence to the cells. In this study, we expressed and purified recombinant Bartonella henselae (B. henselae) α-enolase. And we found that B. henselae α-enolase is highly conserved in Bartonella species. The interacting protein partners of B. henselae α-enolase were showed by String-11. The interactions between B. henselae α-enolase and human plasminogen were subsequently confirmed by ELISA, pull down, T7 phage display and molecular docking assays. And the plasminogen-binding sites of B. henselae α-enolase are predicted at 247FYKNGSYFY255. These findings will help elucidate and improve the understanding of the molecular mechanisms of Bartonella infection.

Featured Species-Specific Loops Are Found in the Crystal Structure of Mhp Eno, a Cell Surface Adhesin From Mycoplasma hyopneumoniae.

Enolase is an evolutionarily conserved enzyme involved in the processes of glycolysis and gluconeogenesis. Mycoplasma hyopneumoniae belongs to Mycoplasma, whose species are wall-less and among the smallest self-replicating bacteria, and is an important colonizing respiratory pathogen in the pig industry worldwide. Mycoplasma hyopneumoniae enolase (Mhp Eno) expression is significantly increased after infection and was previously found to be a virulence factor candidate. Our studies show that Mhp Eno is a cell surface-localized protein that can adhere to swine tracheal epithelial cells (STECs). Adhesion to STECs can be specifically inhibited by an Mhp Eno antibody. Mhp Eno can recognize and interact with plasminogen with high affinity. Here, the first crystal structure of the mycoplasmal enolase from Mycoplasma hyopneumoniae was determined. The structure showed unique features of Mhp Eno in the S3/H1, H6/S6, H7/H8, and H13 regions. All of these regions were longer than those of other enolases and were exposed on the Mhp Eno surface, making them accessible to host molecules. These results show that Mhp Eno has specific structural characteristics and acts as a multifunctional adhesin on the Mycoplasma hyopneumoniae cell surface.

Black phosphorus based fiber optic biosensor for ultrasensitive cancer diagnosis.

We propose the first black phosphorus (BP) - fiber optic biosensor for ultrasensitive diagnosis of human neuron-specific enolase (NSE) cancer biomarkers. A novel optical-nano configuration has been exploited by integrating BP nanosheets with a largely tilted fiber grating (BP-TFG), where the BP is bio-functionalized by the poly-L-lysine acting as a critical cross-linker to facilitate bio-nano-photonic interface with extremely enhanced light-matter interaction. BP nanosheets are synthesized by a liquid ultrasonication-based exfoliation and deposited on fiber device by an in-situ layer-by-layer method. The BP-induced optical modulation effects in terms of thickness-tunable feature, polarization-dependence and enhanced light-matter interaction are experimentally investigated. The anti-NSE immobilized BP-TFG biosensor has been implemented to detect NSE biomarkers demonstrating ultrahigh sensitivity with limit of detection down to 1.0 pg/mL, which is 4 orders magnitude lower than NSE cut-off value of small cell lung cancer. The enhanced sensitivity of BP-TFG is 100-fold higher than graphene oxide or AuNPs based biosensors. We believe that BP-fiber optic configuration opens a new bio-nano-photonic platform for the applications in healthcare, biomedical, food safety and environmental monitoring.

QCQuan: A Web Tool for the Automated Assessment of Protein Expression and Data Quality of Labeled Mass Spectrometry Experiments.

In the context of omics disciplines and especially proteomics and biomarker discovery, the analysis of a clinical sample using label-based tandem mass spectrometry (MS) can be affected by sample preparation effects or by the measurement process itself, resulting in an incorrect outcome. Detection and correction of these mistakes using state-of-the-art methods based on mixed models can use large amounts of (computing) time. MS-based proteomics laboratories are high-throughput and need to avoid a bottleneck in their quantitative pipeline by quickly discriminating between high- and low-quality data. To this end we developed an easy-to-use web-tool called QCQuan (available at qcquan.net ) which is built around the CONSTANd normalization algorithm. It automatically provides the user with exploratory and quality control information as well as a differential expression analysis based on conservative, simple statistics. In this document we describe in detail the scientifically relevant steps that constitute the workflow and assess its qualitative and quantitative performance on three reference data sets. We find that QCQuan provides clear and accurate indications about the scientific value of both a high- and a low-quality data set. Moreover, it performed quantitatively better on a third data set than a comparable workflow assembled using established, reliable software.

Recognition of protein biomarkers using epitope-mediated molecularly imprinted films: Histidine or cysteine modified epitopes?

This research aims to engineer molecularly imprinted polymer (MIP)-based synthetic receptors for the molecular recognition of neuron specific enolase (NSE) biomarker. The synthetic peptide derived from the NSE was synthesized along with its cysteine and histidine modified versions. The modified peptides were utilized as templates for molecular imprinting, which was achieved by combination of epitope- and electrochemical surface imprinting strategy. The subsequently generated imprinted cavities were used for the detection of the NSE derived peptide and NSE. The imprints created with cysteine (CME) and histidine modified epitopes (HME) could detect the peptide in a concentration range of 2-128 µM and 15.6 nM to 128 µM, respectively. The recognition of NSE was achieved by the same imprints in a linear range of 1-64 ng mL-1 (CME) and 0.25-64 ng mL-1 (HME), respectively. The target molecules bound to the control polymer very weakly, confirming the high selectivity of the MIP cavities. Selectivity studies resulted in imprinting factors of 8.8 and 11 for the CME and HME imprints, respectively. The affinity analyses provided dissociation constants of 2.3 × 10-10 M and 3 × 10-11 M for NSE recognition using the corresponding epitope imprints. Cross-reactivity studies with non-specific molecules proved high specificity of the artificial receptors for the targets.

Molecular cloning of enolase from Trichinella spiralis and the protective immunity in mice.

Trichinella spiralis, the main pathogen of trichinosis, infects a wide range of mammalian hosts and is one of the most widespread parasites worldwide. For parasites, glycolysis is the most important way to generate energy. Previous studies showed that some enzymes involved in the glycolytic pathway play roles in regulation the host immunity. In this paper, enolase from T. spiralis was cloned and the protective potentials were studied. One hundred and sixty ICR mice were divided into four groups and vaccinated with recombinant enolase (pET-ENO), eukaryotic recombinant plasmid encoding enolase (pVAX1-ENO) and negative controls (pVAXl and PBS), respectively. Two weeks after the second immunization, each mouse was challenged orally with 200 muscle larvae (MLs) of T. spiralis. Results showed that mice vaccinated with pET-ENO and pVAX1-ENO induced specific antibodies of IgG, IgA, IgM, but no IgE. Subclasses of IgG antibodies showed that mice immunized with recombinant protein and recombinant plasmids induced a Th1/Th2 immune response. Concentrations of serum cytokines were detected and showed significant increase of IFN-γ, IL-4 and TGFß1, while IL-17 in each group was not significantly different. Flow cytometric analysis showed significant increase of CD4+ and CD8+ T lymphocytes in the groups immunized with recombinant protein and recombinant plasmids. Challenge infection demonstrated that immunized groups had a reduced number of worm burdens. The reductions of larvae per gram muscle (LPG) in pET-ENO and pVAX1-ENO group were 17.7% and 15.8% when compared with PBS control.

Expression, purification, and biological characterization of Anaplasma phagocytophilum enolase.

The obligate intracellular bacteria Anaplasma phagocytophilum is the etiological agent of human granulocytic anaplasmosis (HGA), an acute febrile tick-borne disease. A. phagocytophilum has a complex lifecycle within both vertebrate reservoirs and tick vectors, and employs a range of different molecules to infect and multiply within the host cells. Enolase is an essential glycolytic enzyme in intracellular glucose metabolism, but is also a multifunctional protein expressed on the pathogen surface, that binds to and promotes plasminogen conversion to plasmin. In this study, we generated recombinant ApEno protein (rApEno), and confirmed that rApEno retains its enzymatic activity. Furthermore, we demonstrated that rApEno binds to human plasminogen, and that this binding could be significantly reduced in the presence of lysine analogs (ε-aminocaproic acid). Additionally, rApEno promotes plasminogen to plasmin conversion in the presence of plasminogen activator. In conclusion, A. phagocytophilum enolase is a multifunctional protein which can catalyze the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate, and facilitate binding to host plasminogen.

Sensing platform for neuron specific enolase based on molecularly imprinted polymerized ionic liquids in between gold nanoarrays.

A molecularly imprinted electrochemical sensor for neuron specific enolase (NSE) was developed by electrochemical polymerizing ionic liquid, which was functionalized with pyrrole moiety, in between gold nanoarrays. A well-defined 3D structured gold nanoarray was fabricated on a glassy carbon electrode (GCE) surface by using template-assisted electrochemical deposition technique. 1-(3-mercaptopropyl)-3-vinyl-imidazolium tetrafluoroborate was self-assembled onto the surface of gold nanoarrays to produce active sites for anchoring the molecularly imprinted film. Subsequently, an electrochemical polymerization procedure was carried out in an aqueous solution containing 1,3-di(3-N-pyrrolpropyl)imidazolium bromine ionic liquid and neuron specific enolase (NSE). After removing NSE templates, a molecularly imprinted sensor was successfully fabricated. The sensor showed high selectivity and sensitivity towards NSE, produced a linear response in the concentration range from 0.01 to 1.0ngmL-1 and had a detection limit of 2.6pgmL-1 with an incubation time of 15min. The developed sensor was demonstrated successful in determining NSE in clinical serum samples.

Employing proteomic analysis to compare Paracoccidioides lutzii yeast and mycelium cell wall proteins.

Paracoccidioidomycosis is an important systemic mycosis caused by thermodimorphic fungi of the Paracoccidioides genus. During the infective process, the cell wall acts at the interface between the fungus and the host. In this way, the cell wall has a key role in growth, environment sensing and interaction, as well as morphogenesis of the fungus. Since the cell wall is absent in mammals, it may present molecules that are described as target sites for new antifungal drugs. Despite its importance, up to now few studies have been conducted employing proteomics in for the identification of cell wall proteins in Paracoccidioides spp. Here, a detailed proteomic approach, including cell wall-fractionation coupled to NanoUPLC-MSE, was used to study and compare the cell wall fractions from Paracoccidioides lutzii mycelia and yeast cells. The analyzed samples consisted of cell wall proteins extracted by hot SDS followed by extraction by mild alkali. In summary, 512 proteins constituting different cell wall fractions were identified, including 7 predicted GPI-dependent cell wall proteins that are potentially involved in cell wall metabolism. Adhesins previously described in Paracoccidioides spp. such as enolase, glyceraldehyde-3-phosphate dehydrogenase were identified. Comparing the proteins in mycelium and yeast cells, we detected some that are common to both fungal phases, such as Ecm33, and some specific proteins, as glucanase Crf1. All of those proteins were described in the metabolism of cell wall. Our study provides an important elucidation of cell wall composition of fractions in Paracoccidioides, opening a way to understand the fungus cell wall architecture.

A wireless point-of-care testing system for the detection of neuron-specific enolase with microfluidic paper-based analytical devices.

Neuron-specific enolase (NSE) had clinical significance on diagnosis, staging, monitoring effect and judging prognosis of small cell lung cancer. Thus, there had a growing demand for the on-site testing of NSE. Here, a wireless point-of-care testing (POCT) system with electrochemical measurement for NSE detection was developed and verified. The wireless POCT system consisted of microfluidic paper-based analytical devices (µPADs), electrochemical detector and Android's smartphone. Differential pulse voltammetry (DPV) measurement was adopted by means of electrochemical detector which including a potentiostat and current-to-voltage converter. µPADs were modified with nanocomposites synthesized by Amino functional graphene, thionine and gold nanoparticles (NH2-G/Thi/AuNPs) as immunosensors for NSE detection. Combined with µPADs, the performance of the wireless POCT system was evaluated. The peak currents showed good linear relationship of the logarithm of NSE concentration ranging from 1 to 500ngmL-1 with the limit of detection (LOD) of 10pgmL-1. The detection results were automatically stored in EEPROM memory and could be displayed on Android's smartphone through Bluetooth in real time. The detection results were comparable to those measured by a commercial electrochemical workstation. The wireless POCT system had the potential for on-site testing of other tumor markers.

Identification of proteins involved in the adhesionof Candida species to different medical devices.

Adhesion is the first step for Candida species to form biofilms on medical devices implanted in the human host. Both the physicochemical nature of the biomaterial and cell wall proteins (CWP) of the pathogen play a determinant role in the process. While it is true that some CWP have been identified in vitro, little is known about the CWP of pathogenic species of Candida involved in adhesion. On this background, we considered it important to investigate the potential role of CWP of C. albicans, C. glabrata, C. krusei and C. parapsilosis in adhesion to different medical devices. Our results indicate that the four species strongly adher to polyvinyl chloride (PVC) devices, followed by polyurethane and finally by silicone. It was interesting to identify fructose-bisphosphate aldolase (Fba1) and enolase 1 (Eno1) as the CWP involved in adhesion of C. albicans, C. glabrata and C. krusei to PVC devices whereas phosphoglycerate kinase (Pgk) and Eno1 allow C. parapsilosis to adher to silicone-made implants. Results presented here suggest that these CWP participate in the initial event of adhesion and are probably followed by other proteins that covalently bind to the biomaterial thus providing conditions for biofilm formation and eventually the onset of infection.

A T4SS Effector Targets Host Cell Alpha-Enolase Contributing to Brucella abortus Intracellular Lifestyle.

Brucella abortus, the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus, ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus.

Identification and characterization of an immunogenic antigen, enolase 2, among excretory/secretory antigens (ESA) of Toxoplasma gondii.

An immunogenic protein, enolase 2, was identified among the secreted excretory/secretory antigens (ESAs) from Toxoplasma gondii strain RH using immunoproteomics based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Enolase 2 was cloned, sequenced, and heterologously expressed. BLAST analysis revealed 75-96% similarity with enolases from other parasites. Immunoblotting demonstrated good immunoreactivity of recombinant T. gondii enolase (Tg-enolase 2) to T. gondii-infected animal serum. Purified Tg-enolase 2 was found to catalyze dehydration of 2-phospho-d-glycerate to phosphoenolpyruvate. In vitro studies revealed maximal activity at pH 7.5 and 37 °C, and activity was inhibited by K(+), Ni(2+), Al(3+), Na(+), Cu(2+) and Cr(3+). A monoclonal antibody against Tg-enolase 2 was prepared, 1D6, with the isotype IgG2a/κ. Western blotting revealed that 1D6 reacts with Tg-enolase 2 and native enolase 2, present among T. gondii ESAs. The indirect immunofluorescence assays showed that enolase 2 could be specifically detected on the growing T. gondii tachyzoites. Immunoelectron microscopy revealed the surface and intracellular locations of enolase 2 on T. gondii cells. In conclusion, our results clearly show that the enzymatic activity of T. gondii enolase 2 is ion dependent and that it could be influenced by environmental factors. We also provide evidence that enolase 2 is an important immunogenic protein of ESAs from T. gondii and that it is a surface-exposed protein with strong antigenicity and immunogenicity. Our findings indicate that enolase 2 could play important roles in metabolism, immunogenicity and pathogenicity and that it may serve as a novel drug target and candidate vaccine against T. gondii infection.

Production of bioactive conjugated linoleic acid by the multifunctional enolase from Lactobacillus plantarum.

Lactobacillus plantarum α-enolase, a multifunctional-anchorless-surface protein belonging to the conserved family of enolases with a central role in glycolytic metabolism, was characterized to have a side role in the intricate metabolism of biohydrogenation of linoleic acid, catalyzing the formation of bioactive 9-cis-11-trans-CLA through dehydration and isomerization of 10-hydroxy-12-cis-octadecenoic acid. The identity of the enolase was confirmed through mass spectrometric analysis that showed the characteristic 442 amino acid sequence with a molecular mass of 48.03kDa. The enolase was not capable of using linoleic acid directly as a substrate but instead uses its hydroxyl derivative 10-hydroxi-12-cis-octadecenoic acid to finally form bioactive conjugated linoleic acid. Biochemical optimization studies were carried out to elucidate the conditions for maximum production of 9-cis-11-trans-CLA and maximum stability of α-enolase when catalyzing this reaction. Furthermore, through structural analysis of the protein, we propose the binding sites of substrate and product molecules that were characterized as two hydrophobic superficial pockets located at opposite ends of the enolase connected through a channel where the catalysis of dehydration and isomerization might occur. These results prove that multifunctional α-enolase also plays a role in cell detoxification from polyunsaturated fatty acids such as linoleic acid, along with the linoleate isomerase complex.

Trypanosoma evansi contains two auxiliary enzymes of glycolytic metabolism: Phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase.

Trypanosoma evansi is a monomorphic protist that can infect horses and other animal species of economic importance for man. Like the bloodstream form of the closely related species Trypanosoma brucei, T. evansi depends exclusively on glycolysis for its free-energy generation. In T. evansi as in other kinetoplastid organisms, the enzymes of the major part of the glycolytic pathway are present within organelles called glycosomes, which are authentic but specialized peroxisomes. Since T. evansi does not undergo stage-dependent differentiations, it occurs only as bloodstream forms, it has been assumed that the metabolic pattern of this parasite is identical to that of the bloodstream form of T. brucei. However, we report here the presence of two additional enzymes, phosphoenolpyruvate carboxykinase and PPi-dependent pyruvate phosphate dikinase in T. evansi glycosomes. Their colocalization with glycolytic enzymes within the glycosomes of this parasite has not been reported before. Both enzymes can make use of PEP for contributing to the production of ATP within the organelles. The activity of these enzymes in T. evansi glycosomes drastically changes the model assumed for the oxidation of glucose by this parasite.