The Bordetella adenylate cyclase-hemolysin (CyaA, ACT, or AC-Hly) is a multifunctional toxin. Simultaneously with promoting calcium ion entry, CyaA delivers into host cells an adenylate cyclase enzyme (AC) and permeabilizes cell membrane by forming small cation-selective pores. Indirect evidence suggested that these two activities were accomplished by different membrane-inserted CyaA conformers, one acting as an AC-delivering monomer and the other as an uncharacterized pore-forming oligomer. We tested this model by directly detecting toxin oligomers in cell membrane and by assessing oligomerization of specific mutants with altered pore-forming properties. CyaA oligomers were revealed in sheep erythrocyte membranes by immunogold labeling and directly demonstrated by pulldown of membrane-inserted CyaA together with biotinylated CyaA-AC(-) toxoid. Membrane oligomers of CyaA could also be resolved by nondenaturing electrophoresis of mild detergent extracts of erythrocytes. Furthermore, CyaA mutants exhibiting enhanced (E581K) or reduced (E570K+E581P) specific hemolytic and pore-forming activity were found to exhibit also a correspondingly enhanced or reduced propensity to form oligomers in erythrocyte membranes. On the other hand, processed CyaA, with the AC domain cleaved off by erythrocyte proteases, was detected only in a monomeric form excluded from the oligomers of unprocessed CyaA. These results provide the first direct evidence that oligomerization is involved in formation of CyaA pores in target membranes and that translocation of the AC domain across cell membrane may be accomplished by monomeric CyaA.

• MeSH
• adenylátcyklasový toxin farmakokinetika   metabolismus   MeSH
• Bordetella enzymologie   MeSH
• endocytóza MeSH
• erytrocyty MeSH
• hemolýza účinky léků   MeSH
• missense mutace MeSH
• multimerizace proteinu MeSH
• ovce MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

BACKGROUND: The first systematic study of small non-coding RNAs (sRNA, ncRNA) in Streptomyces is presented. Except for a few exceptions, the Streptomyces sRNAs, as well as the sRNAs in other genera of the Actinomyces group, have remained unstudied. This study was based on sequence conservation in intergenic regions of Streptomyces, localization of transcription termination factors, and genomic arrangement of genes flanking the predicted sRNAs. RESULTS: Thirty-two potential sRNAs in Streptomyces were predicted. Of these, expression of 20 was detected by microarrays and RT-PCR. The prediction was validated by a structure based computational approach. Two predicted sRNAs were found to be terminated by transcription termination factors different from the Rho-independent terminators. One predicted sRNA was identified computationally with high probability as a Streptomyces 6S RNA. Out of the 32 predicted sRNAs, 24 were found to be structurally dissimilar from known sRNAs. CONCLUSION: Streptomyces is the largest genus of Actinomyces, whose sRNAs have not been studied. The Actinomyces is a group of bacterial species with unique genomes and phenotypes. Therefore, in Actinomyces, new unique bacterial sRNAs may be identified. The sequence and structural dissimilarity of the predicted Streptomyces sRNAs demonstrated by this study serve as the first evidence of the uniqueness of Actinomyces sRNAs.

• MeSH
• algoritmy MeSH
• bakteriální RNA genetika   chemie   MeSH
• druhová specificita MeSH
• financování organizované MeSH
• genom bakteriální MeSH
• intergenová DNA MeSH
• konformace nukleové kyseliny MeSH
• molekulární modely MeSH
• nekódující RNA genetika   chemie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• Streptomyces coelicolor genetika   MeSH
• Streptomyces genetika   MeSH
• terminátorové oblasti (genetika) MeSH
• výpočetní biologie MeSH

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenylátcyklasový toxin genetika   chemie   izolace a purifikace   metabolismus   MeSH
• adenylátcyklasy metabolismus   MeSH
• AMP cyklický metabolismus   MeSH
• antigeny CD11b fyziologie   MeSH
• biologický transport MeSH
• buněčná membrána fyziologie   MeSH
• buněčné linie MeSH
• financování organizované MeSH
• hemolýza MeSH
• katalýza MeSH
• makrofágy fyziologie   MeSH
• monocyty fyziologie   MeSH
• mutageneze cílená MeSH
• myši MeSH
• ovce MeSH
• polymerázová řetězová reakce MeSH
• rekombinantní proteiny chemie   izolace a purifikace   metabolismus   MeSH
• substituce aminokyselin MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH

Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic alpha-helix(502-522) containing a pair of negatively charged Glu(509) and Glu(516) residues. Another predicted transmembrane alpha-helix(565-591) comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu(516) increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu(516) by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu(570) and Glu(581) by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA

• MeSH
• adenylátcyklasový toxin farmakologie   genetika   metabolismus   MeSH
• bakteriální proteiny farmakologie   genetika   metabolismus   MeSH
• Bordetella enzymologie   genetika   MeSH
• erytrocytární membrána metabolismus   MeSH
• financování organizované MeSH
• hemolýza genetika   účinky léků   MeSH
• missense mutace MeSH
• ovce MeSH
• signální transdukce genetika   účinky léků   MeSH
• substituce aminokyselin MeSH
• transport proteinů genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

• Publikační typ
• abstrakt z konference MeSH

Signal transduction pathways in both prokaryotes and eukaryotes utilize protein phosphorylation as a key regulatory mechanism. Recent studies have proven that eukaryotic-type serine/threonine protein kinases (Hank's type) are widespread in many bacteria, although little is known regarding the cellular processes they control. In this study, we have attempted to establish the role of a single eukaryotic-type protein kinase, StkP of Streptococcus pneumoniae, in bacterial survival. Our results indicate that the expression of StkP is important for the resistance of S. pneumoniae to various stress conditions. To investigate the impact of StkP on this phenotype, we compared the whole-genome expression profiles of the wild-type and DeltastkP mutant strains by microarray technology. This analysis revealed that StkP positively controls the transcription of a set of genes encoding functions involved in cell wall metabolism, pyrimidine biosynthesis, DNA repair, iron uptake, and oxidative stress response. Despite the reduced transformability of the stkP mutant, we found that the competence regulon was derepressed in the stkP mutant under conditions that normally repress natural competence development. Furthermore, the competence regulon was expressed independently of exogenous competence-stimulating peptide. In summary, our studies show that a eukaryotic-type serine/threonine protein kinase functions as a global regulator of gene expression in S. pneumoniae.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• delece genu MeSH
• eukaryotické buňky enzymologie   MeSH
• fenotyp MeSH
• financování organizované MeSH
• koncentrace vodíkových iontů MeSH
• mikrobiální viabilita genetika   účinky léků   MeSH
• mutace MeSH
• osmotický tlak MeSH
• oxidační stres MeSH
• peroxid vodíku farmakologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• regulace genové exprese u bakterií MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• Streptococcus pneumoniae enzymologie   genetika   růst a vývoj   MeSH
• testy genetické komplementace MeSH
• vysoká teplota MeSH

• Publikační typ
• abstrakt z konference MeSH

Neisseria meningitidis colonizes the human nasopharynx and occasionally causes lethal or damaging septicemia and meningitis. Here, we examined the adherence-mediated signaling of meningococci to human cells by comparing gene expression profiles of human umbilical vein endothelial cells (HUVEC) infected by adherent wild-type, frpC-deficient mutant, or the nonadherent (DeltapilD) N. meningitidis. Pili-mediated adhesion of meningococci resulted in alterations of expression levels of human genes known to regulate apoptosis, cell proliferation, inflammatory response, adhesion and genes for signaling pathway proteins such as TGF-beta/Smad, Wnt/beta-catenin and Notch/Jagged. This reveals that adhering piliated meningocci manipulate host signaling pathways controlling cell proliferation while establishing a commensal relationship.

• MeSH
• apoptóza imunologie   MeSH
• bakteriální adheze MeSH
• bakteriální proteiny genetika   MeSH
• buněčné kultury MeSH
• cytoprotekce genetika   MeSH
• embryo savčí metabolismus   MeSH
• endoteliální buňky mikrobiologie   MeSH
• financování organizované MeSH
• lidé MeSH
• membránové proteiny genetika   nedostatek   MeSH
• Neisseria meningitidis patogenita   MeSH
• proteiny fimbrií genetika   nedostatek   MeSH
• průtoková cytometrie MeSH
• RNA komplementární MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• signální transdukce MeSH
• stanovení celkové genové exprese MeSH
• upregulace MeSH
• venae umbilicales cytologie   MeSH
• zánět genetika   MeSH
• Check Tag
• lidé MeSH

Bordetella adenylate cyclase (AC) toxin-hemolysin (CyaA) targets myeloid phagocytes expressing the alphaMbeta2 integrin (CD11b/CD18) and delivers into their cytosol an AC enzyme that converts ATP into cyclic AMP (cAMP). In parallel, CyaA acts as a hemolysin, forming small membrane pores. Using specific mutations, we dissected the contributions of the two activities to cytolytic potency of CyaA on J774A.1 murine monocytes. The capacity of AC to penetrate cells and deplete cytosolic ATP was essential for promoting lysis and the enzymatically inactive but fully hemolytic CyaA-AC- toxoid exhibited a 15-fold-lower cytolytic capacity on J774A.1 cells than intact CyaA. Moreover, a two- or fourfold drop of specific hemolytic activity of the CyaA-E570Q and CyaA-E581P mutants was overpowered by an intact capacity to dissipate cytosolic ATP into cAMP, allowing the less hemolytic proteins to promote lysis of J774A.1 cells as efficiently as intact CyaA. However, an increased hemolytic activity, due to lysine substitutions of glutamates 509, 516, and 581 in the pore-forming domain, conferred on AC- toxoids a correspondingly enhanced cytolytic potency. Moreover, a threefold increase in hemolytic activity could override a fourfold drop in capacity to convert cellular ATP to cAMP, conferring on the CyaA-E581K construct an overall twofold increased cytolytic potency. Hence, although appearing auxiliary in cytolytic action of the toxin on nucleated cells, the pore-forming activity can synergize with ATP-depleting activity of the cell-invasive AC enzyme and complement its action toward maximal cytotoxicity.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenylátcyklasový toxin toxicita   MeSH
• AMP cyklický metabolismus   MeSH
• antigeny CD11b metabolismus   MeSH
• antigeny CD18 metabolismus   MeSH
• Bordetella pertussis enzymologie   imunologie   MeSH
• buněčná smrt imunologie   MeSH
• buněčné linie MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• cytotoxicita imunologická MeSH
• erytrocyty metabolismus   MeSH
• financování organizované MeSH
• křečci praví MeSH
• monocyty enzymologie   imunologie   MeSH
• myši MeSH
• ovce MeSH
• permeabilita buněčné membrány imunologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• myši MeSH
• zvířata MeSH

The Bordetella adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) forms cation-selective membrane channels and delivers into the cytosol of target cells an adenylate cyclase domain (AC) that catalyzes uncontrolled conversion of cellular ATP to cAMP. Both toxin activities were previously shown to depend on post-translational activation of proCyaA to CyaA by covalent palmitoylation of the internal Lys983 residue (K983). CyaA, however, harbors a second RTX acylation site at residue Lys860 (K860), and the role of K860 acylation in toxin activity is unclear. We produced in E. coli the CyaA-K860R and CyaA-K983R toxin variants having the Lys860 and Lys983 acylation sites individually ablated by arginine substitutions. When examined for capacity to form membrane channels and to penetrate sheep erythrocytes, the CyaA-K860R acylated on Lys983 was about 1 order of magnitude more active than CyaA-K983R acylated on Lys860, although, in comparison to intact CyaA, both monoacylated constructs exhibited markedly reduced activities in erythrocytes. Channels formed in lipid bilayers by CyaA-K983R were importantly less selective for cations than channels formed by CyaA-K860R, intact CyaA, or proCyaA, showing that, independent of its acylation status, the Lys983 residue may play a role in toxin structures that determine the distribution of charged residues at the entry or inside of the CyaA channel. While necessary for activity on erythrocytes, acylation of Lys983 was also sufficient for the full activity of CyaA on CD11b+ J774A.1 monocytes. In turn, acylation of Lys860 alone did not permit toxin activity on erythrocytes, while it fully supported the high-affinity binding of CyaA-K983R to the toxin receptor CD11b/CD18 and conferred on CyaA-K983R a reduced but substantial capacity to penetrate and kill the CD11b+ cells. This is the first evidence that acylation of Lys860 may play a role in the biological activity of CyaA, even if redundant to the acylation of Lys983.

• MeSH
• acylace MeSH
• adenylátcyklasový toxin chemie   toxicita   MeSH
• antigeny CD11b metabolismus   MeSH
• buněčné linie MeSH
• erytrocyty účinky léků   MeSH
• financování organizované MeSH
• iontové kanály chemie   MeSH
• kationty MeSH
• kompetitivní vazba MeSH
• křečci praví MeSH
• lysin chemie   MeSH
• makrofágy účinky léků   MeSH
• myši MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• abstrakty MeSH