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Intact-mass spectrometry has huge potential for clinical application, as it enables both quantitative and qualitative analysis of intact proteins and possibly unlocks additional pathophysiological information via, e.g., detection of specific post-translational modifications (PTMs). Such valuable and clinically useful selectivity is typically lost during conventional bottom-up mass spectrometry. We demonstrate an innovative immunoprecipitation protein enrichment assay coupled to ultrahigh performance liquid chromatography quadrupole time-of-flight high resolution mass spectrometry (UPLC-QToF-HRMS) for the fast and simple identification of the protein tumor marker Neuron Specific Enolase Gamma (NSEγ) at low endogenous concentrations in human serum. Additionally, using the combination of immunoaffinity purification with intact mass spectrometry, the presence of NSEγ in an acetylated form in human serum was detected. This highlights the unique potential of immunoaffinity intact mass spectrometry in clinical diagnostics.

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., (© 2021. The Author(s).)

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 ( 252 FYNKENGMY 260 ) 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., Competing Interests: The authors declared there are no competing financial interests., (Copyright © 2021 Yesenia Osorio-Aguilar et al.)

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 K m, but approximately 20-25% reduction in maximum reaction velocity (V max ) 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 Mg 2+ , Zn 2+ and Mn 2+ . 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.

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 (Ab 2 ) 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 H 2 O 2 , 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 H 2 O 2 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 247 FYKNGSYFY 255 . These findings will help elucidate and improve the understanding of the molecular mechanisms of Bartonella infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 (K m = 0.23 +/- 0.013 x 10 -3 M; V max = 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 Mg 2+ stimulates the activity of rSaeno while the rest of the divalent cations (Zn 2+ , Mn 2+ , Fe 2+ , Cu 2+ , Ni 2+ and Ca 2+ ) lead to a dose-dependent loss in the activity with a total loss of activity in the presence of Hg 2+ and Cr 2+ . The circular dichroism data indicate that other than Hg 2+ , Ni 2+ and to a certain extent Cu 2+ , 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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 TGFβ1, 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.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

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-MS E , 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 (NH 2 -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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 .

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Blackleg is a highly fatal disease of cattle and sheep, caused by Clostridium chauvoei, a Gram positive, anaerobic, spore forming bacteria. Cell surface-associated proteins play a major role in inducing the protective immunity. However, the identity of a majority of cell surface-associated proteins of C. chauvoei is not known. In the present investigation, we have used SDS-PAGE, 2D-gel electrophoresis and Western blotting followed by mass spectrometry to identify cell surface-associated proteins of C. chauvoei. Among the identified proteins, which have shown to offer protective antigencity in other bacteria, Enolase, Chaperonin, Ribosomal protein L10, Glycosyl Hydrolase and Flavoprotein were characterized by sequencing and their overexpression in Escherichia coli. In conclusion, cell surface-associated proteins were identified using proteomic approach and the genes for the immunoreactive proteins were expressed, which may prove to be potential diagnostic or vaccine candidates., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sporotrichosis is a subcutaneous mycosis caused by several closely related thermo-dimorphic fungi of the Sporothrix schenckii species complex, affecting humans and other mammals. In the last few years, new strategies have been proposed for controlling sporotrichosis owning to concerns about its growing incidence in humans, cats, and dogs in Brazil, as well as the toxicity and limited efficacy of conventional antifungal drugs. In this study, we assessed the immunogenicity and protective properties of two aluminum hydroxide (AH)-adsorbed S. schenckii cell wall protein (ssCWP)-based vaccine formulations in a mouse model of systemic S. schenckii infection. Fractioning by SDS-PAGE revealed nine protein bands, two of which were functionally characterized: a 44kDa peptide hydrolase and a 47kDa enolase, which was predicted to be an adhesin. Sera from immunized mice recognized the 47kDa enolase and another unidentified 71kDa protein, whereas serum from S. schenckii-infected mice recognized both these proteins plus another unidentified 9.4kDa protein. Furthermore, opsonization with the anti-ssCWP sera led to markedly increased phagocytosis and was able to strongly inhibit the fungus' adhesion to fibroblasts. Immunization with the higher-dose AH-adjuvanted formulation led to increased ex vivo release of IL-12, IFN-γ, IL-4, and IL-17, whereas only IL-12 and IFN-γ were induced by the higher-dose non-adjuvanted formulation. Lastly, passive transference of the higher-dose AH-adjuvanted formulation's anti-ssCWP serum was able to afford in vivo protection in a subsequent challenge with S. schenckii, becoming a viable vaccine candidate for further testing., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

Collagen is a target of pathogens for adhesion, colonization, and invasion of host tissue. Probiotic bacteria can mimic the same mechanism as used by the pathogens in the colonization process, expressing cell surface proteins that specifically interact with extracellular matrix component proteins. The capability to bind collagen is expressed by several Lactobacillus isolates, including some Lactobacillus plantarum strains. In this study we report the involvement of the L. plantarum EnoA1 alfa-enolase in type I collagen (CnI) binding. By adhesion assays, we show that the mutant strain LM3-CC1, carrying a null mutation in the enoA1 gene, binds to immobilized collagen less efficiently than wild type strain. CnI overlay assay and Elisa tests, performed on the purified EnoA1, show that this protein can bind collagen both under denaturing and native conditions. By using truncated recombinant enolase proteins, we also show that the region spanning from 73rd to the 140th amino acid residues is involved in CnI binding., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aim: Previously, we reported that visual arrestin co-purified with glycolytic enzymes. The aim of this study was to analyze the co-purification of arrestin like proteins (ALP) in bovine cardiac tissues with enolases., Methods: The soluble extract of bovine myocardial tissues from different regions such as left and right atriums and ventricles of the bovine heart (n = 3) was analyzed by ACA-34 gel filtration, immuno-affinity column, SDS-PAGE, ELISA, western blot and a sandwich immune assay for quantification of ALP and sequence analysis., Results: We observed that; 1) The cardiac muscle contained a 50 kDa ALP at a concentration of 751 pg/mg of soluble protein extract, 2) ALP purified, by immunoaffinity, contained alpha-enolase of 48 kDa confirmed by protein sequence analysis; 3) Cardiomyocyte cells exposed to anti arrestin and anti enolase monoclonal antibodies showed decreased proliferation in vitro, 4) High level of autoantibodies were detected by ELISA (3.57% for arrestin and 9.12% for α-enolase) in serum of patients with infarcted heart disease., Conclusion: We suggest a possible interaction between ALP and alpha-enolases yielding a complex that may be involved in the induction of cardiac autoimmune diseases., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Cryopreservation is essential for long-term storage of cells and tissues, which can be used for clinical applications such as drug toxicity testing, human transplantation, reproductive, regenerative and transfusion medicine. It requires use of cryoprotectants (e.g. dimethyl disulfoxide (DMSO), glycerol) that protect cells and tissues from dehydration and damage caused by formation of intracellular ice during freezing. As an alternative to these cytotoxic cryoprotectants, we are developing new technology using natural substances produced by plants that survive freezing conditions. We previously showed that soluble protein extracts such as wheat protein extract (WPE) prepared from winter wheat plants can substitute for DMSO as a cryoprotectant for certain mammalian cell types. To identify novel cryoactive proteins, WPE was separated using different chromatographic procedures and cryoactive fractions were analyzed by mass spectrometry. The analysis revealed enolase as a potential wheat protein candidate. A recombinant enolase protein was prepared and was able to successfully cryopreserve rat hepatocytes and insulin-secreting INS832/13 pancreatic cells. Post-thaw cells had high viability and levels of metabolic activities. Cryopreserved cells were plateable and had good adherence and morphological properties. These results indicate that individual plant proteins such as enolase have promising potential as new, non-toxic technology for cryopreservation protocols used for clinical applications., (Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fusarium oxysporum f. sp. lycopersici is a fungus responsible for the tomato disease known as fusariosis. Enolase, which is the enzyme that catalyzes the reaction of 2-phosphoglycerate to phosphoenolpyruvate, is present during glycolysis. Enolase genes have been isolated from bacteria and fungi, among other organisms. In this research, a large portion of the enolase, eno, gene sequence was isolated from F. oxysporum and compared with those of other microorganisms, revealing a similarity of 51-69 %. We analyzed the copy number of the eno gene and determined that only a single copy is present in F. oxysporum, as in several fungi, such as Candida albicans and Aspergillus oryzae. We also detected the expression of the eno gene by reverse transcription-polymerase chain reaction during in vitro growth under two growth conditions where glucose was used as the carbon source, and we observed the same eno gene expression levels under both growth conditions.

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti-α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti-α-enolase/low anti-annexin AI IgG2 and patients with low anti-α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti-α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum., (Copyright © 2014 by the American Society of Nephrology.)

Aim: A study was performed on the articular disk and periarticular tissues of the temporo-mandibular joint (TMJ) with immunohistochemical techniques to give evidence to the presence of neuroreceptors (NRec) in these sites., Methods: The study was carried out on tissue samples obtained from 10 subjects without TMJ disease and from 7 patients with severe TMJ arthritis and arthrosis. We use antibodies directed against following antigens: Gliofibrillary Acidic Protein (GFAP), Leu-7, Myelin Basic Protein (MBP), Neurofilaments 68 kD (NF), Neuron Specific Enolase (NSE), S-100 protein (S-100) and Synaptophysin (SYN)., Results: This study revealed that Ruffini's-like, Pacini's-like and Golgi's-like receptors can be demonstrated in TMJ periarticular tissues and that free nervous endings are present in the subsynovial tissues but not within the articular disk. We observed elongated cytoplamic processes of chondrocytes that demonstrated strong S-100 immunoreactivity but they were unreactive with all other antibodies. These cytoplamic processes were more abundant and thicker in the samples obtained from patients with disease TMJ., Conclusion: The results of this study confirm that different Nrec are detectable in TMJ periarticular tissues but they are absent within the articular disk. In the latter site, only condrocytic processes are evident, especially in diseased TMJ, and they might have been confused with nervous endings in previous morphological studies. Nevertheless the absence of immunoreactivity for NF, NSE and SYN proves that they are not of neural origin.

Novel synthetic peptides represent smart molecules for antigen-antibody interactions in several bioanalytics applications, from purification to serum screening. Their immobilization onto a solid phase is considered a key point for sensitivity increasing. In this view, we exploited Quartz Crystal Microbalance with simultaneous frequency and dissipation monitoring (QCM-D) with a double aim, specifically, as investigative tool for spacers monolayer assembling and its functional evaluation, as well as high sensitive method for specific immunosorbent assays. The method was applied to pancreatic ductal adenocarcinoma (PDAC) detection by studying the interactions between synthetic phosphorylated and un-phosphorylated α-enolase peptides with sera of healthy and PDAC patients. The synthetic peptides were immobilized on the gold surface of the QCM-D sensor via a self-assembled alkanethiol monolayer. The presented experimental results can be applied to the development of surfaces less sensitive to non-specific interactions with the final target to suggest specific protocols for detecting PDAC markers with un-labeled biosensors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The enolase EnoA1 of Lactobacillus plantarum is here shown to interact with human plasminogen (Plg). By sequence alignment of EnoA1 with Streptococcus pneumoniae and Bifidobacterium lactis enolases, we identified BS1 and BS2 Plg-binding sites. A structure prediction of EnoA1 showed lysine residues in position 255 (BS2), and 422 (BS1) exposed on protein surface. A lysine residue in position 259 was as well identified as surface-exposed amino acid. The enoA1 gene was site directed-mutagenized to generate four mutated proteins, carrying K255A, K259A, K422A and K259A/K422A substitutions. The functional role of these lysine residues was assessed evaluating specific Plg-binding activity of the mutated proteins. While the binding activity of the mutated proteins was drastically reduced, the residual enzymatic activity was more than 50% of EnoA1. Our results show that L. plantarum EnoA1 exhibits the Plg-BS1, and the Plg-BS2 extending up to the lysine residue in position 259, therefore consisting of 12-aa residues instead of 9-aa residues described in S. pneumoniae. A test performed on whole cells of L. plantarum, demonstrated that after inducing conversion of the cell-bound plasminogen to plasmin, this was released into the medium, unlike the mechanism reported for most pathogens, that retained plasmin bound to the cell surface., (Copyright © 2012. Published by Elsevier GmbH.)

Infertility or subfertility of bovine spermatozoa may lead to disintegration of the breeding system and large economic losses. Recently, proteomics have identified candidates for the sperm fertility biomarkers, but no definite studies have clearly identified the relationship between the proteome and sperm fertility after proteomic study. Therefore, to determine the clinical value of the protein markers identified by proteomic study, we first compared the protein expression profiles of spermatozoa from high and low fertility bulls using 2-dimensional electrophoresis. We then investigated the relationship between protein expression and the fertility of individual bulls as assessed by Western blot analysis. Five proteins, enolase 1 (ENO1), ATP synthase H+ transporting mitochondrial F1 complex beta subunit, apoptosis-stimulating of p53 protein 2, alpha-2-HS-glycoprotein, and phospholipid hydroperoxide glutathione peroxide, were more highly represented in high fertility bulls, whereas three proteins, voltage dependent anion channel 2 (VDAC2), ropporin-1, and ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2), were more highly represented in low fertility bulls. Among those proteins, ENO1, VDAC2, and UQCRC2 were significantly correlated with individual fertility. Therefore, these results suggest that concurrent comparisons between protein expression and other fertility assays may represent a good in vitro assay to determine sperm fertility.

We performed proteomics analysis of the cerebrospinal fluid (CSF) of healthy dogs and dogs with meningoencephalitis of unknown etiology (MUE). By comparing two-dimensional electrophoreses (2DE), an upregulated spot was found in MUE dogs. This protein was identified as a neuron-specific enolase (NSE) by analysis with MALDI-TOF mass spectrometry. In comparing dot blots using an antibody against NSE, the NSE levels in the CSF of MUE dogs was significantly higher than that of the controls. NSE is a diagnostic marker of neuroendocrine tumors, brain injury and spinal cord trauma in humans. It seems that the NSE concentration in the CSF is increased by cellular destruction in canine encephalitis. Though elevation of NSE may not be specific in canine encephalitis because the NSE level was increased in other CNS diseases, further study including measurement with serum is necessary.

Paenibacillus larvae is a gram-positive spore-forming bacteria, causative agent of American Foulbrood (AFB), a severe disease affecting larvae of the honeybee Apis mellifera. In an attempt to detect potential virulence factors secreted by P. larvae, we identified an enolase among different secreted proteins. Although this protein is a cytosolic enzyme involved in glycolytic pathways, it has been related to virulence. The aim of the present work was to evaluate its role during the infection of honeybee larvae. Toxicity assays showed that enolase was highly toxic and immunogenic to honeybee larvae. Its production was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. P. larvae enolase production was also confirmed in vivo, during the infection of honeybee larvae. This protein was able to hydrolyze milk proteins as described for P. larvae, suggesting that could be involved in larval degradation, maybe through the plasmin(ogen) system. These results suggest that P. larvae enolase may have a role in virulence and could contribute to a general insight about insect-pathogen interaction mechanisms., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 3.25 A resolution. The crystals belonged to space group I4, with unit-cell parameters a=b=145.31, c=99.79 A. There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient VM of 2.8 A3 Da(-1), corresponding to 55.2% solvent content.

During oilseed embryo development, carbon from sucrose is utilized for fatty acid synthesis in the plastid. The role of plastidial glycolysis in Arabidopsis embryo oil accumulation was investigated. Genes encoding enolases (ENO) and phosphoglyceromutases (PGlyM) were identified, and activities and subcellular locations were established by expression of recombinant and green fluorescent protein (GFP)-fusion proteins. Mutant Arabidopsis plants lacking putative plastidial isoforms were characterized with respect to isoform composition and embryo oil content. In the developing embryo, ENO1 and ENO2 account for most or all of the plastidial and cytosolic ENO activity, respectively, and PGLYM1 accounts for most or all of the plastidial PGlyM activity. The eno1 and pglym1 mutants, in which plastidic ENO and PGlyM activities were undetectable, had wild-type amounts of seed oil at maturity. It is concluded that although plastids of developing Arabidopsis embryos have the capacity to carry out the lower part of the glycolytic pathway, the cytosolic glycolytic pathway alone is sufficient to support the flux from 3-phosphoglycerate to phosphoenolpyruvate required for oil production. The results highlight the importance for oil production of translocators that facilitate interchange of glycolytic intermediates between the cytosol and the plastid stroma.

Objective: To express enolase gene of Taenia asiatica, investigate the immunoreactivity of the recombinant TaENO protein, and immuno-histo-localize the presence of the recombinant TaENO in adults of T. asiatica., Methods: The gene encoding enolase of T. asiatica (TaENO) was cloned by high throughput sequencing from the cDNA library of adult T. asiatica. The coding region of TaENO was amplified by PCR, and cloned into a prokaryotic expression vector pET-30a (+). The recombinant plasmid was transformed into E. coli BL-21/DE3 and followed by expression of the protein induced by IPTG. The protein was purified by Ni-IDA affinity chromatography, and tested by SDS-PAGE. Its immunoreactivity was examined by Western blotting. The mice were immunized subcutaneously with purified TaENO formulated in Freund's adjuvant. Serum samples were collected and analyzed for specific antibodies by ELISA. The localization of TaENO in adult worms was demonstrated by immunofluorescent technique., Results: The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant TaENO was about Mr 47 000 with a concentration of 0.37 mg/ml. It was recognized by antisera of SD rats immunized with TaENO, sera of taeniasis patients and sera of infected swine. The immunofluorescence assay revealed that TaENO immune serum located in the tegument of T. asiatica adult., Conclusion: The TaENO gene has been expressed with immunoreactivity, and the recombinant TaENO is immunolocalized in the tegument of T. asiatica adult.

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pI 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

The Arabidopsis thaliana gene At1g74030 codes for a putative plastid phosphoenolpyruvate (PEP) enolase (ENO1). The recombinant ENO1 protein exhibited enolase activity and its kinetic properties were determined. ENO1 is localized to plastids and expressed in most heterotrophic tissues including trichomes and non-root-hair cells, but not in the mesophyll of leaves. Two T-DNA insertion eno1 mutants exhibited distorted trichomes and reduced numbers of root hairs as the only visible phenotype. The essential role of ENO1 in PEP provision for anabolic processes within plastids, such as the shikimate pathway, is discussed with respect to plastid transporters, such as the PEP/phosphate translocator.

Streptococcus suis serotype 2 (SS2) is a porcine and human pathogen with adhesive and invasive properties. The absence of suitable vaccine or virulent marker can be the bottleneck to control SS2 infection. In the present study, a novel immunogenic Enolase identified in the previous study was inducibly overexpressed in Escherichia coli, and the purified recombinant protein could elicit a significant humoral antibody response and confer efficient immunity against challenge with lethal dose of SS2 or SS7 infection in mouse model. The roles Enolase plays in pathogenicity of SS2 were also explored as reasons for which Enolase could be a protective antigen. The Enolase was an in vivo-induced antigen confirmed by the real-time PCR and could adhere to the Hep-2 cells by the indirect immunofluorescent assay and the inhibition assay. These suggested that Enolase could play important roles in pathogenicity and may serve as a novel vaccine candidate against SS2 infection.

Multiple reaction monitoring (MRM) mass spectrometry identifies and quantifies specific peptides in a complex mixture with very high sensitivity and speed and thus has promise for the high throughput screening of clinical samples for candidate biomarkers. We have developed an interactive software platform, called MRMer, for managing highly complex MRM-MS experiments, including quantitative analyses using heavy/light isotopic peptide pairs. MRMer parses and extracts information from MS files encoded in the platform-independent mzXML data format. It extracts and infers precursor-product ion transition pairings, computes integrated ion intensities, and permits rapid visual curation for analyses exceeding 1000 precursor-product pairs. Results can be easily output for quantitative comparison of consecutive runs. Additionally MRMer incorporates features that permit the quantitative analysis experiments including heavy and light isotopic peptide pairs. MRMer is open source and provided under the Apache 2.0 license.

Streptococcus suis is an important swine pathogen that causes meningitis, endocarditis, arthritis and septicaemia. As a zoonotic agent, S. suis also causes similar diseases in humans. Binding of pathogenic bacteria to extracellular matrix components enhances their adhesion to and invasion of host cells. In the present study we isolated and identified a novel fibronectin-binding protein from S. suis. The native protein (designated SsEno) possessed not only high homology with other bacterial enolases but also enolase activity. We cloned, expressed and purified SsEno and showed that it is ubiquitously expressed by all S. suis serotypes and we identified its surface localization using immunoelectron microscopy. ELISA demonstrated that SsEno binds specifically to fibronectin and plasminogen in a lysine-dependent manner. Additional surface plasmon resonance assays demonstrated that SsEno binds to fibronectin or plasminogen with low nanomolar affinity. Inhibition experiments with anti-SsEno antibodies also showed that bacterial SsEno is important for the adhesion to and invasion of brain microvascular endothelial cells by S. suis. Overall, the present work is the first study, to our knowledge, to demonstrate a fibronectin-binding activity of a bacterial enolase, and shows that, similar to other bacterial fibronectin-binding proteins, SsEno may contribute to the virulence of S. suis.

alpha-enolase of Bacillus anthracis has recently been classified as an immunodominant antigen and a potent virulence factor determinant. alpha-enolase (2-phospho-d-glycerate hydrolase (EC 4.2.1.11), a key glycolytic metalloenzyme catalyzes the dehydration of d-(+)-2-phosphoglyceric acid to phosphoenolpyruvate. Interaction of surface bound alpha-enolase with plasminogen has been incriminated in tissue invasion for pathogenesis. B. anthracis alpha-enolase was expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity that exhibited a K(m) of 3.3 mM for phosphoenolpyruvate and a V(max) of 0.506 microM min(- 1) mg(-1). B. anthracis whole cells and membrane vesicles probed with anti-enolase antibodies confirmed the surface localization of alpha-enolase. The specific interaction of alpha-enolase with human plasminogen (but not plasmin) evident from ELISA and the retardation in the native gel reinforced its role in plasminogen binding. Putative plasminogen receptors in B. anthracis other than enolase were also observed. This binding was found to be carboxypeptidase sensitive implicating the role of C-terminal lysine residues. The recombinant enolase displayed in vitro laminin binding, an important mammalian extracellular matrix protein. Plasminogen interaction conferred B. anthracis with a potential to in vitro degrade fibronectin and exhibit fibrinolytic phenotype. Therefore, by virtue of its interaction to host plasminogen and extracellular matrix proteins, alpha-enolase may contribute in augmenting the invasive potential of B. anthracis.

Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell-cell interactions, since only small amounts of arginine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine.

Antigenic proteins of Echinostoma caproni (Trematoda) against mouse IgM, IgA, IgG, IgG1 and IgG2a were investigated by immunoproteomics. Excretory/secretory products (ESP) of E. caproni separated by two-dimensional (2D) gel electrophoresis were transferred to nitrocellulose membranes and probed with the different mouse immunoglobulin classes. A total of four proteins (enolase, 70 kDa heat-shock protein (HSP-70), actin and aldolase) were accurately identified. Enolase was recognized in eight different spots of which seven of them were detected in the expected molecular weight and were recognized by IgA, IgG or IgG and IgG1. Another spot identified as enolase at 72 kDa was only recognized by IgM. Digestion with N-glycosidase F of the 72 kDa band rendered a polypeptide with an apparent molecular weight similar to that expected for enolase recognized by Western immunoblotting using anti-enolase antibodies. This suggests that glycosylated forms of enolase may be involved in the early thymus-independent responses against E. caproni. Early IgM responses were also generated by actin and the HSP-70 which suggests that these proteins are exposed early to the host and may be of importance in the parasite establishment. The IgA responses also appear to be mediated by the HSP-70 and aldolase which could be related with the close contact of these proteins with the host mucosal surface after secretion.

Trichomonas vaginalis is a protist that causes the most common human sexually transmitted infection. A T. vaginalis cDNA expression library was screened with pooled sera from patients with trichomoniasis. A highly reactive cDNA clone of 1,428 bp encoded a trichomonad protein of 472 amino acids with sequence identity to alpha-enolase (tv-eno1). The sequence alignment confirmed the highly conserved nature of the enzyme with 65% to 84% identity among organisms. The expression of tv-eno1 was up-regulated by contact of parasites with vaginal epithelial cells, and this is the first report demonstrating up-regulation by cytoadherence of a plasminogen-binding alpha-enolase in T. vaginalis. Immunofluorescence with monoclonal antibody of nonpermeabilized trichomonads showed tv-ENO1 on the surface. The recombinant tv-ENO1 was expressed in Escherichia coli as a glutathione S-transferase (GST)::tv-ENO1 fusion protein, which was cleaved using thrombin to obtain affinity-purified recombinant tv-ENO1 protein (tv-rENO1) detectable in immunoblots by sera of patients. Immobilized tv-rENO1 bound human plasminogen in a dose-dependent manner, and plasminogen binding by tv-rENO1 was confirmed in a ligand blot assay. The plasminogen-specific inhibitor epsilon-aminocaproic acid blocked the tv-rENO1-plasminogen association, indicating that lysines play a role in binding to tv-rENO1. Further, both parasites and tv-rENO1 activate plasminogen to plasmin that is mediated by tissue plasminogen activator. These data indicate that as with other bacterial pathogens, tv-ENO1 is an anchorless, surface-associated glycolytic enzyme of T. vaginalis.

Enolase occurs as a cytoplasmic and a surface-associated protein in bacteria. Enolases of the bacterial pathogens Streptococcus pyogenes, Streptococcus pneumoniae and Staphylococcus aureus, as well as of the commensal lactic acid bacteria, Lactobacillus crispatus and Lactobacillus johnsonii, were purified as His(6)-fusion proteins from recombinant Escherichia coli. The fusion proteins were compared for putative virulence-associated functions, i.e., binding of human plasminogen, enhancement of plasminogen activation by human plasminogen activators, as well as binding to immobilized laminin, fibronectin and collagens. The individual enolases showed varying efficiencies in these functions. In particular, highly and equally effective interactions with plasminogen and laminin were seen with lactobacillar and staphylococcal enolases.

Plasminogen (Plg) binding to the cell surface of Mycoplasma fermentans results in a marked increase in the maximal adherence of the organism to HeLa cells, enhanced Plg activation by the urokinase-type Plg activator, and the induction of the internalization of M. fermentans by eukaryotic host cells (A. Yavlovich, A. Katzenell, M. Tarshis, A. A. Higazi, and S. Rottem, Infect. Immun. 72:5004-5011, 2004). In this study, the M. fermentans Plg binding protein was isolated by affinity chromatography of Triton X-100-solubilized M. fermentans membranes by utilizing a column of a Plg-biotin complex attached to avidin that was eluted with epsilon-aminocaproic acid. The eluted approximately 50-kDa protein was identified by mass spectrometric techniques as alpha-enolase. The possibility that alpha-enolase, a key cytoplasmatic glycolytic enzyme, resides also on the cell surface of M. fermentans was supported by an immunoblot analysis using polyclonal anti-alpha-enolase antiserum, which showed that alpha-enolase was present in a purified M. fermentans membrane preparation, as well as by immunochemical criteria and by immunoelectron microscopy analysis. Our observation that Plg blocked the binding of anti-alpha-enolase antibodies to a 50-kDa polypeptide band resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of M. fermentans membrane or soluble preparations further supports our notion that mycoplasmal surface alpha-enolase is a major Plg binding protein of M. fermentans.