RESUMO
Type III protein-secretion machines are essential for the interactions of many pathogenic or symbiotic bacterial species with their respective eukaryotic hosts. The core component of these machines is the injectisome, a multiprotein complex that mediates the selection of substrates, their passage through the bacterial envelope, and ultimately their delivery into eukaryotic target cells. The injectisome is composed of a large cytoplasmic complex or sorting platform, a multiring base embedded in the bacterial envelope, and a needle-like filament that protrudes several nanometers from the bacterial surface and is capped at its distal end by the tip complex. A characteristic feature of these machines is that their activity is stimulated by contact with target host cells. The sensing of target cells, thought to be mediated by the distal tip of the needle filament, generates an activating signal that must be transduced to the secretion machine by the needle filament. Here, through a multidisciplinary approach, including solid-state NMR (SSNMR) and cryo electron microscopy (cryo-EM) analyses, we have identified critical residues of the needle filament protein of a Salmonella Typhimurium type III secretion system that are involved in the regulation of the activity of the secretion machine. We found that mutations in the needle filament protein result in various specific phenotypes associated with different steps in the type III secretion process. More specifically, these studies reveal an important role for a polymorphic helix of the needle filament protein and the residues that line the lumen of its central channel in the control of type III secretion.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Complexos Multiproteicos/metabolismo , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/química , Sistemas de Secreção Bacterianos/genética , Microscopia Crioeletrônica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Mutação , Conformação Proteica , Transporte Proteico/genética , Salmonella typhimurium/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/ultraestruturaRESUMO
Little is known about the mechanisms by which Treponema pallidum (Tp), the causative agent of syphilis, copes with oxidative stress as it establishes persistent infection within its obligate human host. The Tp genomic sequence indicates that the bacterium's antioxidant defenses do not include glutathione and are limited to just a few proteins, with only one, TP0509, offering direct defense against peroxides. Although this Tp peroxiredoxin (Prx) closely resembles AhpC-like Prxs, Tp lacks AhpF, the typical reductant for such enzymes. Functionally, TpAhpC resembles largely eukaryotic, nonAhpC typical 2-Cys Prx proteins in using thioredoxin (Trx, TP0919) as an efficient electron donor and exhibiting broad specificity toward hydroperoxide substrates. Unlike many of the eukaryotic Prxs, however, TpAhpC is relatively resistant to inactivation during turnover with hydroperoxide substrates. As is often observed in typical 2-Cys Prxs, TpAhpC undergoes redox-sensitive oligomer formation. Quantitative immunoblotting revealed that TpTrx and TpAhpC are present at very high levels (over 100 and 300 microM, respectively) in treponemes infecting rabbit testes; their redox potentials, at -242 +/- 1 and -192 +/- 2 mV, respectively, are consistent with the role of TpTrx as the cellular reductant of TpAhpC. Transcriptional analysis of select antioxidant genes confirmed the presence of high mRNA levels for ahpC and trx which diminish greatly when spirochetes replicate under in vitro growth conditions. Thus, T. pallidum has evolved an extraordinarily robust, broad-spectrum AhpC as its sole mechanism for peroxide defense to combat this significant threat to treponemal growth and survival during infection.
Assuntos
Antioxidantes/metabolismo , Peroxirredoxinas/metabolismo , Tiorredoxinas/metabolismo , Treponema pallidum/metabolismo , Sequência de Aminoácidos , Animais , Genoma Bacteriano , Dados de Sequência Molecular , Oxirredução , Peroxirredoxinas/química , Coelhos , Alinhamento de Sequência , Especificidade por Substrato , Transcrição Gênica , Treponema pallidum/genéticaRESUMO
The molecular architecture and composition of the outer membrane (OM) of Treponema pallidum (Tp), the noncultivable agent of venereal syphilis, differ considerably from those of typical Gram-negative bacteria. Several years ago we described TP0453, the only lipoprotein associated with the inner leaflet of the Tp OM. Whereas polypeptides of other treponemal lipoproteins are hydrophilic, non-lipidated TP0453 can integrate into membranes, a property attributed to its multiple amphipathic helices (AHs). Furthermore, membrane integration of the TP0453 polypeptide was found to increase membrane permeability, suggesting the molecule functions in a porin-like manner. To better understand the mechanism of membrane integration of TP0453 and its physiological role in Tp OM biogenesis, we solved its crystal structure and used mutagenesis to identify membrane insertion elements. The crystal structure of TP0453 consists of an α/ß/α-fold and includes five stably folded AHs. In high concentrations of detergent, TP0453 transitions from a closed to open conformation by lateral movement of two groups of AHs, exposing a large hydrophobic cavity. Triton X-114 phase partitioning, liposome floatation assay, and bis-1-anilino-8-naphthalenesulfonate binding revealed that two adjacent AHs are critical for membrane sensing/integration. Using terbium-dipicolinic acid complex-loaded large unilamellar vesicles, we found that TP0453 increased efflux of fluorophore only at acidic pH. Gel filtration and cross-linking experiments demonstrated that one AH critical for membrane sensing/insertion also forms a dimeric interface. Based on structural dynamics and comparison with Mycobacterium tuberculosis lipoproteins LprG and LppX, we propose that TP0453 functions as a carrier of lipids, glycolipids, and/or derivatives during OM biogenesis.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Permeabilidade da Membrana Celular , Membrana Celular/química , Multimerização Proteica , Treponema pallidum/química , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/química , Lipossomos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Coelhos , Sífilis/genética , Sífilis/metabolismo , Treponema pallidum/genética , Treponema pallidum/metabolismoRESUMO
Definitive identification of Treponema pallidum rare outer membrane proteins (OMPs) has long eluded researchers. TP0326, the sole protein in T. pallidum with sequence homology to a Gram-negative OMP, belongs to the BamA family of proteins essential for OM biogenesis. Structural modelling predicted that five polypeptide transport-associated (POTRA) domains comprise the N-terminus of TP0326, while the C-terminus forms an 18-stranded amphipathic ß-barrel. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning and liposome incorporation supported these topological predictions and confirmed that the ß-barrel is responsible for the native protein's amphiphilicity. Expression analyses revealed that native TP0326 is expressed at low abundance, while a protease-surface accessibility assay confirmed surface exposure. Size-exclusion chromatography and blue native polyacrylamide gel electrophoresis revealed a modular Bam complex in T. pallidum larger than that of Escherichia coli. Non-orthologous ancillary factors and self-association of TP0326 via its ß-barrel may both contribute to the Bam complex. T. pallidum-infected rabbits mount a vigorous antibody response to both POTRA and ß-barrel portions of TP0326, whereas humans with secondary syphilis respond predominantly to POTRA. The syphilis spirochaete appears to have devised a stratagem for harnessing the Bam pathway while satisfying its need to limit surface antigenicity.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/microbiologia , Sífilis/microbiologia , Treponema pallidum/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Sífilis/metabolismo , Treponema pallidum/química , Treponema pallidum/genéticaRESUMO
In a microarray analysis of the RpoS regulon in mammalian host-adapted Borrelia burgdorferi, bb0728 (cdr) was found to be dually transcribed by the sigma factors σ(70) and RpoS. The cdr gene encodes a coenzyme A disulphide reductase (CoADR) that reduces CoA-disulphides to CoA in an NADH-dependent manner. Based on the abundance of CoA in B. burgdorferi and the biochemistry of the enzyme, CoADR has been proposed to play a role in the spirochaete's response to reactive oxygen species. To better understand the physiologic function(s) of BbCoADR, we generated a B. burgdorferi mutant in which the cdr gene was disrupted. RT-PCR and 5'-RACE analysis revealed that cdr and bb0729 are co-transcribed from a single transcriptional start site upstream of the bb0729 coding sequence; a shuttle vector containing the bb0729-cdr operon and upstream promoter element was used to complement the cdr mutant. Although the mutant was no more sensitive to hydrogen peroxide than its parent, it did exhibit increased sensitivity to high concentrations of t-butyl-hydroperoxide, an oxidizing compound that damages spirochetal membranes. Characterization of the mutant during standard (15% oxygen, 6% CO(2)) and anaerobic (< 1% O(2) , 9-13% CO(2)) cultivation at 37°C revealed a growth defect under both conditions that was particularly striking during anaerobiosis. The mutant was avirulent by needle inoculation and showed decreased survival in feeding nymphs, but displayed no survival defect in unfed flat nymphs. Based on these results, we propose that BbCoADR is necessary to maintain optimal redox ratios for CoA/CoA-disulphide and NAD(+) /NADH during periods of rapid replication throughout the enzootic cycle, to support thiol-disulphide homeostasis, and to indirectly protect the spirochaete against peroxide-mediated membrane damage; one or more of these functions are essential for infection of the mammalian host by B. burgdorferi.
Assuntos
Borrelia burgdorferi/enzimologia , Borrelia burgdorferi/crescimento & desenvolvimento , Coenzima A/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fatores de Virulência/metabolismo , Aerobiose , Sequência de Aminoácidos , Anaerobiose , Animais , Antibacterianos/toxicidade , Artrite/microbiologia , Artrite/patologia , Infecções por Borrelia/microbiologia , Infecções por Borrelia/patologia , Borrelia burgdorferi/efeitos dos fármacos , Borrelia burgdorferi/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Ixodes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , NAD/metabolismo , NADH NADPH Oxirredutases/genética , Ninfa/microbiologia , Oxidantes/toxicidade , Homologia de Sequência , Análise de Sobrevida , Transcrição Gênica , VirulênciaRESUMO
Two-component systems (TCS) are principal mechanisms by which bacteria adapt to their surroundings. Borrelia burgdorferi encodes only two TCS. One is comprised of a histidine kinase, Hk2, and the response regulator Rrp2. While the contribution of Hk2 remains unclear, Rrp2 is part of a regulatory pathway involving the spirochete's alternate sigma factors, RpoN and RpoS. Genes within the Rrp2/RpoN/RpoS regulon function to promote tick transmission and early infection. The other TCS consists of a hybrid histidine kinase, Hk1, and the response regulator Rrp1. Hk1 is composed of two periplasmic sensor domains (D1 and D2), followed by conserved cytoplasmic histidine kinase core, REC, and Hpt domains. In addition to its REC domain, Rrp1 contains a GGDEF motif characteristic of diguanylate cyclases. To investigate the role of Hk1 during the enzootic cycle, we inactivated this gene in two virulent backgrounds. Extensive characterization of the resulting mutants revealed a dramatic phenotype whereby Hk1-deficient spirochetes are virulent in mice and able to migrate out of the bite site during feeding but are killed within the midgut following acquisition. We hypothesize that the phosphorelay between Hk1 and Rrp1 is initiated by the binding of feeding-specific ligand(s) to Hk1 sensor domain D1 and/or D2. Once activated, Rrp1 directs the synthesis of cyclic dimeric GMP (c-di-GMP), which, in turn, modulates the expression and/or activity of gene products required for survival within feeding ticks. In contrast to the Rrp2/RpoN/RpoS pathway, which is active only within feeding nymphs, the Hk1/Rrp1 TCS is essential for survival during both larval and nymphal blood meals.
Assuntos
Borrelia burgdorferi/enzimologia , Borrelia burgdorferi/fisiologia , Ixodes/microbiologia , Viabilidade Microbiana , Proteínas Quinases/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Histidina Quinase , Doença de Lyme/microbiologia , Camundongos , Camundongos Endogâmicos C3H , Proteínas Quinases/deficiência , Doenças dos Roedores/microbiologia , Transdução de Sinais , Estresse Fisiológico , VirulênciaRESUMO
Borrelia burgdorferi outer surface protein C (ospC) is required for the establishment of infection in mammals. However, its precise function remains controversial. The biologically active form of OspC appears to be a homodimer. Alpha helix 1 and 1' of the apposing monomers form a solvent-accessible pocket at the dimeric interface that presents a putative ligand-binding domain (LBD1). Here we employ site-directed and allelic-exchange mutagenesis to test the hypothesis that LBD1 is a determinant of OspC function in the mammalian environment. Substitution of residues K60, E61 and E63 which line LBD1 resulted in the loss of infectivity or influenced dissemination. Analyses of the corresponding recombinant proteins demonstrated that the loss of function was not due to structural perturbation, impaired dimer formation or the loss of plasminogen binding. This study is the first to assess the involvement of individual residues and domains of OspC in its in vivo function. The data support the hypothesis that OspC interacts with a mammalian derived ligand that is critical for survival during early infection. These results shed new light on the structure-functions relationships of OspC and challenge existing hypotheses regarding OspC function in mammals.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Borrelia burgdorferi/patogenicidade , Fatores de Virulência/metabolismo , Substituição de Aminoácidos/genética , Animais , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Borrelia burgdorferi/genética , Dimerização , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos C3H , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Plasminogênio/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Fatores de Virulência/genéticaRESUMO
Treponema pallidum reacts poorly with the antibodies present in rabbit and human syphilitic sera, a property attributed to the paucity of proteins in its outer membrane. To better understand the basis for the syphilis spirochete's "stealth pathogenicity," we used a dual-label, 3-step amplified assay in which treponemes encapsulated in gel microdroplets were probed with syphilitic sera in parallel with anti-FlaA antibodies. A small (approximately 5 to 10%) but reproducible fraction of intact treponemes bound IgG and/or IgM antibodies. Three lines of evidence supported the notion that the surface antigens were likely ß-barrel-forming outer membrane proteins (OMPs): (i) surface labeling with anti-lipoidal (VDRL) antibodies was not observed, (ii) immunoblot analysis confirmed prior results showing that T. pallidum glycolipids are not immunoreactive, and (iii) labeling of intact organisms was not appreciably affected by proteinase K (PK) treatment. With this method, we also demonstrate that TprK (TP0897), an extensively studied candidate OMP, and TP0136, a lipoprotein recently reported to be surface exposed, are both periplasmic. Consistent with the immunolabeling studies, TprK was also found to lack amphiphilicity, a characteristic property of ß-barrel-forming proteins. Using a consensus computational framework that combined subcellular localization and ß-barrel structural prediction tools, we generated ranked groups of candidate rare OMPs, the predicted T. pallidum outer membrane proteome (OMPeome), which we postulate includes the surface-exposed molecules detected by our enhanced gel microdroplet assay. In addition to underscoring the syphilis spirochete's remarkably poor surface antigenicity, our findings help to explain the complex and shifting balance between pathogen and host defenses that characterizes syphilitic infection.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Treponema pallidum/imunologia , Animais , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Clonagem Molecular , Biologia Computacional/métodos , Eletroforese em Gel de Poliacrilamida , Imunofluorescência/métodos , Immunoblotting , Masculino , Coelhos , Sífilis/imunologia , Sífilis/microbiologia , Treponema pallidum/metabolismoRESUMO
Little is known about Zn homeostasis in Yersinia pestis, the plague bacillus. The Znu ABC transporter is essential for zinc (Zn) uptake and virulence in a number of bacterial pathogens. Bioinformatics analysis identified ZnuABC as the only apparent high-affinity Zn uptake system in Y. pestis. Mutation of znuACB caused a growth defect in Chelex-100-treated PMH2 growth medium, which was alleviated by supplementation with submicromolar concentrations of Zn. Use of transcriptional reporters confirmed that Zur mediated Zn-dependent repression and that it can repress gene expression in response to Zn even in the absence of Znu. Virulence testing in mouse models of bubonic and pneumonic plague found only a modest increase in survival in low-dose infections by the znuACB mutant. Previous studies of cluster 9 (C9) transporters suggested that Yfe, a well-characterized C9 importer for manganese (Mn) and iron in Y. pestis, might function as a second, high-affinity Zn uptake system. Isothermal titration calorimetry revealed that YfeA, the solute-binding protein component of Yfe, binds Mn and Zn with comparably high affinities (dissociation constants of 17.8 ± 4.4 nM and 6.6 ± 1.2 nM, respectively), although the complete Yfe transporter could not compensate for the loss of Znu in in vitro growth studies. Unexpectedly, overexpression of Yfe interfered with the znu mutant's ability to grow in low concentrations of Zn, while excess Zn interfered with the ability of Yfe to import iron at low concentrations; these results suggest that YfeA can bind Zn in the bacterial cell but that Yfe is incompetent for transport of the metal. In addition to Yfe, we have now eliminated MntH, FetMP, Efe, Feo, a substrate-binding protein, and a putative nickel transporter as the unidentified, secondary Zn transporter in Y. pestis. Unlike other bacterial pathogens, Y. pestis does not require Znu for high-level infectivity and virulence; instead, it appears to possess a novel class of transporter, which can satisfy the bacterium's Zn requirements under in vivo metal-limiting conditions. Our studies also underscore the need for bacterial cells to balance binding and transporter specificities within the periplasm in order to maintain transition metal homeostasis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Peste/microbiologia , Yersinia pestis/patogenicidade , Zinco/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/fisiologia , Camundongos , Oriente Médio , Mutação , Virulência/genética , Virulência/fisiologia , Yersinia pestis/genética , Yersinia pestis/crescimento & desenvolvimento , Yersinia pestis/fisiologia , Zinco/fisiologiaRESUMO
Treponema pallidum rapidly disseminates from a genital site of inoculation to diverse organs where it establishes persistent infection. T. pallidum has long been regarded as a stealth pathogen because of its poorly antigenic and non-inflammatory surface. There is now increasing evidence that antigenic variation also contributes to the ability of the spirochaete to evade host defences. Among the small number of proteins encoded by the T. pallidum genome with sequence similarity to well-characterized transcription factors is TP0262, an orthologue for cAMP regulatory protein (CRP) of Escherichia coli. Giacani and co-workers identified sequences matching the CRP consensus-binding motif upstream of the promoters of tprE, tprG and tprJ, three members of the T. pallidum repeat (tpr) gene family (subfamily II). Using electrophoretic mobility shift assay, DNaseI footprinting and an E. coli-based reporter system, they demonstrated that TP0262 specifically recognizes the putative binding sequences and that DNA binding is cAMP-dependent. Their report, a major methodological advance for syphilis research, suggests that T. pallidum has appropriated a paradigmatic global regulator of metabolic processes in heterotrophic bacteria to further its capacity for immune evasion in its obligate human host.
Assuntos
Proteínas de Bactérias/genética , Proteína Receptora de AMP Cíclico/genética , Regiões Promotoras Genéticas , Treponema pallidum/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Treponema pallidum/metabolismoRESUMO
Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure's fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete's complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.
Assuntos
Tomografia com Microscopia Eletrônica , Treponema pallidum/ultraestrutura , Sequência de Aminoácidos , Animais , Aderência Bacteriana , Proteínas de Bactérias/genética , Membrana Celular/ultraestrutura , Parede Celular/ultraestrutura , Células Epiteliais/microbiologia , Flagelos/ultraestrutura , Humanos , Imageamento Tridimensional , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Organelas/ultraestrutura , Estrutura Terciária de Proteína , Coelhos , Alinhamento de Sequência , Treponema pallidum/fisiologiaRESUMO
The frequently observed ankyrin repeat motif represents a structural scaffold evolved for mediating protein-protein interactions. As such, these repeats modulate a diverse range of cellular functions. We thermodynamically characterized the heterodimeric GA-binding protein (GABP) alphabeta complex and focused specifically on the interaction mediated by the ankyrin repeat domain of the GABPbeta. Our isothermal titration calorimetric analysis of the interaction between the GABP subunits determined an association constant (K(A)) of 6.0 x 10(8) M(-1) and that the association is favorably driven by a significant change in enthalpy (DeltaH) and a minor change in entropy (-TDeltaS). A total of 16 GABPbeta interface residues were chosen for alanine scanning mutagenesis. The calorimetrically measured differences in the free energy of binding were compared to computationally calculated values resulting in a correlation coefficient r = 0.71. We identified three spatially contiguous hydrophobic and aromatic residues that form a binding free energy hot spot (DeltaDeltaG > 2.0 kcal/mol). One residue provides structural support to the hot spot residues. Three non-hot spot residues are intermediate contributors (DeltaDeltaG approximately 1.0 kcal/mol) and create a canopy-like structure over the hot spot residues to possibly occlude solvent and orientate the subunits. The remaining interface residues are located peripherally and have weak contributions. Finally, our mutational analysis revealed a significant entropy-enthalpy compensation for this interaction.
Assuntos
Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Mapeamento de Interação de Proteínas , Animais , Repetição de Anquirina , Sítios de Ligação , Calorimetria , Dicroísmo Circular , Clonagem Molecular , Entropia , Fator de Transcrição de Proteínas de Ligação GA/química , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , TermodinâmicaRESUMO
The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein-protein interaction module is involved in a diverse set of cellular functions, and consequently, defects in ankyrin repeat proteins have been found in a number of human diseases. Recent biophysical, crystallographic, and NMR studies have been used to measure the stability and define the various topological features of this motif in an effort to understand the structural basis of ankyrin repeat-mediated protein-protein interactions. Characterization of the folding and assembly pathways suggests that ankyrin repeat domains generally undergo a two-state folding transition despite their modular structure. Also, the large number of available sequences has allowed the ankyrin repeat to be used as a template for consensus-based protein design. Such projects have been successful in revealing positions responsible for structure and function in the ankyrin repeat as well as creating a potential universal scaffold for molecular recognition.
Assuntos
Repetição de Anquirina , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Simulação por Computador , Dados de Sequência Molecular , Ligação ProteicaRESUMO
The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% alpha-helix with little beta-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Proteínas Periplásmicas/química , Porinas/química , Proteínas da Membrana Bacteriana Externa/análise , Proteínas de Bactérias/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Proteínas Periplásmicas/análise , Porinas/fisiologia , Estrutura Secundária de ProteínaRESUMO
Borrelia burgdorferi survives in an enzootic cycle, and Dps proteins protect DNA against damage during starvation or oxidative stress. The role of a Dps homologue encoded by Borrelia in spirochaete survival was assessed. Dps-deficient spirochaetes were infectious in mice via needle-inoculation at the dose of 10(5) spirochaetes. Larval ticks successfully acquired Dps-deficient spirochaetes via a blood meal on mice. However, after extended periods within unfed nymphs, the Dps-deficient spirochaetes failed to be transmitted to a new host when nymphs fed. Our data suggest that Dps functions to protect the spirochaetes during dormancy in unfed ticks, and in its absence, the spirochaetes become susceptible during tick feeding. dps is differentially expressed in vivo- low in mice and high in ticks - but constitutively expressed in vitro, showing little change during growth or in response to oxidative stress. Borrelia Dps forms a dodecameric complex capable of sequestering iron. The Dps-deficient spirochaetes showed no defect in starvation and oxidative stress assays, perhaps due to the lack of iron in spirochaetes grown in vitro. Dps is critical for spirochaete persistence within ticks, and strategies to interfere with Dps could potentially reduce Borrelia populations in nature and thereby influence the incidence of Lyme disease.
Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/fisiologia , Proteínas de Ligação a DNA/metabolismo , Ixodes/microbiologia , Doença de Lyme/microbiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Teste de Complementação Genética , Ixodes/crescimento & desenvolvimento , Doença de Lyme/transmissão , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ninfa/microbiologia , Fenótipo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transformação GenéticaRESUMO
Acquisition of transition metals is central to the struggle between a bacterial pathogen and its mammalian host. Previous studies demonstrated that Treponema pallidum encodes a cluster-9 (C9) ABC transporter (troABCD) whose solute-binding protein component (TroA) ligands Zn(2+) and Mn(2+) with essentially equal affinities. Bioinformatic analysis revealed that T. pallidum encodes an additional C9 transporter (tp0034-36) orthologous to Zn(2+)-uptake (Znu) systems in other bacteria; the binding protein component, ZnuA, contains a His-rich tract characteristic of C9 Zn(2+)-binding proteins. Metal analysis and metal-reconstitution studies demonstrated that ZnuA is a Zn(2+)-binding protein; parallel studies confirmed that TroA binds Zn(2+), Mn(2+) and Fe. Circular dichroism showed that ZnuA, but not TroA, undergoes conformational changes in the presence of Zn(2+). Using isothermal titration calorimetry (ITC), we demonstrated that TroA binds Zn(2+) and Mn(2+) with affinities approximately 100-fold greater than those previously reported. ITC analysis revealed that ZnuA contains multiple Zn(2+)-binding sites, two of which are high-affinity and presumed to be located within the binding pocket and His-rich loop. Quantitative reverse transcription polymerase chain reaction of tro and znu transcripts combined with immunoblot analysis of TroA and ZnuA confirmed that both transporters are simultaneously expressed in T. pallidum and that TroA is expressed at much greater levels than ZnuA. Collectively, our findings indicate that T. pallidum procures transition metals via the concerted utilization of its general metal (Tro) and Zn(2+) (Znu) transporters. Sequestration of periplasmic Zn(2+) by ZnuA may free up TroA binding capacity for the importation of Fe and Mn(2+).
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Manganês/metabolismo , Treponema pallidum/metabolismo , Zinco/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Masculino , Manganês/farmacologia , Modelos Moleculares , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Conformação Proteica , Coelhos , Treponema pallidum/genética , Zinco/farmacologiaRESUMO
The outer membrane of Treponema pallidum, the non-cultivable agent of venereal syphilis, contains a paucity of protein(s) which has yet to be definitively identified. In contrast, the outer membranes of gram-negative bacteria contain abundant immunogenic membrane-spanning beta-barrel proteins mainly involved in nutrient transport. The absence of orthologs of gram-negative porins and outer membrane nutrient-specific transporters in the T. pallidum genome predicts that nutrient transport across the outer membrane must differ fundamentally in T. pallidum and gram-negative bacteria. Here we describe a T. pallidum outer membrane protein (TP0453) that, in contrast to all integral outer membrane proteins of known structure, lacks extensive beta-sheet structure and does not traverse the outer membrane to become surface exposed. TP0453 is a lipoprotein with an amphiphilic polypeptide containing multiple membrane-inserting, amphipathic alpha-helices. Insertion of the recombinant, non-lipidated protein into artificial membranes results in bilayer destabilization and enhanced permeability. Our findings lead us to hypothesize that TP0453 is a novel type of bacterial outer membrane protein which may render the T. pallidum outer membrane permeable to nutrients while remaining inaccessible to antibody.