An alternative vaccine target for bovine Anaplasmosis based on enolase, a moonlighting protein
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PROVIDER: S-EPMC10556744 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Project description:BackgroundStreptococcus thermophilus is an important food starter and receiving more attention to serve as cell factories for production of high-valued metabolites. However, the low yields of intracellular or extracellular expression of biotechnological and biomedical proteins limit its practical applications.ResultsHere, an enolase EnoM was identified from S. thermophilus CGMCC7.179 with about 94% identities to the surface-located enolases from other Streptococcus spp. strains. The EnoM was used as an anchor to achieve surface display in S. thermophilus using GFP as a reporter. After respectively mixing the GFP-EnoM fusion protein or GFP with S. thermophilus cells in vitro, the relative fluorescence units (RFU) of the S. thermophilus cells with GFP-EnoM was 80-folds higher than that with purified GFP. The sharp decrease in the RFU of sodium dodecyl sulfate (SDS) pretreated cells compared to those of non-pretreated cells demonstrated that the membrane proteins were the binding ligand of EnoM. Furthermore, an engineered β-galactosidase (β-Gal) was also successfully displayed on the cell surface of S. thermophilus CGMCC7.179 and the relative activity of the immobilized β-Gal remained up to 64% after reused 8 times. Finally, we also demonstrated that EnoM could be used as an anchor for surface display in L. casei, L. bulgaricus, L. lactis and Leuconostoc lactis.ConclusionTo our knowledge, EnoM from S. thermophilus was firstly identified as an anchor and successfully achieved surface display in LAB. The EnoM-based surface display system provided a novel strategy for the enzyme immobilization.
| S-EPMC7301973 | biostudies-literature
Project description:Tick-borne Anaplasma species are obligate, intracellular, bacterial pathogens that cause important diseases globally in people, agricultural animals, and dogs. Targeted mutagenesis methods are yet to be developed to define genes essential for these pathogens. In addition, vaccines conferring protection against diseases caused by Anaplasma species are not available. Here, we describe a targeted mutagenesis method for deletion of the phage head-to-tail connector protein (phtcp) gene in Anaplasma marginale. The mutant did not cause disease and exhibited attenuated growth in its natural host (cattle). We then assessed its ability to confer protection against wild-type A. marginale infection challenge. Additionally, we compared vaccine protection with the mutant to that of whole cell A. marginale inactivated antigens as a vaccine (WCAV) candidate. Upon infection challenge, non-vaccinated control cattle developed severe disease, with an average 57% drop in packed cell volume (PCV) between days 26-31 post infection, an 11% peak in erythrocytic infection, and apparent anisocytosis. Conversely, following challenge, all animals receiving the live mutant did not develop clinical signs or anemia, or erythrocyte infection. In contrast, the WCAV vaccinees developed similar disease as the non-vaccinees following A. marginale infection, though the peak erythrocyte infection reduced to 6% and the PCV dropped 43%. This is the first study describing targeted mutagenesis and its application in determining in vivo virulence and vaccine development for an Anaplasma species pathogen. This study will pave the way for similar research in related Anaplasma pathogens impacting multiple hosts.
| S-EPMC9135337 | biostudies-literature
Project description:During maturation and the acquisition of their fertilization potential, male germ cells are subjected to various sequential modifications that occur in the epididymis. Protein addition, reorganization or withdrawal, comprise some of these modifications. Dicarbonyl L-xylulose reductase (DCXR), a multifunctional protein involved in various enzymatic and protein interaction processes in different physiological systems, is one of the proteins added to spermatozoa in the epididymis. DCXR is a well-conserved protein with multiple characteristics including enzymatic activities and mediation of cell-cell interaction. In this study, we characterized the DCXR gene and protein expression in the bovine epididymis. Dicarbonyl L-xylulose reductase mRNA is differentially expressed in the caput, corpus, and cauda epididymide epithelial cells with a higher level observed in the cauda region. Tissue protein expression follows the same pattern as the corresponding mRNA expression with a cytoplasmic and apical distribution in the corpus and cauda epithelial cells, respectively. The protein can also be found with a nuclear localization in cauda epididymidis epithelial cells. Dicarbonyl L-xylulose reductase is secreted in the epididymis luminal compartment in the soluble fraction and is associated with microvesicular elements named epididymosomes. In spermatozoa, the DCXR protein was found in the cytoplasmic and membranous fractions. Expression of the DCXR protein is higher on caput spermatozoa but finally shows a weak detection in semen. These data describe DCXR in the bovine epididymis and reveal that its behavior differs from that found in humans. It seems that, in this model, the DCXR protein might have a questionable involvement in the fertilization process.
| S-EPMC4376396 | biostudies-literature
Project description:Bovine anaplasmosis is a tick-borne disease with zoonotic potential, caused by the obligate intracellular bacterium Anaplasma marginale. The disease is distributed worldwide in tropical and subtropical regions. The economic losses from anaplasmosis in animals is of significant importance because it causes severe morbidity and mortality in cattle. Recovered animals may become persistent carriers. Epidemiological information on the actual status of bovine anaplasmosis in Egypt is scarce. Thus, this study aimed to determine anti-Anaplasma antibody and DNA in serum samples using ELISA and PCR, respectively. In total, 758 bovine sera were collected from cattle farms located in 24 Egyptian governorates in 2015 to 2016. Sera were analyzed with the commercially available 'Anaplasma antibody competitive ELISA v2' kit and 'AmpliTest Anaplasma/Ehrlichia spp. real time TaqMan TM PCR. Anaplasma spp. antibodies were detected in 140 (18.5%) (CI: 15.8-21.4%) of the investigated sera by ELISA, and Anaplasma/Ehrlichia-DNA was detected in 40 (5.3%) (CI: 3.8-7.1%) of the positive sera by real time PCR. Co-detection of both Anaplasma spp. and Coxiella burnetii-specific antibodies was proven in 30 (4%) of the investigated sera. The results of this work confirm the significant prevalence of bovine anaplasmosis in Egypt. Raising awareness in decision makers of the public health, veterinarians and animal owners is required to reduce the spread of infection.
| S-EPMC7168636 | biostudies-literature
Project description:The opportunistic fungal pathogen Aspergillus fumigatus can cause severe infections, particularly in immunocompromised individuals. Upon infection, A. fumigatus faces the powerful and directly acting immune defense of the human host. The mechanisms on how A. fumigatus evades innate immune attack and complement are still poorly understood. Here, we identify A. fumigatus enolase, AfEno1, which was also characterized as fungal allergen, as a surface ligand for human plasma complement regulators. AfEno1 binds factor H, factor-H-like protein 1 (FHL-1), C4b binding protein (C4BP), and plasminogen. Factor H attaches to AfEno1 via two regions, via short conserved repeats (SCRs) 6-7 and 19-20, and FHL-1 contacts AfEno1 via SCRs 6-7. Both regulators when bound to AfEno1 retain cofactor activity and assist in C3b inactivation. Similarly, the classical pathway regulator C4BP binds to AfEno1 and bound to AfEno1; C4BP assists in C4b inactivation. Plasminogen which binds to AfEno1 via lysine residues is accessible for the tissue-type plasminogen activator (tPA), and active plasmin cleaves the chromogenic substrate S2251, degrades fibrinogen, and inactivates C3 and C3b. Plasmin attached to swollen A. fumigatus conidia damages human A549 lung epithelial cells, reduces the cellular metabolic activity, and induces cell retraction, which results in exposure of the extracellular matrix. Thus, A. fumigatus AfEno1 is a moonlighting protein and virulence factor which recruits several human regulators. The attached human regulators allow the fungal pathogen to control complement at the level of C3 and to damage endothelial cell layers and tissue components.
| S-EPMC6883375 | biostudies-literature
Project description:The coagulase-negative staphylococcal (CoNS) species Staphylococcus lugdunensis is unique in causing serious infections in humans that resemble those of Staphylococcus aureus rather than those of other CoNS species. The colonization and invasion of host tissue presupposes the presence of adherence factors, but only a few proteins mediating adhesion of S. lugdunensis to biotic surfaces are known yet. Here, we report on the functionality of the S. lugdunensis enolase (SlEno), which performs two distinct roles, first, as the metabolic enzyme of the glycolysis, and second, as an adherence factor to the extracellular matrix (ECM) of cells. Phylogenetic analyses of the SlEno confirmed their high conservation to enolases of other species and revealed a closer relationship to Staphylococcus epidermidis than to S. aureus. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry and Western blot experiments, we identified SlEno to be located in the cytoplasm as well as on the cell surface of S. lugdunensis. Recombinantly generated and surface-associated SlEno showed the usual enolase activity by catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate but, in addition, also displayed strong binding to immobilized laminin, fibronectin, fibrinogen, and collagen type IV in a dose-dependent manner. We also showed a strong binding of SlEno to plasminogen (Plg) and observed a tissue plasminogen activator (tPA)-dependent conversion of Plg to plasmin (Pln) whereby the Plg activation significantly increased in the presence of SlEno. This interaction might be dependent on lysines of the SlEno protein as binding to Plg was inhibited by ε-aminocaproic acid. Furthermore, the enhanced activation of the Plg/Pln system by SlEno enabled S. lugdunensis to migrate through a fibrin matrix. This migration was about 10-fold higher than without exogenously added SlEno. Finally, we observed a significantly higher clearance of S. lugdunensis by freshly prepared granulocytes and in the presence of anti-SlEno antibodies. In conclusion, these data demonstrate for the first time a moonlighting function of the S. lugdunensis enolase, which is an underrated virulence factor for colonization and invasion of tissues. Hence, SlEno might be a potential vaccine candidate to prevent severe infections caused by this pathogen.
| S-EPMC8928124 | biostudies-literature
Project description:Aberrantly upregulated choline phospholipid metabolism is a novel emerging hallmark of cancer, and choline kinase α (CHKα), a key enzyme for phosphatidylcholine production, is overexpressed in many types of human cancer through undefined mechanisms. Here, we demonstrate that the expression levels of the glycolytic enzyme enolase-1 (ENO1) are positively correlated with CHKα expression levels in human glioblastoma specimens and that ENO1 tightly governs CHKα expression via posttranslational regulation. Mechanistically, we reveal that both ENO1 and the ubiquitin E3 ligase TRIM25 are associated with CHKα. Highly expressed ENO1 in tumor cells binds to I199/F200 of CHKα, thereby abrogating the interaction between CHKα and TRIM25. This abrogation leads to the inhibition of TRIM25-mediated polyubiquitylation of CHKα at K195, increased stability of CHKα, enhanced choline metabolism in glioblastoma cells, and accelerated brain tumor growth. In addition, the expression levels of both ENO1 and CHKα are associated with poor prognosis in glioblastoma patients. These findings highlight a critical moonlighting function of ENO1 in choline phospholipid metabolism and provide unprecedented insight into the integrated regulation of cancer metabolism by crosstalk between glycolytic and lipidic enzymes.
| S-EPMC10104498 | biostudies-literature
Project description:Introduction: Hydatid disease is a ubiquitous parasitic zoonotic disease, which causes different medical, economic and serious public health problems in some parts of the world. The causal organism is a multi-stage parasite named Echinococcus granulosus whose life cycle is dependent on two types of mammalian hosts viz definitive and intermediate hosts. Methods: In this study, enolase, as a key functional enzyme in the metabolism of E. granulosus (EgEnolase), was targeted through a comprehensive in silico modeling analysis and designing a host-specific multi-epitope vaccine. Three-dimensional (3D) structure of enolase was modeled using MODELLER v9.18 software. The B-cell epitopes (BEs) were predicted based on the multi-method approach and via some authentic online predictors. ClusPro v2.0 server was used for docking-based T-helper epitope prediction. The 3D structure of the vaccine was modeled using the RaptorX server. The designed vaccine was evaluated for its immunogenicity, physicochemical properties, and allergenicity. The codon optimization of the vaccine sequence was performed based on the codon usage table of E. coli K12. Finally, the energy minimization and molecular docking were implemented for simulating the vaccine binding affinity to the TLR-2 and TLR-4 and the complex stability. Results: The designed multi-epitope vaccine was found to induce anti-EgEnolase immunity which may have the potential to prevent the survival and proliferation of E. granulosus into the definitive host. Conclusion: Based on the results, this step-by-step immunoinformatics approach could be considered as a rational platform for designing vaccines against such multi-stage parasites. Furthermore, it is proposed that this multi-epitope vaccine is served as a promising preventive anti-echinococcosis agent.
| S-EPMC6726745 | biostudies-literature
Project description:Transcriptional rewiring generates phenotypic novelty, acting as an important mechanism contributing to evolutionary development, speciation, and adaptation in all organisms. The phenotypic outcomes (functions) of transcription factor (TF) activity are determined by the combined effects of all target genes in the TF's regulatory network. Plastic rewiring of target genes accumulates during species divergence and ultimately alters phenotypes, indicating a TF functional switch. We define this phenomenon as 'disruptive rewiring', where the rewiring process disrupts the link between a TF and its original target genes that determine phenotypes. Here, we investigate if 'complete' disruptive rewiring is a prerequisite for a TF functional switch by employing chromatin immunoprecipitation sequencing, RNA expression, and phenotypic assays across yeast species. In yeasts where Sef1 targets TCA (tricarboxylic acid) cycle genes, we demonstrate that Sef1 orthologs can promote and inhibit respiratory growth by modulating the moonlighting function of their conserved target, NDE1. This modulation occurs without changing the overall association of Sef1 with TCA cycle genes. We propose that phenotypic masking by NDE1 promotes 'deceptive' disruptive rewiring of the Sef1 regulatory network in Saccharomyces cerevisiae, thereby potentially constraining future evolutionary trajectories.
| S-EPMC11662654 | biostudies-literature
Project description:Streptococcus suis is a serious pathogen in the pig industry with zoonotic potential. With respect to the current effort to reduce antibiotic use in animals, a prophylactic measure is needed to control the disease burden. Unfortunately, immunization against streptococcal pathogens is challenging due to nature of the interaction between the pathogen and the host immune system, but vaccines based on conjugates of capsular polysaccharide (CPS) and carrier protein were proved to be efficient. The main obstacle of these vaccines is manufacturing cost, limiting their use in animals. In this work, we tested an experimental vaccine against Streptococcus suis serotype 2 based on capsular polysaccharide conjugated to chicken ovalbumin (OVA) and compared its immunogenicity and protectivity with a vaccine based on CRM197 conjugate. Ovalbumin was selected as a cheap alternative to recombinant carrier proteins widely used in vaccines for human use. We found that the ovalbumin-based experimental vaccine successfully induced immune response in pigs, and the IgG antibody response was even higher than after immunization with capsular polysaccharide-CRM197 conjugate. Protectivity of vaccination against infection was evaluated in the challenge experiment and was found promising for both conjugates.
| S-EPMC9612095 | biostudies-literature