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.

• Keywords
• Francisella, glyceraldehyde-3-phosphate dehydrogenase, infection, interacting partners, multitasking, pleiotropy, secretion,
• MeSH
• Cytokines metabolism   MeSH
• Gene Expression MeSH
• Francisella tularensis * genetics   metabolism   MeSH
• Glyceraldehyde-3-Phosphate Dehydrogenases genetics   metabolism   MeSH
• Proteomics MeSH
• Virulence genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokines MeSH
• Glyceraldehyde-3-Phosphate Dehydrogenases MeSH

Phosphoinositides are glycerol-based phospholipids, and they play essential roles in cellular signalling, membrane and cytoskeletal dynamics, cell movement, and the modulation of ion channels and transporters. Phosphoinositides are also associated with fundamental nuclear processes through their nuclear protein-binding partners, even though membranes do not exist inside of the nucleus. Phosphatidylinositol 4-phosphate (PI(4)P) is one of the most abundant cellular phosphoinositides; however, its functions in the nucleus are still poorly understood. In this study, we describe PI(4)P localisation in the cell nucleus by super-resolution light and electron microscopy, and employ immunoprecipitation with a specific anti-PI(4)P antibody and subsequent mass spectrometry analysis to determine PI(4)P's interaction partners. We show that PI(4)P is present at the nuclear envelope, in nuclear lamina, in nuclear speckles and in nucleoli and also forms multiple small foci in the nucleoplasm. Nuclear PI(4)P undergoes re-localisation to the cytoplasm during cell division; it does not localise to chromosomes, nucleolar organising regions or mitotic interchromatin granules. When PI(4)P and PI(4,5)P2 are compared, they have different nuclear localisations during interphase and mitosis, pointing to their functional differences in the cell nucleus. Mass spectrometry identified hundreds of proteins, including 12 potentially novel PI(4)P interactors, most of them functioning in vital nuclear processes such as pre-mRNA splicing, transcription or nuclear transport, thus extending the current knowledge of PI(4)P's interaction partners. Based on these data, we propose that PI(4)P also plays a role in essential nuclear processes as a part of protein-lipid complexes. Altogether, these observations provide a novel insight into the role of PI(4)P in nuclear functions and provide a direction for further investigation.

• Keywords
• PI(4)P, nucleus, phosphoinositides,
• MeSH
• Cell Nucleolus metabolism   ultrastructure   MeSH
• Cell Nucleus metabolism   ultrastructure   MeSH
• Cell Cycle MeSH
• Phosphatidylinositol Phosphates metabolism   MeSH
• Nuclear Proteins metabolism   MeSH
• Nuclear Envelope metabolism   ultrastructure   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Proteome metabolism   MeSH
• Cluster Analysis MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphatidylinositol Phosphates MeSH
• Nuclear Proteins MeSH
• phosphatidylinositol 4-phosphate MeSH Browser
• Proteome 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.

• Keywords
• DsbA, Francisella tularensis, SILAC, glyceraldehyde-3-phosphate dehydrogenase, moonlighting,
• 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
• Names of Substances
• Virulence Factors MeSH
• Glyceraldehyde-3-Phosphate Dehydrogenases MeSH
• Blood Proteins MeSH
• Protein Disulfide-Isomerases MeSH
• Proteome MeSH

Using quantitative evaluation of immuno-gold labeling and antigen content, we evaluated various automated freeze-substitution protocols used in preparation of biological samples for immunoelectron microscopy. Protein extraction from cryoimmobilized cells was identified as a critical point during the freeze-substitution. The loss of antigens (potentially available for subsequent immuno-gold labeling) was not significantly affected by freezing, while the cryosubstitution with an organic solvent caused a significant loss of antigens. While addition of water can improve visibility of some cell structures, it strengthened the negative effect of cryosubstitution on antigen loss by extraction. This was, however, significantly reversed in the presence of 0.5% glutaraldehyde in the substitution medium. Furthermore, we showed that the level of these changes was antigen-dependent. In conclusion, low concentrations of glutaraldehyde can be generally recommended for cryosubstitution rather than the use of pure solvent, but the exact conditions need to be elaborated individually for certain antigens.

• MeSH
• Antigens, Nuclear metabolism   MeSH
• Glutaral MeSH
• HeLa Cells MeSH
• Microscopy, Immunoelectron MeSH
• Humans MeSH
• Freeze Substitution methods   MeSH
• Solvents MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Nuclear MeSH
• Glutaral MeSH
• Solvents MeSH

The best available approach of biological sample preparation for transmission electron microscopy currently includes cryoimmobilization by high-pressure freezing (HPF) followed by freeze-substitution (FS). This method has been well established for interphase cells; however, a reliable and easy procedure is still missing for mitotic cells especially because of their fragility and sensitivity to treatments. Here, we present a fast and effective method for HPF/automated FS and LR White embedding of mitotic cells which allows for their controlled and reproducible quality processing. It should be useful in various ultrastructural studies on mitotic cells especially in combination with immunocytochemistry.

• MeSH
• HeLa Cells MeSH
• Histological Techniques methods   MeSH
• Immunohistochemistry * MeSH
• Interphase MeSH
• Cryopreservation * MeSH
• Humans MeSH
• Mitosis * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.

• MeSH
• Acrylic Resins MeSH
• Microscopy, Electron MeSH
• HeLa Cells ultrastructure   MeSH
• Histological Techniques methods   MeSH
• Immunohistochemistry methods   MeSH
• Humans MeSH
• Freeze Substitution methods   MeSH
• Preservation, Biological methods   MeSH
• Pressure MeSH
• Tissue Embedding methods   MeSH
• Freezing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Acrylic Resins MeSH
• LR white MeSH Browser