RESUMO
Listeria monocytogenes and other pathogenic bacteria modify their peptidoglycan to protect it against enzymatic attack through the host innate immune system, such as the cell wall hydrolase lysozyme. During our studies on GpsB, a late cell division protein that controls activity of the bi-functional penicillin binding protein PBP A1, we discovered that GpsB influences lysozyme resistance of L. monocytogenes as mutant strains lacking gpsB showed an increased lysozyme resistance. Deletion of pbpA1 corrected this effect, demonstrating that PBP A1 is also involved in this. Susceptibility to lysozyme mainly depends on two peptidoglycan modifying enzymes: The peptidoglycan N-deacetylase PgdA and the peptidoglycan O-acetyltransferase OatA. Genetic and biochemical experiments consistently demonstrated that the increased lysozyme resistance of the ΔgpsB mutant was PgdA-dependent and OatA-independent. Protein-protein interaction studies supported the idea that GpsB, PBP A1 and PgdA form a complex in L. monocytogenes and identified the regions in PBP A1 and PgdA required for complex formation. These results establish a physiological connection between GpsB, PBP A1 and the peptidoglycan modifying enzyme PgdA. To our knowledge, this is the first reported link between a GpsB-like cell division protein and factors important for escape from the host immune system.
Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Listeria monocytogenes/enzimologia , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/metabolismo , Acetiltransferases/metabolismo , Proteínas de Bactérias/genética , Parede Celular/efeitos dos fármacos , Parede Celular/enzimologia , Farmacorresistência Bacteriana/genética , Humanos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/patogenicidade , Muramidase/farmacologia , Proteínas de Ligação às Penicilinas/genética , Plasmídeos/genéticaRESUMO
Streptococcus pneumoniae is a major cause of life-threatening diseases worldwide. Here we provide an in-depth functional characterization of LytB, the peptidoglycan hydrolase responsible for physical separation of daughter cells. Identified herein as an N-acetylglucosaminidase, LytB is involved also in colonization and invasion of the nasopharynx, biofilm formation and evasion of host immunity as previously demonstrated. We have shown that LytB cleaves the GlcNAc-ß-(1,4)-MurNAc glycosidic bond of peptidoglycan building units. The hydrolysis occurs at sites with fully acetylated GlcNAc moieties, with preference for uncross-linked muropeptides. The necessity of GlcN acetylation and the presence of a single acidic moiety (Glu585) essential for catalysis strongly suggest a substrate-assisted mechanism with anchimeric assistance of the acetamido group of GlcNAc moieties. Additionally, modelling of the catalytic region bound to a hexasaccharide tripentapeptide provided insights into substrate-binding subsites and peptidoglycan recognition. Besides, cell-wall digestion products and solubilisation rates might indicate a tight control of LytB activity to prevent unrestrained breakdown of the cell wall. Choline-independent localization at the poles of the cell, mediated by the choline-binding domain, peptidoglycan modification, and choline-mediated (lipo)teichoic-acid attachment contribute to the high selectivity of LytB. Moreover, so far unknown chitin hydrolase and glycosyltransferase activities were detected using GlcNAc oligomers as substrate.
Assuntos
N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/metabolismo , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidade , Acetilglucosaminidase/metabolismo , Catálise , Domínio Catalítico/fisiologia , Parede Celular/metabolismo , Quitina/metabolismo , Colina/metabolismo , Glicosiltransferases/metabolismo , Hidrolases/metabolismo , Hidrólise , Nasofaringe/microbiologia , Especificidade por Substrato , Ácidos Teicoicos/metabolismo , VirulênciaRESUMO
CodY, a nutritional regulator highly conserved in low G+C Gram-positive bacteria, is essential in Streptococcus pneumoniae (the pneumococcus). A published codY mutant possessed suppressing mutations inactivating the fatC and amiC genes, respectively belonging to iron (Fat/Fec) and oligopeptide (Ami) ABC permease operons, which are directly repressed by CodY. Here we analyzed two additional published codY mutants to further explore the essentiality of CodY. We show that one, in which the regulator of glutamine/glutamate metabolism glnR had been inactivated by design, had only a suppressor in fecE (a gene in the fat/fec operon), while the other possessed both fecE and amiC mutations. Independent isolation of three different fat/fec suppressors thus establishes that reduction of iron import is crucial for survival without CodY. We refer to these as primary suppressors, while inactivation of ami, which is not essential for survival of codY mutants and acquired after initial fat/fec inactivation, can be regarded as a secondary suppressor. The availability of codY- ami+ cells allowed us to establish that CodY activates competence for genetic transformation indirectly, presumably by repressing ami which is known to antagonize competence. The glnR codY fecE mutant was then found to be only partially viable on solid medium and hypersensitive to peptidoglycan (PG) targeting agents such as the antibiotic cefotaxime and the muramidase lysozyme. While analysis of PG and teichoic acid composition uncovered no alteration in the glnR codY fecE mutant compared to wildtype, electron microscopy revealed altered ultrastructure of the cell wall in the mutant, establishing that co-inactivation of GlnR and CodY regulators impacts pneumococcal cell wall physiology. In light of rising levels of resistance to PG-targeting antibiotics of natural pneumococcal isolates, GlnR and CodY constitute potential alternative therapeutic targets to combat this debilitating pathogen, as co-inactivation of these regulators renders pneumococci sensitive to iron and PG-targeting agents.
Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/metabolismo , Amidoidrolases/metabolismo , Proteínas de Bactérias/genética , Cefotaxima/farmacologia , Parede Celular/efeitos dos fármacos , Hidrólise , Mutação , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Transformação GenéticaRESUMO
Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) ß-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis.
Assuntos
Amidoidrolases/metabolismo , Antraz/microbiologia , Bacillus anthracis/citologia , Bacillus anthracis/enzimologia , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Osmose/fisiologia , Estresse Fisiológico , Amidoidrolases/química , Amidoidrolases/genética , Sequência de Aminoácidos , Antraz/genética , Antraz/metabolismo , Bacillus anthracis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Western Blotting , Clonagem Molecular , Cristalografia por Raios X , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Dados de Sequência Molecular , Peptidoglicano/metabolismo , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tolerância ao Sal , Homologia de Sequência de AminoácidosRESUMO
Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with ß-lactam antibiotics. These target Listeria's five high molecular weight penicillin-binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi-functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross-linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)-PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod-shape and PBP B2 for cross-wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased ß-lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross-linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence.
Assuntos
Listeria monocytogenes/fisiologia , Listeria monocytogenes/patogenicidade , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Células 3T3 , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Parede Celular/química , Parede Celular/fisiologia , Células HeLa , Humanos , Listeria monocytogenes/citologia , Listeria monocytogenes/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Mutação , Peptidoglicano/metabolismo , Virulência/genética , beta-Lactamas/farmacologiaRESUMO
Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host-pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB-regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2-associated responses were preserved. Experiments using leukocytes from IL-1R-associated kinase-4-deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R-associated kinase-4-mediated inflammatory responses to S. pneumoniae.
Assuntos
Proteínas de Bactérias/imunologia , Regulação Bacteriana da Expressão Gênica/imunologia , Lipoproteínas/imunologia , Pneumonia Pneumocócica/imunologia , Streptococcus pneumoniae/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Regulação Bacteriana da Expressão Gênica/genética , Células HEK293 , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/imunologia , Lipoproteínas/genética , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , Pneumonia Pneumocócica/genética , Pneumonia Pneumocócica/patologia , Doenças da Imunodeficiência Primária , Streptococcus pneumoniae/genética , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Peptidoglycan surrounds the bacterial cytoplasmic membrane to protect the cell against osmolysis. The biosynthesis of peptidoglycan, made of glycan strands crosslinked by short peptides, is the target of antibiotics like ß-lactams and glycopeptides. Nascent peptidoglycan contains pentapeptides that are trimmed by carboxypeptidases to tetra- and tripeptides. The well-characterized DD-carboxypeptidases hydrolyze the terminal D-alanine from the stem pentapeptide to produce a tetrapeptide. However, few LD-carboxypeptidases that produce tripeptides have been identified, and nothing is known about substrate specificity in these enzymes. We report biochemical properties and crystal structures of the LD-carboxypeptidases LdcB from Streptococcus pneumoniae, Bacillus anthracis, and Bacillus subtilis. The enzymes are active against bacterial cell wall tetrapeptides and adopt a zinc-carboxypeptidase fold characteristic of the LAS superfamily. We have also solved the structure of S. pneumoniae LdcB with a product mimic, elucidating the residues essential for peptidoglycan recognition and the conformational changes that occur on ligand binding.
Assuntos
Proteínas de Bactérias/química , Carboxipeptidases/química , Peptidoglicano/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Bacillus anthracis/enzimologia , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Configuração de Carboidratos , Carboxipeptidases/metabolismo , Cristalografia por Raios X , Cinética , Modelos Moleculares , Peptidoglicano/metabolismo , Ligação Proteica , Streptococcus pneumoniae/enzimologiaRESUMO
The vancomycin resistance operons from Enterococci, Staphylococci and Actinomycetes encode a VanRS two-component signal transduction system (TCS) and a suite of enzymes to modify the peptidoglycan biosynthetic precursor lipid II and to eliminate the D-Ala-D-Ala from the cell. Commingling of these regulatory and enzymatic activities with host functions has the potential to significantly impact host gene expression and cell wall metabolism. Here we report the effects of individually expressing the VanR(B) S(B) TCS and the VanY(B) WH(B) BX(B) resistance proteins in Bacillus subtilis. VanY(B) WH(B) BX(B) expression confers resistance to 2 µg ml(-1) of vancomycin with concomitant reduced Van-FL staining and leads to a cell division defect. In contrast to E. faecalis and S. aureus, VanS(B) is active in B. subtilis without vancomycin addition. Individual expression of the VanR(B) S(B) TCS and the VanY(B) WH(B) BX(B) resistance proteins repress and increase, respectively, expression of PhoPR regulon genes in the phosphate-limited state. When vancomycin-resistant cells are exposed to elevated vancomycin levels, mutant strains with increased resistance to vancomycin and a growth dependency on vanY(B) WH(B) BX(B) expression frequently arise. Mutation of the endogenous Ddl ligase is the necessary and sufficient cause of both phenotypes. We discuss how these effects may influence establishment of van operons in new host bacteria.
Assuntos
Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Parede Celular/química , Expressão Gênica , Resistência a Vancomicina , Vancomicina/farmacologia , Antibacterianos/farmacologia , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Enterococcus faecalis/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus/genéticaRESUMO
Bacterial flagella play key roles in surface attachment and host-bacterial interactions as well as driving motility. Here, we have investigated the ability of Caulobacter crescentus to assemble its flagellar filament from six flagellins: FljJ, FljK, FljL, FljM, FljN, and FljO. Flagellin gene deletion combinations exhibited a range of phenotypes from no motility or impaired motility to full motility. Characterization of the mutant collection showed the following: (i) that there is no strict requirement for any one of the six flagellins to assemble a filament; (ii) that there is a correlation between slower swimming speeds and shorter filament lengths in ΔfljK ΔfljM mutants; (iii) that the flagellins FljM to FljO are less stable than FljJ to FljL; and (iv) that the flagellins FljK, FljL, FljM, FljN, and FljO alone are able to assemble a filament.
Assuntos
Caulobacter crescentus/fisiologia , Flagelos/metabolismo , Flagelina/genética , Flagelina/metabolismo , Substâncias Macromoleculares/metabolismo , Caulobacter crescentus/genética , Flagelos/ultraestrutura , Deleção de Genes , Genes Bacterianos , Locomoção , Substâncias Macromoleculares/ultraestrutura , Microscopia EletrônicaRESUMO
The flagellar genes in Salmonella enterica are expressed in a temporal hierarchy that mirrors the assembly process itself. The σ(28)-FlgM regulatory circuit plays a key role in controlling this temporal hierarchy. This circuit ensures that the class 3 genes are expressed only when the hook-basal body (HBB), a key intermediate in flagellar assembly, is complete. In this work, we investigated the role of the σ(28)-FlgM regulatory circuit in controlling the timing and magnitude of class 3 gene expression using a combination of mathematical modelling and experimental analysis. Analysis of the model predicted that this circuit continuously controls class 3 gene expression in response to HBB abundance. We experimentally validated these predictions by eliminating different components of the σ(28)-FlgM regulatory system and also by rewiring the transcriptional hierarchy. Based on these results, we conclude that the σ(28)-FlgM regulatory circuit continuously senses the HBB assembly process and regulates class 3 gene expression and possibly flagellar numbers in response.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelina/biossíntese , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/fisiologia , Fator sigma/metabolismo , Flagelina/genética , Perfilação da Expressão Gênica , Modelos Teóricos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Fatores de TempoRESUMO
Each Salmonella enterica serovar Typhimurium cell produces a discrete number of complete flagella. Flagellar assembly responds to changes in growth rates through FlhD(4) C(2) activity. FlhD(4) C(2) activity is negatively regulated by the type 3 secretion chaperone FliT. FliT is known to interact with the flagellar filament cap protein FliD as well as components of the flagellar type 3 secretion apparatus. FliD is proposed to act as an anti-regulator, in a manner similar to FlgM inhibition of σ(28) activity. We have found that efficient growth-dependent regulation of FlhD(4) C(2) requires FliT regulation. In turn, FliD regulation of FliT modulates the response. We also show that, unlike other flagellar-specific regulatory circuits, deletion of fliT or fliD did not lead to an all-or-nothing response in FlhD(4) C(2) activity. To investigate why, we characterized the biochemical interactions in the FliT : FliD : FlhD(4) C(2) circuit. When FlhD(4) C(2) was not bound to DNA, FliT disrupted the FlhD(4) C(2) complex. Interestingly, when FlhD(4) C(2) was bound to DNA it was insensitive to FliT regulation. This suggests that the FliT circuit regulates FlhD(4) C(2) activity by preventing the formation of the FlhD(4) C(2) :DNA complex. Our data would suggest that this level of endogenous regulation of FlhD(4) C(2) activity allows the flagellar system to efficiently respond to external signals.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/metabolismo , Proteínas de Bactérias/genética , Flagelos/genética , Ligação Proteica , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimentoRESUMO
FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica. To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Salmonella enterica/genética , Salmonella enterica/metabolismo , Proteínas de Bactérias/genética , Flagelos/genética , Cinética , Fatores de TempoRESUMO
This is the first structural elucidation of the lipooligosaccharide (LOS) endotoxin isolated from Burkholderia vietnamiensis, a clinically important member of Burkholderia cepacia complex, a group of over 10 opportunistic species that are highly problematic in cystic fibrosis. We have characterized a novel LOS structure extracted from two clonal strains of B. vietnamiensis isolated from a cystic fibrosis patient who underwent lung transplantation. Strains were selected from the pretransplantation and post-transplantation periods and endotoxin was extracted. Subsequent analysis interestingly revealed identical oligosaccharidic sequences, but variation in lipid A moieties. Further, both LOS fractions were tested for their immunostimulatory activity on human myelomonocytic U937 cells and for signaling on an HEK293 cell line stably expressing both TLR 4 and MD-2. We observed an increase in lipid A acylation and a resultant increase in biological activity in bio-reporter assays of TNF-alpha secretion in the post-transplantation strain.
Assuntos
Burkholderia/química , Fibrose Cística/microbiologia , Lipopolissacarídeos/química , Transplante de Pulmão , Burkholderia/isolamento & purificação , Configuração de Carboidratos , Sequência de Carboidratos , Humanos , Lipopolissacarídeos/isolamento & purificação , Dados de Sequência MolecularRESUMO
Flagellar gene expression is temporally regulated in response to the assembly state of the growing flagellum. The key mechanism for enforcing this temporal hierarchy in Salmonella enterica serovar Typhimurium is the sigma(28)-FlgM checkpoint, which couples the expression of the late flagellar (P(class3)) genes to the completion of the hook-basal body. This checkpoint is triggered when FlgM is secreted from the cell. In addition to the sigma(28)-FlgM checkpoint, a number of other regulatory mechanisms respond to the secretion of late proteins. In this work, we examined how middle (P(class2)) and late (P(class3)) gene expression is affected by late protein secretion. Dynamic analysis of flagellar gene expression identified a novel mechanism where induction of P(class2) activity is delayed either when late protein secretion is abolished or when late protein secretion is increased. Using a number of different approaches, we were able to show that this mechanism did not involve any known flagellar regulator. Furthermore, the changes in P(class2) activity were not correlated with the associated changes in P(class3) activity, which was found to be proportional to late protein secretion rates. Our data indicate that both P(class2) and P(class3) promoters are continuously regulated in response to assembly and late protein secretion rates. These results suggest that flagellar regulation is more complex than previously thought.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/genética , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/genética , Proteínas de Bactérias/genética , Retroalimentação Fisiológica , Flagelos/metabolismo , Genes Bacterianos , Genes Reporter , Mutação , Fenótipo , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Salmonella typhimurium/metabolismo , Fatores de Tempo , Transcrição GênicaRESUMO
Signaling-protein mRNAs tend to have long untranslated regions (UTRs) containing binding sites for RNA-binding proteins regulating gene expression. Here we show that a PUF-family RNA-binding protein, Mpt5, represses the yeast MAP-kinase pathway controlling differentiation to the filamentous form. Mpt5 represses the protein levels of two pathway components, the Ste7 MAP-kinase kinase and the Tec1 transcriptional activator, and negatively regulates the kinase activity of the Kss1 MAP kinase. Moreover, Mpt5 specifically inhibits the output of the pathway in the absence of stimuli, and thereby prevents inappropriate cell differentiation. The results provide an example of what may be a genome-scale level of regulation at the interface of signaling networks and protein-RNA binding networks.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas Repressoras/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Adesão Celular , Forma Celular , Proteínas de Ligação a DNA/fisiologia , Retroalimentação Fisiológica , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fenótipo , Fosforilação , Proteínas Quinases/fisiologia , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de Transcrição/fisiologiaRESUMO
Members of genus Burkholderia include opportunistic Gram-negative bacteria that are responsible for serious infections in immunocompromised and cystic fibrosis (CF) patients. The Burkholderia cepacia complex is a group of microorganisms composed of at least nine closely related genomovars. Among these, B. cenocepacia is widely recognized to cause epidemics associated with excessive mortality. Species that belong to this strain are problematic CF pathogens because of their high resistance to antibiotics, which makes respiratory infections difficult to treat and impossible to eradicate. Infection by these bacteria is associated with higher mortality in CF and poor outcomes following lung transplantation. One virulence factor contributing to this is the pro-inflammatory lipopolysaccharide (LPS) molecules. Thus, the knowledge of the lipopolysaccharide structure is an essential prerequisite to the understanding of the molecular mechanisms involved in the inflammatory process. Such data are instrumental in aiding the design of antimicrobial compounds and for developing therapeutic strategies against the inflammatory cascade. In particular, defining the structure of the LPS from B. cenocepacia ET-12 clone LMG 16656 (also known as J2315) is extremely important given the recent completion of the sequencing project at the Sanger Centre using this specific strain. In this paper we address this issue by defining the pro-inflammatory activity of the pure lipopolysaccharide, and by describing its full primary structure. The activity of the lipopolysaccharide was tested as a stimulant in human myelomonocytic U937 cells. The structural analysis was carried out by compositional analysis, mass spectrometry and 2D NMR spectroscopy on the intact lipooligosacchride (LOS) and its fragments, which were obtained by selective chemical degradations.
Assuntos
Infecções por Burkholderia/patologia , Burkholderia cepacia/patogenicidade , Fibrose Cística/patologia , Inflamação/patologia , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Infecções por Burkholderia/epidemiologia , Infecções por Burkholderia/microbiologia , Burkholderia cepacia/genética , Burkholderia cepacia/imunologia , Fibrose Cística/epidemiologia , Fibrose Cística/microbiologia , Surtos de Doenças , Humanos , Lipopolissacarídeos/imunologia , Espectroscopia de Ressonância Magnética , Células U937/efeitos dos fármacos , Células U937/patologia , VirulênciaRESUMO
The sigma(28) protein is a member of the bacterial sigma(70)-family of transcription factors that directs RNA polymerase to flagellar late (class 3) promoters. The sigma(28) protein is regulated in response to flagellar assembly by the anti-sigma(28) factor FlgM. FlgM inhibits sigma(28)-dependent transcription of genes whose products are needed late in assembly until the flagellar basal motor structure, the hook-basal body (HBB), is constructed. A second function for the sigma(28) transcription factor has been discovered: sigma(28) facilitates the secretion of FlgM through the HBB, acting as the FlgM Type III secretion chaperone. Transcription-specific mutants in sigma(28) were isolated that remained competent for FlgM-facilitated secretion separating the transcription and secretion-facilitation activities of sigma (28). Conversely, we also describe the isolation of mutants in sigma(28) that are specific for FlgM-facilitated secretion. The data demonstrate that sigma(28) is the Type III secretion chaperone for its own anti-sigma factor FlgM. Thus, a novel role for a sigma(70)-family transcription factor is described.
Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Chaperonas Moleculares/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Fator sigma/metabolismo , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Flagelos/genética , Mutagênese , Mutação , Reação em Cadeia da Polimerase , Salmonella typhimurium/citologia , Fator sigma/genética , beta-Galactosidase/metabolismoRESUMO
On solid growth media with limiting nitrogen source, diploid budding-yeast cells differentiate from the yeast form to a filamentous, adhesive, and invasive form. Genomic profiles of mRNA levels in Saccharomyces cerevisiae yeast-form and filamentous-form cells were compared. Disparate data types, including genes implicated by expression change, filamentation genes known previously through a phenotype, protein-protein interaction data, and protein-metabolite interaction data were integrated as the nodes and edges of a filamentation-network graph. Application of a network-clustering method revealed 47 clusters in the data. The correspondence of the clusters to modules is supported by significant coordinated expression change among cluster co-member genes, and the quantitative identification of collective functions controlling cell properties. The modular abstraction of the filamentation network enables the association of filamentous-form cell properties with the activation or repression of specific biological processes, and suggests hypotheses. A module-derived hypothesis was tested. It was found that the 26S proteasome regulates filamentous-form growth.