Strasková, A*
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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
The intracellular pathogens have the unique capacity to sense the host cell environment and to respond to it by alteration in gene expression and protein synthesis. Proteomic analysis of bacteria exposed directly to the host cell milieu might thus greatly contribute to the elucidation of processes leading to bacterial adaptation and proliferation inside the host cell. Here we have performed a global proteome analysis of a virulent Francisella tularensis subsp. holarctica strain during its intracellular cycle within the macrophage-like murine cell line J774.2 using the metabolic pulse-labeling of bacterial proteins with (35)S-methionine and (35)S-cysteine in various periods of infection. The two-dimensional gel analysis revealed macrophage-induced bacterial proteome changes in which 64 identified proteins were differentially expressed in comparison to controls grown in tissue culture medium. Nevertheless, activation of macrophages with interferon gamma before in vitro infection decreased the number of detected alterations in protein levels. Thus, these proteomic data indicate the F. tularensis ability to adapt to the intracellular hostile environment that is, however, diminished by prior interferon gamma treatment of host cells.
- MeSH
- 2D gelová elektroforéza MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- Francisella tularensis chemie genetika fyziologie MeSH
- interakce hostitele a patogenu * MeSH
- lidé MeSH
- makrofágy mikrobiologie MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- proteomika MeSH
- tularemie mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Francisella tularensis subspecies tularensis is a highly virulent intracellular bacterial pathogen, causing the disease tularemia. However, a safe and effective vaccine for routine application against F. tularensis has not yet been developed. We have recently constructed the deletion mutants for the DsbA homolog protein (ΔdsbA/FSC200) and a hypothetical protein IglH (ΔiglH/FSC200) in the type B F. tularensis subsp. holarctica FSC200 strain, which exerted different protection capacity against parental virulent strain. In this study, we further investigated the immunological correlates for these different levels of protection provided by ΔdsbA/FSC200 and ΔiglH/FSC200 mutants. Our results show that ΔdsbA/FSC200 mutant, but not ΔiglH/FSC200 mutant, induces an early innate inflammatory response leading to strong Th1-like antibody response. Furthermore, vaccination with ΔdsbA/FSC200 mutant, but not with ΔiglH/FSC200, elicited protection against the subsequent challenge with type A SCHU S4 strain in mice. An immunoproteomic approach was used to map a spectrum of antigens targeted by Th1-like specific antibodies, and more than 80 bacterial antigens, including novel ones, were identified. Comparison of tularemic antigens recognized by the ΔdsbA/FSC200 post-vaccination and the SCHU S4 post-challenge sera then revealed the existence of 22 novel SCHU S4 specific antibody clones.
- MeSH
- atenuované vakcíny aplikace a dávkování genetika imunologie MeSH
- bakteriální vakcíny aplikace a dávkování genetika imunologie MeSH
- cytokiny sekrece MeSH
- faktory virulence nedostatek MeSH
- Francisella tularensis klasifikace enzymologie imunologie MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední BALB C MeSH
- proteindisulfidisomerasy nedostatek MeSH
- Th1 buňky imunologie MeSH
- tularemie imunologie prevence a kontrola MeSH
- tvorba protilátek * MeSH
- zkřížená ochrana * MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Thylakoids are the place of the light-photosynthetic reactions. To gain maximal efficiency, these reactions are conditional to proper pigment-pigment and protein-protein interactions. In higher plants thylakoids, the interactions lead to a lateral asymmetry in localization of protein complexes (i.e. granal/stromal thylakoids) that have been defined as a domain-like structures characteristic by different biochemical composition and function (Albertsson P-Å. 2001,Trends Plant Science 6: 349-354). We explored this complex organization of thylakoid pigment-proteins at single cell level in the cyanobacterium Synechocystis sp. PCC 6803. Our 3D confocal images captured heterogeneous distribution of all main photosynthetic pigment-protein complexes (PPCs), Photosystem I (fluorescently tagged by YFP), Photosystem II and Phycobilisomes. The acquired images depicted cyanobacterial thylakoid membrane as a stable, mosaic-like structure formed by microdomains (MDs). These microcompartments are of sub-micrometer in sizes (~0.5-1.5 μm), typical by particular PPCs ratios and importantly without full segregation of observed complexes. The most prevailing MD is represented by MD with high Photosystem I content which allows also partial separation of Photosystems like in higher plants thylakoids. We assume that MDs stability (in minutes) provides optimal conditions for efficient excitation/electron transfer. The cyanobacterial MDs thus define thylakoid membrane organization as a system controlled by co-localization of three main PPCs leading to formation of thylakoid membrane mosaic. This organization might represent evolutional and functional precursor for the granal/stromal spatial heterogeneity in photosystems that is typical for higher plant thylakoids.
- MeSH
- bakteriální proteiny metabolismus MeSH
- fotosyntéza fyziologie MeSH
- fotosystém I (proteinový komplex) metabolismus MeSH
- fotosystém II (proteinový komplex) metabolismus MeSH
- fykobilizomy metabolismus MeSH
- konfokální mikroskopie MeSH
- membránové mikrodomény metabolismus MeSH
- Synechocystis MeSH
- tylakoidy metabolismus MeSH
- zobrazování trojrozměrné MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The tetratricopeptide repeat (TPR) structural motif is known to occur in a wide variety of proteins present in prokaryotic and eukaryotic organisms. The TPR motif represents an elegant module for the assembly of various multiprotein complexes, and thus, TPR-containing proteins often play roles in vital cell processes. As the TPR profile is well defined, the complete TPR protein repertoire of a bacterium with a known genomic sequence can be predicted. This provides a tremendous opportunity for investigators to identify new TPR-containing proteins and study them in detail. In the past decade, TPR-containing proteins of bacterial pathogens have been reported to be directly related to virulence-associated functions. In this minireview, we summarize the current knowledge of the TPR-containing proteins involved in virulence mechanisms of bacterial pathogens while highlighting the importance of TPR motifs for the proper functioning of class II chaperones of a type III secretion system in the pathogenesis of Yersinia, Pseudomonas, and Shigella.
Francisella tularensis, the causative agent of tularemia, is a highly infectious intracellular pathogen with no licensed vaccine available today. The recent search for genome sequences involved in F. tularensis virulence mechanisms led to the identification of the 30-kb region defined as a Francisella pathogenicity island (FPI). In our previous iTRAQ study we described the concerted upregulation of some FPI proteins in different F. tularensis strains cultivated under stress conditions. Among them we identified the IglH protein whose role in Francisella virulence has not been characterized yet. In this work we deleted the iglH gene in a European clinical isolate of F. tularensis subsp. holarctica FSC200. We showed that the iglH gene is necessary for intracellular growth and escape of F. tularensis from phagosomes. We also showed that the iglH mutant is avirulent in a mouse model of infection and persists in the organs for about three weeks after infection. Importantly, mice vaccinated by infection with the iglH mutant were protected against subcutaneous challenge with the fully virulent parental FSC200 strain. This is the first report of a defined subsp. holarctica FPI deletion strain that provides protective immunity against subsequent subcutaneous challenge with a virulent isolate of F. tularensis subsp. holarctica.
- MeSH
- atenuované vakcíny genetika imunologie MeSH
- bakteriální proteiny genetika imunologie MeSH
- bakteriální vakcíny genetika imunologie MeSH
- fagozomy mikrobiologie MeSH
- Francisella tularensis genetika imunologie patogenita MeSH
- makrofágy mikrobiologie MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- sekvenční delece MeSH
- testy genetické komplementace MeSH
- tularemie imunologie mikrobiologie prevence a kontrola MeSH
- virulence MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Francisella tularensis is a highly infectious facultative intracellular bacterium and aetiological agent of tularaemia. The conserved hypothetical lipoprotein with homology to thiol/disulphide oxidoreductase proteins (FtDsbA) is an essential virulence factor in F. tularensis. Its protein sequence has two different domains: the DsbA_Com1_like domain (DSBA), with the highly conserved catalytically active site CXXC and cis-proline residue; and the domain amino-terminal to FKBP-type peptidyl-prolyl isomerases (FKBP_N). To establish the role of both domains in tularaemia infection models, site-directed and deletion mutagenesis affecting the active site (AXXA), the cis-proline (P286T) and the FKBP_N domain (ΔFKBP_N) were performed. The generated mutations led to high attenuation with the ability to induce full or partial host protective immunity. Recombinant protein analysis revealed that the active site CXXC as well as the cis-proline residue and the FKBP_N domain are necessary for correct thiol/disulphide oxidoreductase activity. By contrast, only the DSBA domain (and not the FKBP_N domain) seems to be responsible for the in vitro chaperone activity of the FtDsbA protein.
- MeSH
- faktory virulence genetika metabolismus MeSH
- Francisella tularensis enzymologie genetika MeSH
- molekulární chaperony genetika metabolismus MeSH
- mutační analýza DNA MeSH
- mutageneze cílená MeSH
- mutantní proteiny genetika metabolismus MeSH
- proteindisulfidisomerasy genetika metabolismus MeSH
- sekvenční delece MeSH
- thioredoxiny genetika metabolismus MeSH
- virulence MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Intracellular pathogen F. tularensis is a causative agent of tularemia disease and belongs to the most hazardeous pathogen worldwide, categorized by the Center for Disease Control and Prevention, USA (CDC) as a category A agent. However, no safe and licensed vaccine for prevention a F. tularensis infection is available for vaccination. Tularemia is manifested by several forms depending on a route of infection and virulence of a F. tularensis strain. Essential to a development of the disease is the ability to infect, survive and proliferate inside the mononuclear phagocytes, such as macrophages or dendritic cells. Therefore, this review will discuss aspects of F. tularensis intracellular fate within host macrophages, modulate host signaling pathways to benefit Francisella infection and finally, summarize bacterial determinats involved in the process of phagosomal escape and intracellular replication.
- MeSH
- bakteriální proteiny genetika chemie MeSH
- buněčné linie MeSH
- fagozomy chemie mikrobiologie MeSH
- faktory virulence genetika MeSH
- financování organizované MeSH
- Francisella tularensis genetika patogenita růst a vývoj MeSH
- genomové ostrovy genetika MeSH
- lidé MeSH
- makrofágy MeSH
- mutace MeSH
- regulace genové exprese u bakterií MeSH
- savci MeSH
- signální transdukce MeSH
- stanovení celkové genové exprese MeSH
- tularemie genetika mikrobiologie patofyziologie MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
