Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known for its multifunctionality in several pathogenic bacteria. Our previously reported data suggest that the GAPDH homologue of Francisella tularensis, GapA, might also be involved in other processes beyond metabolism. In the present study, we explored GapA's potential implication in pathogenic processes at the host cell level. Using immunoelectron microscopy, we demonstrated the localization of this bacterial protein inside infected macrophages and its peripheral distribution in bacterial cells increasing with infection time. A quantitative proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) combined with pull-down assay enabled the identification of several of GapA's potential interacting partners within the host cell proteome. Two of these partners were further confirmed by alternative methods. We also investigated the impact of gapA deletion on the transcription of selected cytokine genes and the activation of the main signaling pathways. Our results show that ∆gapA-induced transcription of genes encoding several cytokines whose expressions were not affected in cells infected with a fully virulent wild-type strain. That might be caused, at least in part, by the detected differences in ERK/MAPK signaling activation. The experimental observations together demonstrate that the F. tularensis GAPDH homologue is directly implicated in multiple host cellular processes and, thereby, that it participates in several molecular mechanisms of pathogenesis.

• Klíčová slova
• Francisella, glyceraldehyde-3-phosphate dehydrogenase, infection, interacting partners, multitasking, pleiotropy, secretion,
• MeSH
• cytokiny metabolismus   MeSH
• exprese genu MeSH
• Francisella tularensis * genetika   metabolismus   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy genetika   metabolismus   MeSH
• proteomika MeSH
• virulence genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny MeSH
• glyceraldehyd-3-fosfátdehydrogenasy MeSH

Bacterial proteins exhibiting two or more unrelated functions, referred to as moonlighting proteins, are suggested to contribute to full virulence manifestation in pathogens. An expanding number of published studies have revealed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to be a multitasking protein with virulence impact in a number of pathogenic bacteria. This protein can be detected on the bacterial surface or outside the bacterial cell, where it interacts with host proteins. In this way, GAPDH is able to modulate various pathogenic processes. Moreover, it has been shown to be involved in non-enzymatic processes inside the bacterial cell. In this mini review, we summarize main findings concerning the multiple localization and protein interactions of GAPDH derived from bacterial pathogens of humans. We also briefly discuss problems associated with using GAPDH as a vaccine antigen and endeavor to inspire further research to fill gaps in the existing knowledge.

• Klíčová slova
• glyceraldehyde-3-phosphate dehydrogenase, localization, moonlighting proteins, pathogenic bacteria, protein-protein interaction (PPI),
• MeSH
• Bacteria enzymologie   patogenita   MeSH
• bakteriální infekce mikrobiologie   prevence a kontrola   MeSH
• bakteriální proteiny metabolismus   MeSH
• bakteriální vakcíny imunologie   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy imunologie   metabolismus   MeSH
• lidé MeSH
• proteiny metabolismus   MeSH
• vazba proteinů MeSH
• virulence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bakteriální vakcíny MeSH
• glyceraldehyd-3-fosfátdehydrogenasy MeSH
• proteiny MeSH

The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.

• Klíčová slova
• DsbA, Francisella tularensis, SILAC, glyceraldehyde-3-phosphate dehydrogenase, moonlighting,
• MeSH
• delece genu * MeSH
• faktory virulence analýza   MeSH
• Francisella tularensis enzymologie   imunologie   patogenita   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy nedostatek   metabolismus   MeSH
• krevní proteiny metabolismus   MeSH
• mikrobiální viabilita MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• proteindisulfidisomerasy nedostatek   MeSH
• proteom analýza   MeSH
• salmonelová infekce u zvířat mikrobiologie   patologie   MeSH
• vazba proteinů MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktory virulence MeSH
• glyceraldehyd-3-fosfátdehydrogenasy MeSH
• krevní proteiny MeSH
• proteindisulfidisomerasy MeSH
• proteom MeSH

We have previously reported that Schistosoma mansoni larvae emerging from host lung at pH 7.5-7.8 and then fixed with diluted formaldehyde (HCHO) readily bind radiation-attenuated cercariae (RA) vaccine serum antibodies, as assessed by indirect membrane immunofluorescence (IF). Here we show that S. mansoni schistosomula emerging from lung pieces under 5% CO2 (pH < or = 7.3) readily bind RA vaccine serum antibodies, provided they have been incubated for 12 h at pH 7.5-7.8 in foetal calf serum-free RPMI medium, and fixed with diluted HCHO. Ex vivo larvae exposed during incubation to GW4869, a specific inhibitor of tegument-bound, neutral sphingomyelinase (nSMase) displayed significantly diminished binding of RA vaccine serum antibodies, thus suggesting that nSMase activity at pH > or = 7.5 leads to exposure of lung-stage larvae surface membrane antigens to specific antibody detection. More importantly, ex vivo larvae readily bound antibodies directed to dipeptidic multiple antigen peptide constructs, based on S. mansoni-specific sequences in S. mansoni glyceraldehyde 3-phosphate dehydrogenase (SG3PDH). Lung-stage schistosomula IF reactivity was diminished following antiserum absorption with recombinant SG3PDH. The data together indicate that intact ex vivo, as well as, 5-day-old in vitro-grown larvae express SG3PDH on their surface membrane. The findings are discussed in relation to the importance of surface membrane proteins as candidate vaccine antigens.

• MeSH
• antigeny helmintové imunologie   MeSH
• antigeny povrchové imunologie   MeSH
• fluorescenční protilátková technika nepřímá MeSH
• glyceraldehyd-3-fosfátdehydrogenasa (NADP+) imunologie   metabolismus   MeSH
• křečci praví MeSH
• křeček rodu Mesocricetus MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• plíce parazitologie   MeSH
• Schistosoma mansoni enzymologie   MeSH
• schistosomiasis mansoni imunologie   parazitologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny helmintové MeSH
• antigeny povrchové MeSH
• glyceraldehyd-3-fosfátdehydrogenasa (NADP+) MeSH

BACKGROUND: Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. METHODS: Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. RESULTS: Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. CONCLUSION: GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm/oocyte binding.

• MeSH
• akrozom metabolismus   MeSH
• energetický metabolismus MeSH
• flagella metabolismus   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy analýza   genetika   fyziologie   MeSH
• interakce spermie a vajíčka MeSH
• lidé MeSH
• motilita spermií MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• prasata metabolismus   MeSH
• spermatogeneze MeSH
• spermie metabolismus   MeSH
• zona pellucida metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glyceraldehyd-3-fosfátdehydrogenasy MeSH

Four monoepitopic MAPs (MAP A, B, C and E) and one bis-diepitopic MAP B-E derived fromthe primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase, previously tested in BALB/c mice, were examined for their immunogenicity and protective capacity in C57BL/6 mice. Despite multimerization into MAPs, MAP Aand MAP C were poorly immunogenic. In contrast toBALB/c mice, MAP E was non-immunogenic in C57BL/6 mice. Peptide B in the form of MAP B orbis-diepitopic MAPB-E elicited immune responses in C57BL/6 mice that were associated with a significant decrease in worm burden. The MAPs were prepared by the stepwise solid-phase peptide synthesis using Boc/Bzl chemistry, successfully purified on the RP-HPLC column and characterized by RP-HPLC, HPCE and MALDI-TOF MS techniques. A general strategy for MAPs purification is discussed here and the purification of MAP Band MAP E is documented in detail.

• MeSH
• antigeny helmintové chemie   imunologie   MeSH
• cytokiny imunologie   MeSH
• epitopy chemie   imunologie   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy chemie   imunologie   MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• peptidy chemická syntéza   chemie   imunologie   MeSH
• protilátky helmintové imunologie   MeSH
• Schistosoma mansoni enzymologie   imunologie   MeSH
• schistosomiasis mansoni prevence a kontrola   MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny helmintové MeSH
• cytokiny MeSH
• epitopy MeSH
• glyceraldehyd-3-fosfátdehydrogenasy MeSH
• peptidy MeSH
• protilátky helmintové MeSH

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is well known for its involvement in numerous non-metabolic processes inside mammalian cells. Alternative functions of prokaryotic GAPDH are mainly deduced from its extracellular localization ability to bind to selected host proteins. Data on its participation in intracellular bacterial processes are scarce as there has been to date only one study dealing with this issue. We previously have reported several points of evidence that the GAPDH homolog of Francisella tularensis GapA might also exert additional non-enzymatic functions. Following on from our earlier observations we decided to identify GapA's interacting partners within the bacterial proteome to explore its new roles at intracellular level. The quantitative proteomics approach based on stable isotope labeling of amino acids in cell culture (SILAC) in combination with affinity purification mass spectrometry enabled us to identify 18 proteins potentially interacting with GapA. Six of those interactions were further confirmed by alternative methods. Half of the identified proteins were involved in non-metabolic processes. Further analysis together with quantitative label-free comparative analysis of proteomes isolated from the wild-type strain strain with deleted gapA gene suggests that GapA is implicated in DNA repair processes. Absence of GapA promotes secretion of its most potent interaction partner the hypothetical protein with peptidase propeptide domain (PepSY) thereby indicating that it impacts on subcellular distribution of some proteins.

• Klíčová slova
• Francisella tularensis, SILAC, glyceraldehyde-3-phosphate dehydrogenase, multifunctional enzyme, protein–protein interaction,
• Publikační typ
• časopisecké články MeSH

A gapR gene, encoding a protein similar to the AraC/XylS family of bacterial transcriptional regulators, was previously identified upstream of the gap gene, coding for glyceraldehyde-3-phosphate dehydrogenase in Streptomyces aureofaciens. The GapR protein overproduced in Escherichia coli was shown to bind to the gap-P promoter region. Using the gel mobility shift assay with cell-free protein extracts from different developmental stages of S. aureofaciens, we identified several other proteins, in addition to GapR, that specifically bound to the S. aureofaciens gap-P promoter region. When cell-free extracts from S. aureofaciens cultivated in liquid medium with glucose were analyzed, only one complex corresponding to GapR was detected. A new protein interacting with the gap-P promoter was detected in stationary culture of S. aureofaciens grown in the presence of mannitol as carbon sources. The GapR protein was partially purified from S. aureofaciens cultivated in liquid medium containing glucose and used for binding studies. DNA footprinting analysis revealed an identical protected region as previously identified for the GapR protein overproduced from Escherichia coli. The direct role of the GapR protein in the regulation of gap expression in S. aureofaciens in vivo was confirmed but regulation of gap expression seems to be more complex, possibly involving other regulatory protein(s), depending on the developmental stage of S. aureofaciens.

• MeSH
• bakteriální geny * MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• DNA bakterií genetika   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• Escherichia coli genetika   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy genetika   MeSH
• molekulární sekvence - údaje MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u bakterií MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• restrikční mapování MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• Streptomyces aureofaciens genetika   růst a vývoj   metabolismus   MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• vazebná místa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH
• DNA vazebné proteiny MeSH
• GapR protein, Streptomyces aureofaciens MeSH Prohlížeč
• glyceraldehyd-3-fosfátdehydrogenasy MeSH
• rekombinantní proteiny MeSH
• trans-aktivátory MeSH

The antivibrionic activity of crystalline preparations of five enzymes of the glycolytic cycle of animals cells was investigated. Phosphorylase "a" (0.5 mg/ml), aldolase (15 mg/ml) and pyruvate kinase (0.1 mg/ml) were found to inhibit the proliferation of Vibrio cholerae cells; phosphoglucomutase and glyceraldehyde-3-phosphate dehydrogenase at a concentration of 0.25 mg/ml were found to be vibriocidal. A mixture of these enzymes containing 0.062 mg/ml of phosphorylase "a" and 0.125 mg/ml of each phosphoglucomutase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase showed vibriocidal activity.

• MeSH
• aldolasa farmakologie   MeSH
• antibakteriální látky * MeSH
• antisérum farmakologie   MeSH
• fosfoglukomutasa farmakologie   MeSH
• fosforylasa a farmakologie   MeSH
• fosforylasy farmakologie   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy farmakologie   MeSH
• koncentrace vodíkových iontů MeSH
• pyruvátkinasa farmakologie   MeSH
• Vibrio cholerae účinky léků   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aldolasa MeSH
• antibakteriální látky * MeSH
• antisérum MeSH
• fosfoglukomutasa MeSH
• fosforylasa a MeSH
• fosforylasy MeSH
• glyceraldehyd-3-fosfátdehydrogenasy MeSH
• pyruvátkinasa MeSH

More than 100 sequenced genomes were searched for genes coding for the enzymes involved in glycolysis in an effort to find the most frequently occurring ones. Triosephosphate isomerase (TIM), glyceraldehyde-3-phosphate dehydrogenase (GAPD), phosphoglycerate kinase (PGK) and enolase (ENOL) were found to be present in 90 investigated genomes all together. The final set consisted of 80 prokaryotic and 10 eukaryotic genomes. Of the 80 prokaryotic genomes, 73 were from Bacteria, 7 from Archaea. Two microbial genomes were also from Eucarya (yeasts). Eight genomes of nonmicrobial origin were included for comparison. The amino acid sequences of TIMs, GAPDs, PGKs and ENOLs were collected and aligned, and their individual as well as concatenated evolutionary trees were constructed and discussed. The trees clearly demonstrate a closer relatedness between Eucarya and Archaea (especially the concatenated tree) but they do not support the hypothesis that eukaryotic glycolytic enzymes should be closely related to their alpha-proteobacterial counterparts. Phylogenetic analyses further reveal that although the taxonomic groups (e.g., alpha-proteobacteria, gamma-proteobacteria, firmicutes, actinobacteria, etc.) form their more or less compact clusters in the trees, the inter-clade relationships between the trees are not conserved at all. On the other hand, several examples of conservative relatedness separating some clades of the same taxonomic groups were observed, e.g., Buchnera along with Wigglesworthia and the rest of gamma-proteobacteria, or mycoplasmas and the rest of firmicutes. The results support the view that these glycolytic enzymes may have their own evolutionary history.

• MeSH
• fosfoglycerátkinasa genetika   metabolismus   MeSH
• fylogeneze MeSH
• genom archeí MeSH
• genom bakteriální MeSH
• glyceraldehyd-3-fosfátdehydrogenasy genetika   metabolismus   MeSH
• glykolýza MeSH
• molekulární evoluce * MeSH
• triózafosfátizomeráza genetika   metabolismus   MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoglycerátkinasa MeSH
• glyceraldehyd-3-fosfátdehydrogenasy MeSH
• triózafosfátizomeráza MeSH