Francisella tularensis secretes tubular outer membrane vesicles (OMVs) that contain a number of immunoreactive proteins as well as virulence factors. We have reported previously that isolated Francisella OMVs enter macrophages, cumulate inside, and induce a strong pro-inflammatory response. In the current article, we present that OMVs treatment of macrophages also enhances phagocytosis of the bacteria and suppresses their intracellular replication. On the other hand, the subsequent infection with Francisella is able to revert to some extent the strong pro-inflammatory effect induced by OMVs in macrophages. Being derived from the bacterial surface, isolated OMVs may be considered a "non-viable mixture of Francisella antigens" and as such, they present a promising protective material. Immunization of mice with OMVs isolated from a virulent F. tularensis subsp. holarctica strain FSC200 prolonged the survival time but did not fully protect against the infection with a lethal dose of the parent strain. However, the sera of the immunized animals revealed unambiguous cytokine and antibody responses and proved to recognize a set of well-known Francisella immunoreactive proteins. For these reasons, Francisella OMVs present an interesting material for future protective studies.

• Klíčová slova
• FSC200, Francisella tularensis, host-pathogen interaction, outer membrane vesicles, vaccination,
• Publikační typ
• časopisecké články MeSH

Francisella tularensis influences several host molecular/signaling pathways during infection. Ubiquitination and deubiquitination are among the most important regulatory mechanisms and respectively occur through attachment or removal of the ubiquitin molecule. The process is necessary not only to mark molecules for degradation, but also, for example, to the activation of signaling pathways leading to pro-inflammatory host response. Many intracellular pathogens, including Francisella tularensis, have evolved mechanisms of modifying such host immune responses to escape degradation. Here, we describe that F. tularensis interferes with the host's ubiquitination system. We show increased total activity of deubiquitinating enzymes (DUBs) in human macrophages after infection, while confirm reduced enzymatic activities of two specific DUBs (USP10 and UCH-L5), and demonstrate increased activity of USP25. We further reveal the enrichment of these three enzymes in exosomes derived from F. tularensis-infected cells. The obtained results show the regulatory effect on ubiquitination mechanism in macrophages during F. tularensis infection.

• Klíčová slova
• DUBs, Francisella, UCH-L5, USP10, USP25, deubiquitination, exosomes, extracellular vesicles,
• MeSH
• deubikvitinasy metabolismus   MeSH
• Francisella tularensis * MeSH
• gramnegativní bakteriální infekce * metabolismus   MeSH
• lidé MeSH
• makrofágy MeSH
• signální transdukce MeSH
• thiolesterasa ubikvitinu metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deubikvitinasy MeSH
• thiolesterasa ubikvitinu MeSH
• USP10 protein, human MeSH Prohlížeč
• USP25 protein, human MeSH Prohlížeč

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

Francisella tularensis is known to release unusually shaped tubular outer membrane vesicles (OMV) containing a number of previously identified virulence factors and immunomodulatory proteins. In this study, we present that OMV isolated from the F. tularensis subsp. holarctica strain FSC200 enter readily into primary bone marrow-derived macrophages (BMDM) and seem to reside in structures resembling late endosomes in the later intervals. The isolated OMV enter BMDM generally via macropinocytosis and clathrin-dependent endocytosis, with a minor role played by lipid raft-dependent endocytosis. OMVs proved to be non-toxic and had no negative impact on the viability of BMDM. Unlike the parent bacterium itself, isolated OMV induced massive and dose-dependent proinflammatory responses in BMDM. Using transmission electron microscopy, we also evaluated OMV release from the bacterial surface during several stages of the interaction of Francisella with BMDM. During adherence and the early phase of the uptake of bacteria, we observed numerous tubular OMV-like protrusions bulging from the bacteria in close proximity to the macrophage plasma membrane. This suggests a possible role of OMV in the entry of bacteria into host cells. On the contrary, the OMV release from the bacterial surface during its cytosolic phase was negligible. We propose that OMV play some role in the extracellular phase of the interaction of Francisella with the host and that they are involved in the entry mechanism of the bacteria into macrophages.

• Klíčová slova
• FSC200, Francisella tularensis, cell entry, host–pathogen interaction, macrophage, outer membrane vesicles,
• Publikační typ
• časopisecké články MeSH

Immune responses to intracellular pathogens depend largely upon the activation of T helper type 1-dependent mechanisms. The contribution of B cells to establishing protective immunity has long been underestimated. Francisella tularensis, including a number of subspecies, provides a suitable model for the study of immune responses against intracellular bacterial pathogens. We previously demonstrated that Francisella infects B cells and activates B-cell subtypes to produce a number of cytokines and express the activation markers. Recently, we documented the early production of natural antibodies as a consequence of Francisella infection in mice. Here, we summarize current knowledge on the innate and acquired humoral immune responses initiated by Francisella infection and their relationships with the immune defense systems.

• Klíčová slova
• B cells, Francisella tularensis, intracellular pathogen, natural antibodies, natural immunity,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

There remains to this day a great gap in understanding as to the role of B cells and their products-antibodies and cytokines-in mediating the protective response to Francisella tularensis, a Gram-negative coccobacillus belonging to the group of facultative intracellular bacterial pathogens. We previously have demonstrated that Francisella interacts directly with peritoneal B-1a cells. Here, we demonstrate that, as early as 12 h postinfection, germ-free mice infected with Francisella tularensis produce infection-induced antibody clones reacting with Francisella tularensis proteins having orthologs or analogs in eukaryotic cells. Production of some individual clones was limited in time and was influenced by virulence of the Francisella strain used. The phylogenetically stabilized defense mechanism can utilize these early infection-induced antibodies both to recognize components of the invading pathogens and to eliminate molecular residues of infection-damaged self cells.

• MeSH
• B-lymfocyty imunologie   metabolismus   MeSH
• cytokiny metabolismus   MeSH
• Francisella tularensis patogenita   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• tularemie imunologie   mikrobiologie   MeSH
• tvorba protilátek MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny MeSH

This work reports highly selective phosphopeptide enrichment using amorphous TiO2 nanotubes (TiO2NTs) and the same material decorated with superparamagnetic Fe3O4 nanoparticles (TiO2NTs@Fe3O4NPs). TiO2NTs and TiO2NTs@Fe3O4NPs materials were applied for phosphopeptide enrichment both from a simple peptide mixture (tryptic digest of bovine serum albumin and α-casein) and from a complex peptide mixture (tryptic digest of Jurkat T cell lysate). The obtained enrichment efficiency and selectivity for phosphopeptides of TiO2NTs and TiO2NTs@Fe3O4NPs were increased to 28.7 and 25.3%, respectively, as compared to those of the well-established TiO2 microspheres. The enrichment protocol was extended for a second elution step facilitating the identification of additional phosphopeptides. It further turned out that both types of amorphous TiO2 nanotubes provide qualitatively new physicochemical features that are clearly advantageous for highly selective phosphopeptide enrichment. This has been confirmed experimentally resulting in substantial reduction of non-phosphorylated peptides in the enriched samples. In addition, TiO2NTs@Fe3O4NPs combine high selectivity and ease of handling due to the superparamagnetic character of the material. The presented materials and performances are further promising for applications toward a whole range of other types of biomolecules to be treated in a similar fashion.

• Publikační typ
• časopisecké články MeSH

Primary interaction of an intracellular bacterium with its host cell is initiated by activation of multiple signaling pathways in response to bacterium recognition itself or as cellular responses to stress induced by the bacterium. The leading molecules in these processes are cell surface membrane receptors as well as cytosolic pattern recognition receptors recognizing pathogen-associated molecular patterns or damage-associated molecular patterns induced by the invading bacterium. In this review, we demonstrate possible sequences of events leading to recognition of Francisella tularensis, present findings on known mechanisms for manipulating cell responses to protect Francisella from being killed, and discuss newly published data from the perspective of early stages of host-pathogen interaction.

• Klíčová slova
• Francisella tularensis, innate immune recognition, intracellular replication, phagocytosis, signaling pathways,
• MeSH
• alarminy genetika   imunologie   MeSH
• bakteriální proteiny genetika   imunologie   MeSH
• fagocytóza genetika   MeSH
• Francisella tularensis genetika   imunologie   patogenita   MeSH
• interakce hostitele a patogenu genetika   imunologie   MeSH
• lidé MeSH
• makrofágy imunologie   mikrobiologie   MeSH
• PAMP struktury imunologie   metabolismus   MeSH
• přirozená imunita * MeSH
• receptory buněčného povrchu genetika   imunologie   MeSH
• receptory rozpoznávající vzory genetika   imunologie   MeSH
• regulace genové exprese MeSH
• signální transdukce MeSH
• tularemie genetika   imunologie   mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• alarminy MeSH
• bakteriální proteiny MeSH
• PAMP struktury MeSH
• receptory buněčného povrchu MeSH
• receptory rozpoznávající vzory MeSH

Current anti-cancer strategy takes advantage of tumour specific abnormalities in DNA damage response to radio- or chemo-therapy. Inhibition of the ATR/Chk1 pathway has been shown to be synthetically lethal in cells with high levels of oncogene-induced replication stress and in p53- or ATM- deficient cells. In the presented study, we aimed to elucidate molecular mechanisms underlying radiosensitization of T-lymphocyte leukemic MOLT-4 cells by VE-821, a higly potent and specific inhibitor of ATR. We combined multiple approaches: cell biology techniques to reveal the inhibitor-induced phenotypes, and quantitative proteomics, phosphoproteomics, and metabolomics to comprehensively describe drug-induced changes in irradiated cells. VE-821 radiosensitized MOLT-4 cells, and furthermore 10 μM VE-821 significantly affected proliferation of sham-irradiated MOLT-4 cells. We detected 623 differentially regulated phosphorylation sites. We revealed changes not only in DDR-related pathways and kinases, but also in pathways and kinases involved in maintaining cellular metabolism. Notably, we found downregulation of mTOR, the main regulator of cellular metabolism, which was most likely caused by an off-target effect of the inhibitor, and we propose that mTOR inhibition could be one of the factors contributing to the phenotype observed after treating MOLT-4 cells with 10 μM VE-821. In the metabolomic analysis, 206 intermediary metabolites were detected. The data indicated that VE-821 potentiated metabolic disruption induced by irradiation and affected the response to irradiation-induced oxidative stress. Upon irradiation, recovery of damaged deoxynucleotides might be affected by VE-821, hampering DNA repair by their deficiency. Taken together, this is the first study describing a complex scenario of cellular events that might be ATR-dependent or triggered by ATR inhibition in irradiated MOLT-4 cells. Data are available via ProteomeXchange with identifier PXD008925.

• MeSH
• aminokyselinové motivy MeSH
• ATM protein antagonisté a inhibitory   MeSH
• biologické markery MeSH
• fosfoproteiny * chemie   metabolismus   MeSH
• fosforylace MeSH
• genová ontologie MeSH
• inhibitory proteinkinas farmakologie   MeSH
• kontrolní body buněčného cyklu účinky léků   účinky záření   MeSH
• lidé MeSH
• metabolom * MeSH
• metabolomika metody   MeSH
• nádorové buněčné linie MeSH
• proteom * MeSH
• proteomika metody   MeSH
• pyraziny farmakologie   MeSH
• radiosenzibilizující látky farmakologie   MeSH
• signální transdukce MeSH
• sulfony farmakologie   MeSH
• tolerance záření účinky léků   MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• výpočetní biologie metody   MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide MeSH Prohlížeč
• ATM protein MeSH
• ATR protein, human MeSH Prohlížeč
• biologické markery MeSH
• fosfoproteiny * MeSH
• inhibitory proteinkinas MeSH
• MTOR protein, human MeSH Prohlížeč
• proteom * MeSH
• pyraziny MeSH
• radiosenzibilizující látky MeSH
• sulfony MeSH
• TOR serin-threoninkinasy MeSH