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.
Francisella tularensis is a Gram-negative intracellular pathogen causing tularemia. A number of its potential virulence factors have been identified, but their biology and functions are not precisely known. Understanding the biological and immunological functions of these proteins requires adequate genetic tools for homologous and heterologous expression of cloned genes, maintaining both original structure and post-translational modifications. Here, we report the construction of a new multipurpose shuttle plasmid - pEVbr - which can be used for high-level expression in F. tularensis. The pEVbr plasmid has been constructed by modifying the TetR-regulated expression vector pEDL17 (LoVullo, 2012) that includes (i) a strong F. tularensis bfr promoter, and (ii) two tet operator sequences cloned into the promoter. The cloned green fluorescent protein (GFP), used as a reporter, demonstrated almost undetectable basal expression level under uninduced conditions and a highly dynamic dose-dependent response to the inducer. The utility of the system was further confirmed by cloning the gapA and FTT_1676 genes into the pEVbr vector and quantifying proteins expression in F. tularensis LVS, as well as by studying post-translational modification of the cloned genes. This study demonstrates that high levels of recombinant native-like Francisella proteins can be produced in Francisella cells. Hence, this system may be beneficial for the analysis of protein function and the development of new treatments and vaccines.
- MeSH
- Francisella tularensis * genetika MeSH
- lidé MeSH
- plazmidy genetika MeSH
- rekombinantní proteiny genetika MeSH
- tetracyklin farmakologie MeSH
- tularemie * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Targeted proteomics recently proved to be a technique for the detection and absolute quantification of proteins not easily accessible to classical bottom-up approaches. Due to this, it has been considered as a high fidelity tool to detect potential warfare agents in wide spread kinds of biological and environmental matrices. Clostridium perfringens toxins are considered to be potential biological weapons, especially the epsilon toxin which belongs to a group of the most powerful bacterial toxins. Here, the development of a target mass spectrometry method for the detection of C. perfringens protein toxins (alpha, beta, beta2, epsilon, iota) is described. A high-resolution mass spectrometer with a quadrupole-Orbitrap system operating in target acquisition mode (parallel reaction monitoring) was utilized. Because of the lack of commercial protein toxin standards recombinant toxins were prepared within Escherichia coli. The analysis was performed using proteotypic peptides as the target compounds together with their isotopically labeled synthetic analogues as internal standards. Calibration curves were calculated for each peptide in concentrations ranging from 0.635 to 1101 fmol/μL. Limits of detection and quantification were determined for each peptide in blank matrices.
- MeSH
- bakteriální proteiny analýza genetika MeSH
- bakteriální toxiny analýza genetika MeSH
- chromatografie kapalinová MeSH
- Clostridium perfringens * genetika růst a vývoj metabolismus MeSH
- Escherichia coli genetika MeSH
- peptidy analýza genetika MeSH
- proteomika MeSH
- rekombinantní proteiny analýza MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- 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
Francisella tularensis, an intracellular pathogen causing the disease tularemia, utilizes surface glycoconjugates such as lipopolysaccharide, capsule, and capsule-like complex for its protection against inhospitable conditions of the environment. Francisella species also possess a functional glycosylation apparatus by which specific proteins are O-glycosidically modified. We here created a mutant with a nonfunctional FTS_1402 gene encoding for a putative glycan flippase and studied the consequences of its disruption. The mutant strain expressed diminished glycosylation similarly to, but to a lesser extent than, that of the oligosaccharyltransferase-deficient ΔpglA mutant. In contrast to ΔpglA, inactivation of FTS_1402 had a pleiotropic effect, leading to alteration in glycosylation and, importantly, to decrease in lipopolysaccharide, capsule, and/or capsule-like complex production, which were reflected by distinct phenotypes in host-pathogen associated properties and virulence potential of the two mutant strains. Disruption of FTS_1402 resulted in enhanced sensitivity to complement-mediated lysis and reduced virulence in mice that was independent of diminished glycosylation. Importantly, the mutant strain induced a protective immune response against systemic challenge with homologous wild-type FSC200 strain. Targeted disruption of genes shared by multiple metabolic pathways may be considered a novel strategy for constructing effective live, attenuated vaccines.
- MeSH
- ABC transportéry genetika metabolismus MeSH
- bakteriální proteiny genetika metabolismus MeSH
- chromatografie kapalinová MeSH
- Francisella tularensis genetika metabolismus patogenita MeSH
- genetická pleiotropie MeSH
- glykokonjugáty biosyntéza MeSH
- glykosylace MeSH
- hexosyltransferasy genetika metabolismus MeSH
- interakce hostitele a patogenu MeSH
- lipopolysacharidy biosyntéza MeSH
- membránové proteiny genetika metabolismus MeSH
- mutace MeSH
- myši inbrední BALB C MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- regulace genové exprese u bakterií MeSH
- tandemová hmotnostní spektrometrie MeSH
- tularemie mikrobiologie MeSH
- umlčování genů MeSH
- virulence genetika MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- finanční podpora výzkumu jako téma MeSH
- Francisella tularensis patogenita MeSH
- imunotoxiny škodlivé účinky MeSH
- inhalační expozice MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- organofosforové sloučeniny toxicita MeSH
- sarin farmakologie škodlivé účinky MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- MeSH
- B-lymfocyty metabolismus účinky léků MeSH
- buňky NK metabolismus účinky léků MeSH
- chemické bojové látky toxicita MeSH
- finanční podpora výzkumu jako téma MeSH
- imunitní systém metabolismus účinky léků MeSH
- myši MeSH
- počet lymfocytů MeSH
- sarin toxicita MeSH
- T-lymfocyty účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- MeSH
- aplikace inhalační MeSH
- buněčná imunita MeSH
- CD antigeny imunologie MeSH
- chemické bojové látky toxicita MeSH
- cholinesterasové inhibitory aplikace a dávkování toxicita MeSH
- finanční podpora výzkumu jako téma MeSH
- myši MeSH
- sarin aplikace a dávkování toxicita MeSH
- T-lymfocyty imunologie účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- MeSH
- alveolární makrofágy metabolismus účinky léků MeSH
- buňky NK metabolismus účinky léků MeSH
- chemické bojové látky farmakologie MeSH
- finanční podpora výzkumu jako téma MeSH
- imunitní systém imunologie účinky léků MeSH
- inhalační expozice MeSH
- lipopolysacharidy farmakologie MeSH
- myši MeSH
- sarin farmakologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- MeSH
- adenylátcyklasy imunologie MeSH
- antigeny virové genetika imunologie MeSH
- Bordetella pertussis enzymologie MeSH
- CD8-pozitivní T-lymfocyty imunologie MeSH
- cytotoxické T-lymfocyty fyziologie MeSH
- epitopy imunologie MeSH
- myši MeSH
- rekombinantní proteiny farmakologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
