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.
- Publikační typ
- časopisecké články MeSH
Determination of the prognosis and treatment outcomes of dilated cardiomyopathy is a serious problem due to the lack of valid specific protein markers. Using in-depth proteome discovery analysis, we compared 49 plasma samples from patients suffering from dilated cardiomyopathy with plasma samples from their healthy counterparts. In total, we identified 97 proteins exhibiting statistically significant dysregulation in diseased plasma samples. The functional enrichment analysis of differentially expressed proteins uncovered dysregulation in biological processes like inflammatory response, wound healing, complement cascade, blood coagulation, and lipid metabolism in dilated cardiomyopathy patients. The same proteome approach was employed in order to find protein markers whose expression differs between the patients well-responding to therapy and nonresponders. In this case, 45 plasma proteins revealed statistically significant different expression between these two groups. Of them, fructose-1,6-bisphosphate aldolase seems to be a promising biomarker candidate because it accumulates in plasma samples obtained from patients with insufficient treatment response and with worse or fatal outcome. Data are available via ProteomeXchange with the identifier PXD046288.
- MeSH
- biologické markery MeSH
- dilatační kardiomyopatie * terapie MeSH
- hemokoagulace MeSH
- lidé MeSH
- proteom genetika MeSH
- proteomika MeSH
- Check Tag
- lidé MeSH
- 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.
- 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
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 highly infectious Gram-negative coccobacillus which causes the disease tularemia. The potential for its misuse as a biological weapon has led disease control and prevention centers to classify this bacterium as a category A agent. Bacterial outer membrane vesicles (OMVs) are spherical particles 20-250 nm in size produced by all Gram-negative bacteria and constitute one of the major secretory pathways. Bacteria use them in interacting with both other bacterial cells and eukaryotic (host) cells. OMVs of Francisella contain number of its so far described virulence factors and immunomodulatory proteins. Their role in host-pathogen interactions can therefore be presumed, and the possibility exists also for their potential use in a subunit vaccine. Moreover, Francisella microbes produce both usual spherical and unusual tubular OMVs. Because OMVs emerge from the outermost surface of the bacterial cell, we focused on the secretion of OMVs in several mutant Francisella strains with disrupted surface structures (namely the O-antigen). O-antigen in Francisella is not only the structural component of LPS but also forms another important virulence factor: the O-antigen polysaccharide capsule. Mutant strain phenotypes were evaluated by growth curves, vesiculation rates, their sensitivity to the complement contained in serum, and proliferation inside murine bone marrow macrophages. Morphologies of both OMVs and the bacteria were visualized by electron microscopy. The O-antigen mutant strains were considerably attenuated in serum resistance and intracellular proliferation. All the strains showed lower ability to form the tubular OMVs. Some strains formed tubular protrusions from their outer membrane but their stability was weak. Some hypervesiculating strains were revealed that will serve as source of OMVs for further studies of their protective potential. Our results suggest the presence of LPS and the O-antigen capsule on the surface of Francisella to be critical not only for its virulence but also for the exceptional tubular shape of its OMVs.
- MeSH
- Francisella tularensis * genetika MeSH
- gramnegativní bakterie MeSH
- lipopolysacharidy chemie MeSH
- myši MeSH
- O-antigeny MeSH
- proteiny vnější bakteriální membrány genetika metabolismus MeSH
- tularemie * mikrobiologie prevence a kontrola MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference 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.
- Publikační typ
- časopisecké články MeSH
Ubiquitination of proteins, like phosphorylation and acetylation, is an important regulatory aspect influencing numerous and various cell processes, such as immune response signaling and autophagy. The study of ubiquitination has become essential to learning about host-pathogen interactions, and a better understanding of the detailed mechanisms through which pathogens affect ubiquitination processes in host cell will contribute to vaccine development and effective treatment of diseases. Pathogenic bacteria (e.g., Salmonella enterica, Legionella pneumophila and Shigella flexneri) encode many effector proteins, such as deubiquitinating enzymes (DUBs), targeting the host ubiquitin machinery and thus disrupting pertinent ubiquitin-dependent anti-bacterial response. We focus here upon the host ubiquitination system as an integral unit, its interconnection with the regulation of inflammation and autophagy, and primarily while examining pathogens manipulating the host ubiquitination system. Many bacterial effector proteins have already been described as being translocated into the host cell, where they directly regulate host defense processes. Due to their importance in pathogenic bacteria progression within the host, they are regarded as virulence factors essential for bacterial evasion. However, in some cases (e.g., Francisella tularensis) the host ubiquitination system is influenced by bacterial infection, although the responsible bacterial effectors are still unknown.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
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
Release of outer membrane vesicles (OMV) is an important phenomenon in Gram-negative bacteria playing multiple roles in their lifestyle, including in relation to virulence and host-pathogen interaction. Francisella tularensis, unlike other bacteria, releases unusually shaped, tubular OMV. We present a proteomic comparison of OMV and membrane fractions from two F. tularensis strains: moderately virulent subsp. holarctica strain FSC200 and highly virulent subsp. tularensis strain SchuS4. Proteomic comparison studies routinely evaluate samples from the same proteome, but sometimes we must compare samples from closely related organisms. This raises quantification issues. We propose a novel approach to cross-species proteomic comparison based on an intersection protein database from the individual single-species databases. This is less prone to quantification errors arising from differences in the sequences. Consecutively comparing subproteomes of OMV and membranes of the two strains allows distinguishing differences in relative protein amounts caused by global expression changes from those caused by preferential protein packing to OMV or membranes. Among the proteins most differently packed into OMV between the two strains, we detected proteins involved in biosynthesis and metabolism of bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids, as well as some major structural outer membrane proteins. The data are available via ProteomeXchange with identifier PXD022406.
- MeSH
- Francisella tularensis * MeSH
- Francisella MeSH
- lidé MeSH
- proteom genetika MeSH
- proteomika MeSH
- tularemie * MeSH
- virulence MeSH
- vnější bakteriální membrána MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
