Rhodococcus erythropolis CCM2595 is a bacterial strain, which has been studied for its capability to degrade phenol and other toxic aromatic compounds. Its cell wall contains mycolic acids, which are also an attribute of other bacteria of the Mycolata group, such as Corynebacterium and Mycobacterium species. We suppose that many genes upregulated by phenol stress in R. erythropolis are controlled by the alternative sigma factors of RNA polymerase, which are active in response to the cell envelope or oxidative stress. We developed in vitro and in vivo assays to examine the connection between the stress sigma factors and genes activated by various extreme conditions, e.g., heat, cell surface, and oxidative stress. These assays are based on the procedures of such tests carried out in the related species, Corynebacterium glutamicum. We showed that the R. erythropolis CCM2595 genes frmB1 and frmB2, which encode S-formylglutathione hydrolases (named corynomycolyl transferases in C. glutamicum), are controlled by SigD, just like the homologous genes cmt1 and cmt2 in C. glutamicum. The new protocol of the in vivo and in vitro assays will enable us to classify R. erythropolis promoters according to their connection to sigma factors and to assign the genes to the corresponding sigma regulons. The complex stress responses, such as that induced by phenol, could, thus, be analyzed with respect to the gene regulation by sigma factors.
Rhodococci are bacteria which can survive under various extreme conditions, in the presence of toxic compounds, and in other hostile habitats. Their tolerance of unfavorable conditions is associated with the structure of their cell wall and their large array of enzymes, which degrade or detoxify harmful compounds. Their physiological and biotechnological properties, together with tools for their genetic manipulation, enable us to apply them in biotransformations, biodegradation and bioremediation. Many such biotechnological applications cause stresses that positively or negatively affect their efficiency. Whereas numerous reviews on rhodococci described their enzyme activities, the optimization of degradation or production processes, and corresponding technological solutions, only a few reviews discussed some specific effects of stresses on the physiology of rhodococci and biotechnological processes. This review aims to comprehensively describe individual stress responses in Rhodococcus strains, the interconnection of different types of stresses and their consequences for cell physiology. We examine here the responses to (1) environmental stresses (desiccation, heat, cold, osmotic and pH stress), (2) the presence of stress-inducing compounds (metals, organic compounds and antibiotics) in the environment (3) starvation and (4) stresses encountered during biotechnological applications. Adaptations of the cell envelope, the formation of multicellular structures and stresses induced by the interactions of hosts with pathogenic rhodococci are also included. The roles of sigma factors of RNA polymerase in the global regulation of stress responses in rhodococci are described as well. Although the review covers a large number of stressful conditions, our intention was to provide an overview of the selected stress responses and their possible connection to biotechnological processes, not an exhaustive survey of the scientific literature. The findings on stress responses summarized in this review and the demonstration of gaps in current knowledge may motivate researchers working to fill these gaps.
We report results showing that the silencing of carbonic anhydrase I (siCA1) in prostatic (PC3) tumour cells has a significant impact on exosome formation. An increased diameter, concentration and diversity of the produced exosomes were noticed as a consequence of this knock-down. The protein composition of the exosomes' cargo was also altered. Liquid chromatography and mass spectrometry analyses identified 42 proteins significantly altered in PC3 siCA1 exosomes compared with controls. The affected proteins are mainly involved in metabolic processes, biogenesis, cell component organization and defense/immunity. Interestingly, almost all of them have been described as 'enhancers' of tumour development through the promotion of cell proliferation, migration and invasion. Thus, our results indicate that the reduced expression of the CA1 protein enhances the malignant potential of PC3 cells.
- MeSH
- buňky PC-3 MeSH
- energetický metabolismus genetika MeSH
- exozómy genetika metabolismus MeSH
- karboanhydrasa I genetika metabolismus MeSH
- lidé MeSH
- nádory prostaty genetika metabolismus patologie MeSH
- pohyb buněk genetika MeSH
- proliferace buněk genetika MeSH
- regulace genové exprese enzymů * MeSH
- regulace genové exprese u nádorů * MeSH
- RNA interference * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Kingella kingae is a member of the commensal oropharyngeal flora of young children. Improvements in detection methods have led to the recognition of K. kingae as an emerging pathogen that frequently causes osteoarticular infections in children and a severe form of infective endocarditis in children and adults. Kingella kingae secretes a membrane-damaging RTX (Repeat in ToXin) toxin, RtxA, which is implicated in the development of clinical infections. However, the mechanism by which RtxA recognizes and kills host cells is largely unexplored. To facilitate structure-function studies of RtxA, we have developed a procedure for the overproduction and purification of milligram amounts of biologically active recombinant RtxA. Mass spectrometry analysis revealed the activation of RtxA by post-translational fatty acyl modification on the lysine residues 558 and/or 689 by the fatty-acyltransferase RtxC. Acylated RtxA was toxic to various human cells in a calcium-dependent manner and possessed pore-forming activity in planar lipid bilayers. Using various biochemical and biophysical approaches, we demonstrated that cholesterol facilitates the interaction of RtxA with artificial and cell membranes. The results of analyses using RtxA mutant variants suggested that the interaction between the toxin and cholesterol occurs via two cholesterol recognition/interaction amino acid consensus motifs located in the C-terminal portion of the pore-forming domain of the toxin. Based on our observations, we conclude that the cytotoxic activity of RtxA depends on post-translational acylation of the K558 and/or K689 residues and on the toxin binding to cholesterol in the membrane.
- MeSH
- acylace MeSH
- bakteriální toxiny genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- buněčné linie MeSH
- cholesterol metabolismus MeSH
- Kingella kingae enzymologie genetika MeSH
- lidé MeSH
- lysin chemie MeSH
- posttranslační úpravy proteinů * MeSH
- rekombinantní proteiny metabolismus MeSH
- transaminasy genetika metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mammalian lungs are organs exhibiting the cellular and spatial complexity required for gas exchange to support life. The respiratory epithelium internally lining the airways is susceptible to infections due to constant exposure to inhaled microbes. Biomedical research into respiratory bacterial infections in humans has been mostly carried out using small mammalian animal models or two-dimensional, submerged cultures of undifferentiated epithelial cells. These experimental model systems have considerable limitations due to host specificity of bacterial pathogens and lack of cellular and morphological complexity. This review describes the in vitro differentiated and polarized airway epithelial cells of human origin that are used as a model to study respiratory bacterial infections. Overall, these models recapitulate key aspects of the complexity observed in vivo and can help in elucidating the molecular details of disease processes observed during respiratory bacterial infections.
- MeSH
- Bacteria imunologie MeSH
- bakteriální infekce imunologie mikrobiologie patologie MeSH
- biologické modely * MeSH
- interakce hostitele a patogenu imunologie MeSH
- lidé MeSH
- přirozená imunita imunologie MeSH
- respirační sliznice imunologie mikrobiologie patologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The airway epithelium restricts the penetration of inhaled pathogens into the underlying tissue and plays a crucial role in the innate immune defense against respiratory infections. The whooping cough agent, Bordetella pertussis, adheres to ciliated cells of the human airway epithelium and subverts its defense functions through the action of secreted toxins and other virulence factors. We examined the impact of B. pertussis infection and of adenylate cyclase toxin-hemolysin (CyaA) action on the functional integrity of human bronchial epithelial cells cultured at the air-liquid interface (ALI). B. pertussis adhesion to the apical surface of polarized pseudostratified VA10 cell layers provoked a disruption of tight junctions and caused a drop in transepithelial electrical resistance (TEER). The reduction of TEER depended on the capacity of the secreted CyaA toxin to elicit cAMP signaling in epithelial cells through its adenylyl cyclase enzyme activity. Both purified CyaA and cAMP-signaling drugs triggered a decrease in the TEER of VA10 cell layers. Toxin-produced cAMP signaling caused actin cytoskeleton rearrangement and induced mucin 5AC production and interleukin-6 (IL-6) secretion, while it inhibited the IL-17A-induced secretion of the IL-8 chemokine and of the antimicrobial peptide beta-defensin 2. These results indicate that CyaA toxin activity compromises the barrier and innate immune functions of Bordetella-infected airway epithelia.
- MeSH
- adenylátcyklasový toxin genetika metabolismus toxicita MeSH
- AMP cyklický metabolismus MeSH
- Bordetella pertussis genetika metabolismus MeSH
- bronchy cytologie metabolismus mikrobiologie MeSH
- cytoskelet metabolismus MeSH
- epitelové buňky metabolismus mikrobiologie MeSH
- interleukin-6 metabolismus MeSH
- lidé MeSH
- mucin 5AC metabolismus MeSH
- pertuse genetika metabolismus mikrobiologie MeSH
- signální transdukce účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) translocates its adenylate cyclase (AC) enzyme domain into target cells in a step that depends on membrane cholesterol content. We thus examined what role in toxin activities is played by the five putative cholesterol recognition amino acid consensus (CRAC) motifs predicted in CyaA hemolysin moiety. CRAC-disrupting phenylalanine substitutions had no impact on toxin activities and these were not inhibited by free cholesterol, showing that the putative CRAC motifs are not involved in cholesterol binding. However, helix-breaking proline substitutions in these segments uncovered a structural role of the Y632, Y658, Y725 and Y738 residues in AC domain delivery and pore formation by CyaA. Substitutions of Y940 of the fifth motif, conserved in the acylated domains of related RTX toxins, did not impact on fatty-acylation of CyaA by CyaC and the CyaA-Y940F mutant was intact for toxin activities on erythrocytes and myeloid cells. However, the Y940A or Y940P substitutions disrupted the capacity of CyaA to insert into artificial lipid bilayers or target cell membranes. The aromatic ring of tyrosine 940 side chain thus appears to play a key structural role in molecular interactions that initiate CyaA penetration into target membranes.
- MeSH
- adenylátcyklasový toxin genetika metabolismus MeSH
- aminokyselinové motivy MeSH
- buněčná membrána metabolismus MeSH
- buněčné linie MeSH
- cholesterol metabolismus MeSH
- erytrocyty metabolismus MeSH
- makrofágy metabolismus MeSH
- mutační analýza DNA MeSH
- myši MeSH
- substituce aminokyselin MeSH
- transport proteinů MeSH
- tyrosin genetika metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b(+)) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b(-) cells. The nonhemolytic AC(+) Hly(-) bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC(+) Hly(-) mutant also infected mouse lungs as efficiently as the parental AC(+) Hly(+) strain. Hence, elevation of cAMP in CD11b(-) cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>10(7) CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.
- MeSH
- adenylátcyklasový toxin metabolismus MeSH
- AMP cyklický metabolismus MeSH
- antigeny CD11b metabolismus MeSH
- Bordetella pertussis patogenita MeSH
- buněčná membrána metabolismus MeSH
- dendritické buňky imunologie MeSH
- fagocyty imunologie MeSH
- hemolyziny metabolismus MeSH
- makrofágový antigen 1 metabolismus MeSH
- myši inbrední BALB C MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- pertuse mikrobiologie MeSH
- plíce mikrobiologie patologie MeSH
- T-lymfocyty imunologie MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The 'AC to Hly-linking segment' thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins.
- MeSH
- adenylátcyklasový toxin chemie genetika metabolismus MeSH
- adenylátcyklasy chemie genetika MeSH
- AMP cyklický metabolismus MeSH
- Bordetella pertussis chemie patogenita MeSH
- hemolyziny genetika MeSH
- lidé MeSH
- lipidové dvojvrstvy chemie metabolismus MeSH
- perforin chemie MeSH
- permeabilita buněčné membrány účinky léků MeSH
- pertuse genetika mikrobiologie patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
