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.
Degradation of undesirable biogenic amines (BAs) in foodstuffs by microorganisms is considered one of the most effective ways of eliminating their toxicity. In this study, we designed two sets of primers for the detection and quantification of the multicopper oxidase gene (MCO), which encodes an enzyme involved in BAs degradation, and endogenous (glyceraldehyde-3-phosphate dehydrogenase) gene (GAPDH) in Lactobacillus casei group by real-time PCR (qPCR). We tested 15 Lactobacillus strains in the screening assays (thus, MCO gene possessing assay (PCR) and monitoring of BAs degradation by HPLC-UV), in which Lactobacillus casei CCDM 198 exhibited the best degradation abilities. For this strain, we monitored the expression of the target gene (MCO) in time (qPCR), the effect of redox treatments (cysteine, ascorbic acid) on the expression of the gene, and the ability to degrade BAs not only in a modified MRS medium (MRS/2) but also in a real food sample (milk). Moreover, decarboxylase activity (ability to form BAs) of this strain was excluded. According to the results, CCDM 198 significantly (P < 0.05) reduced BAs (putrescine, histamine, tyramine, cadaverine), up to 25% decline in 48 h. The highest level of relative expression of MCO (5.21 ± 0.14) was achieved in MRS/2 media with cysteine.
- MeSH
- Bacterial Proteins genetics metabolism MeSH
- Biogenic Amines analysis metabolism MeSH
- Cysteine analysis metabolism MeSH
- Glyceraldehyde-3-Phosphate Dehydrogenases genetics MeSH
- Culture Media chemistry MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Ascorbic Acid analysis metabolism MeSH
- Lacticaseibacillus casei enzymology genetics growth & development metabolism MeSH
- Lactobacillus enzymology genetics growth & development metabolism MeSH
- Milk chemistry MeSH
- Oxidoreductases genetics metabolism MeSH
- Gene Expression Regulation, Bacterial MeSH
- Chromatography, High Pressure Liquid MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article 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.
- MeSH
- Bacteria enzymology pathogenicity MeSH
- Bacterial Infections microbiology prevention & control MeSH
- Bacterial Proteins metabolism MeSH
- Bacterial Vaccines immunology MeSH
- Glyceraldehyde-3-Phosphate Dehydrogenases immunology metabolism MeSH
- Humans MeSH
- Proteins metabolism MeSH
- Protein Binding MeSH
- Virulence MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Colletotrichum species are known as important pathogens of plants with an impact on crop production. Some of these species are also known as a cause of rare ophthalmic infections in humans. A case of keratitis caused by Colletotrichum dematium after corneal trauma in a 56-year-old woman is presented. Infection was diagnosed based on positive microscopy and culture. The fungal isolate was identified by morphological characteristics and DNA sequencing of the ITS rDNA region, β-tubulin (tub2) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes. The patient responded well to topical therapy with amphotericin B combined with intravenous amphotericin B but improvement was associated with the corneal collagen cross-linking. The review of the literature revealed another 13 cases of C. dematium keratitis, all but one patient having at least one keratitis risk factor in their history. Almost all patients (n = 12) were treated with topical polyene antibiotics (natamycin or amphotericin B), improvement and cure were achieved in eight of them.
- MeSH
- Amphotericin B administration & dosage MeSH
- Antifungal Agents administration & dosage MeSH
- Administration, Topical MeSH
- Colletotrichum classification genetics isolation & purification MeSH
- Molecular Diagnostic Techniques MeSH
- Adult MeSH
- Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) genetics MeSH
- Keratitis diagnosis microbiology pathology MeSH
- Middle Aged MeSH
- Humans MeSH
- DNA, Ribosomal Spacer genetics MeSH
- Microbiological Techniques MeSH
- Adolescent MeSH
- Young Adult MeSH
- Mycoses diagnosis microbiology pathology MeSH
- Eye Injuries complications MeSH
- Sequence Analysis, DNA MeSH
- Tubulin genetics MeSH
- Treatment Outcome MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
- Systematic Review 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.
- MeSH
- Gene Deletion * MeSH
- Virulence Factors analysis MeSH
- Francisella tularensis enzymology immunology pathogenicity MeSH
- Glyceraldehyde-3-Phosphate Dehydrogenases deficiency metabolism MeSH
- Blood Proteins metabolism MeSH
- Microbial Viability MeSH
- Disease Models, Animal MeSH
- Mice MeSH
- Protein Disulfide-Isomerases deficiency MeSH
- Proteome analysis MeSH
- Salmonella Infections, Animal microbiology pathology MeSH
- Protein Binding MeSH
- Virulence MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Potassium nitrate (E252) is widely used as a food preservative and has applications in the treatment of high blood pressure however high doses are carcinogenic. Larvae of Galleria mellonella were administered potassium nitrate to establish whether the acute effects in larvae correlated with those evident in mammals. Intra-haemocoel injection of potassium nitrate resulted in a significant increase in the density of circulating haemocytes and a small change in the relative proportions of haemocytes but haemocytes showed a reduced fungicidal ability. Potassium nitrate administration resulted in increased superoxide dismutase activity and in the abundance of a range of proteins associated with mitochondrial function (e.g. mitochondrial aldehyde dehydrogenase, putative mitochondrial Mn superoxide dismutase), metabolism (e.g. triosephosphate isomerase, glyceraldehyde 3 phosphate dehydrogenase) and nitrate metabolism (e.g. aliphatic nitrilase, glutathione S-transferase). A strong correlation exists between the toxicity of a range of food preservatives when tested in G. mellonella larvae and rats. In this work a correlation between the effect of potassium nitrate in larvae and mammals is shown and opens the way to the utilization of insects for studying the in vivo acute and chronic toxicity of xenobiotics.
- MeSH
- Aldehyde Dehydrogenase metabolism MeSH
- Aminohydrolases metabolism MeSH
- Nitrates metabolism pharmacology toxicity MeSH
- Glutathione Transferase metabolism MeSH
- Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) metabolism MeSH
- Hemocytes drug effects metabolism MeSH
- Insect Proteins metabolism MeSH
- Catalase metabolism MeSH
- Larva drug effects metabolism MeSH
- Mitochondrial Proteins metabolism MeSH
- Moths metabolism MeSH
- Oxidation-Reduction drug effects MeSH
- Proteome metabolism MeSH
- Proteomics MeSH
- Potassium Compounds pharmacology toxicity MeSH
- Superoxide Dismutase metabolism MeSH
- Toxicity Tests, Acute methods MeSH
- Triose-Phosphate Isomerase metabolism MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article 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
- Acrosome metabolism MeSH
- Energy Metabolism MeSH
- Flagella metabolism MeSH
- Glyceraldehyde-3-Phosphate Dehydrogenases analysis genetics physiology MeSH
- Sperm-Ovum Interactions MeSH
- Humans MeSH
- Sperm Motility MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Swine metabolism MeSH
- Spermatogenesis MeSH
- Spermatozoa metabolism MeSH
- Zona Pellucida metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Current standard methods for kinetic and genomic modeling cannot provide deep insight into metabolic regulation. Here, we developed and evaluated a multi-scale kinetic modeling approach applicable to any prokaryote. Specifically, we highlight the primary metabolism of the cyanobacterium Synechococcus elongatus PCC 7942. The model bridges metabolic data sets from cells grown at different CO2 conditions by integrating transcriptomic data and isozymes. Identification of the regulatory roles of isozymes allowed the calculation and explanation of the absolute metabolic concentration of 3-phosphoglycerate. To demonstrate that this method can characterize any isozyme, we determined the function of two glycolytic glyceraldehyde-3-phosphate dehydrogenases: one co-regulates high concentrations of the 3-phosphoglycerate, the other shifts the bifurcation point in hexose regulation, and both improve biomass production. Moreover, the regulatory roles of multiple phosphoglycolate phosphatases were defined for varying (non-steady) CO2 conditions, suggesting their protective role against toxic photorespiratory intermediates.
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Bacterial Proteins chemistry physiology MeSH
- Glyceraldehyde-3-Phosphate Dehydrogenases chemistry physiology MeSH
- Homeostasis MeSH
- Isoenzymes chemistry physiology MeSH
- Kinetics MeSH
- NADP metabolism MeSH
- Carbon Dioxide MeSH
- Oxidation-Reduction MeSH
- Synechococcus enzymology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: High-throughput studies provide a wide spectrum of genes for use as predictive markers during testicular sperm extraction (TESE) in combination with ICSI. In this work, we used the specimens from testicular biopsies of men with non-obstructive azoospermia who underwent TESE to investigate the expression of spermatogenesis-related genes MND1, SPATA22, GAPDHS and ACR. METHODS: Testicular biopsy specimens were subdivided into three groups: hypospermatogenesis (HS); maturation arrest (MA); and Sertoli cell-only syndrome (SCO). The levels of expression of the spermatogenesis-related genes MND1, SPATA22, GAPDHS and ACR in the testes were compared among these three groups using the reverse transcription polymerase chain reaction (RT-PCR) technique. RESULTS: Analysis of the expression of spermatogenic genes in human testes with abnormal spermatogenesis showed different expression patterns in patients from different groups. Fertilization rate for studied set of patients was 66% and pregnancy rate 29%. For HS group fertilization rate was 72% and pregnancy rate 32%, while for MA group fertilization and pregnancy rates were 54% and 26%, respectively. Fertilization rates in relation to the studied genes were uniformly around 70%, pregnancy rates for ACR and GAPDHS genes were surprisingly low at 6% and 8% correspondingly. CONCLUSIONS: Analysis of the expression of genes involved in spermatogenesis can be a fast additional test for the level of spermatogenesis in testicular samples.
- MeSH
- Acrosin genetics MeSH
- Azoospermia genetics pathology MeSH
- Biopsy MeSH
- Adult MeSH
- Fertilization MeSH
- Glyceraldehyde-3-Phosphate Dehydrogenases genetics MeSH
- Sperm Injections, Intracytoplasmic MeSH
- Middle Aged MeSH
- Humans MeSH
- Testicular Diseases genetics pathology MeSH
- Sperm Retrieval MeSH
- Oligospermia genetics pathology MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Cell Cycle Proteins genetics MeSH
- Sertoli Cell-Only Syndrome genetics pathology MeSH
- Spermatogenesis genetics MeSH
- Gene Expression Profiling MeSH
- Pregnancy MeSH
- Testis metabolism pathology MeSH
- Pregnancy Rate MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Pregnancy MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
A trypanosomatid species, designated as Typing Unit 1 (TU1) by sequences of SL RNA gene repeats, has been found in the intestine of pyrrhocorids (Insecta: Heteroptera) in Europe, Mediterranean, Central America and some parts of Asia and Africa. Phylogenetic analysis of the SL repeat sequences has shown that the isolates group in the tree according to their geographic origin. The maximal sequence divergence was observed in parasites from Neotropics suggesting the origin within and subsequent migrations from this region. The global distribution of the parasite could have been facilitated by ubiquity of its hosts that include several genera of the family Pyrrhocoridae. In Europe the TU1 flagellates frequently occur in Pyrrhocoris apterus, the host of Leptomonas pyrrhocorisZotta, 1912, a species that had been insufficiently defined by host and light microscopy level morphology. Herein, the Zotta's species description has been amended to include the TU1 SL RNA repeat, SSU rRNA, glycosomal GAPDH gene sequences, as well as ultrastructure. In addition, Leptomonas scantii n. sp. with an overlapping host range has been described. Moreover, 10 typing units of trypanosomatids found in the pyrrhocorid hosts demonstrate the extent of variability of trypanosomatids occurring in one host family.
- MeSH
- Phylogeography MeSH
- Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) MeSH
- Heteroptera parasitology MeSH
- Molecular Sequence Data MeSH
- DNA, Protozoan chemistry genetics MeSH
- DNA, Ribosomal chemistry genetics MeSH
- RNA, Ribosomal, 18S genetics MeSH
- RNA, Spliced Leader genetics MeSH
- Sequence Analysis, DNA MeSH
- Trypanosomatina classification genetics isolation & purification ultrastructure MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Africa MeSH
- Asia MeSH
- Europe MeSH
- Central America MeSH
