RESUMO
Riboflavin is an essential micronutrient, but its transport and utilization have remained largely understudied among pathogenic spirochetes. Here, we show that Borrelia burgdorferi, the zoonotic spirochete that causes Lyme disease, is able to import riboflavin via products of its rfuABCD-like operon as well as synthesize flavin mononucleotide and flavin adenine dinucleotide despite lacking canonical genes for their synthesis. Additionally, a mutant deficient in the rfuABCD-like operon is resistant to the antimicrobial effect of roseoflavin, a natural riboflavin analog, and is attenuated in a murine model of Lyme borreliosis. Our combined results indicate not only that are riboflavin and the maintenance of flavin pools essential for B. burgdorferi growth but also that flavin utilization and its downstream products (e.g., flavoproteins) may play a more prominent role in B. burgdorferi pathogenesis than previously appreciated.
Assuntos
Proteínas de Bactérias/genética , Borrelia burgdorferi/efeitos dos fármacos , Borrelia burgdorferi/genética , Doença de Lyme/tratamento farmacológico , Doença de Lyme/microbiologia , Óperon/genética , Riboflavina/farmacologia , Animais , Feminino , Mamíferos/microbiologia , Camundongos , Camundongos Endogâmicos C3HRESUMO
It has been known for decades that proteins undergo conformational changes in response to binding ligands. Such changes are usually accompanied by a loss of entropy by the protein, and thus conformational changes are integral to the thermodynamics of ligand association. Methods to detect these alterations are numerous; here, we focus on the sedimentation velocity (SV) mode of AUC, which has several advantages, including ease of use and rigorous data-selection criteria. In SV, it is assumed that conformational changes manifest primarily as differences in the sedimentation coefficient (the s-value). Two methods of determining s-value differences were assessed. The first method used the widely adopted c(s) distribution to gather statistics on the s-value differences to determine whether the observed changes were reliable. In the second method, a decades-old technique called "difference SV" was revived and updated to address its viability in this era of modern instrumentation. Both methods worked well to determine the extent of conformational changes to three model systems. Both simulations and experiments were used to explore the strengths and limitations of the methods. Finally, software incorporating these methodologies was produced.
Assuntos
Ultracentrifugação/métodos , Animais , Bovinos , Hidrodinâmica , Modelos Moleculares , Conformação Proteica , Soroalbumina Bovina/química , Soroalbumina Bovina/isolamento & purificaçãoRESUMO
Treponema pallidum, an obligate parasite of humans and the causative agent of syphilis, has evolved the capacity to exploit host-derived metabolites for its survival. Flavin-containing compounds are essential cofactors that are required for metabolic processes in all living organisms, and riboflavin is a direct precursor of the cofactors FMN and FAD. Unlike many pathogenic bacteria, Treponema pallidum cannot synthesize riboflavin; we recently described a flavin-uptake mechanism composed of an ABC-type transporter. However, there is a paucity of information about flavin utilization in bacterial periplasms. Using a discovery-driven approach, we have identified the TP0796 lipoprotein as a previously uncharacterized Mg(2+)-dependent FAD pyrophosphatase within the ApbE superfamily. TP0796 probably plays a central role in flavin turnover by hydrolyzing exogenously acquired FAD, yielding AMP and FMN. Biochemical and structural investigations revealed that the enzyme has a unique bimetal Mg(2+) catalytic center. Furthermore, the pyrophosphatase activity is product-inhibited by AMP, indicating a possible role for this molecule in modulating FMN and FAD levels in the treponemal periplasm. The ApbE superfamily was previously thought to be involved in thiamine biosynthesis, but our characterization of TP0796 prompts a renaming of this superfamily as a periplasmic flavin-trafficking protein (Ftp). TP0796 is the first structurally and biochemically characterized FAD pyrophosphate enzyme in bacteria. This new paradigm for a bacterial flavin utilization pathway may prove to be useful for future inhibitor design.
Assuntos
Proteínas de Bactérias/química , Flavina-Adenina Dinucleotídeo/química , Lipoproteínas/química , Magnésio/química , Pirofosfatases/química , Treponema pallidum/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Flavina-Adenina Dinucleotídeo/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Homeostase/fisiologia , Lipoproteínas/genética , Lipoproteínas/metabolismo , Magnésio/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo , Treponema pallidum/genéticaRESUMO
Decorin-binding protein A (DbpA) of Borrelia burgdorferi mediates bacterial adhesion to heparin and dermatan sulfate associated with decorin. Lysines K82, K163, and K170 of DbpA are known to be important for in vitro interaction with decorin, and the DbpA structure, initially solved by nuclear magnetic resonance (NMR) spectroscopy, suggests these lysine residues colocalize in a pocket near the C terminus of the protein. In the current study, we solved the structure of DbpA from B. burgdorferi strain 297 using X-ray crystallography and confirmed the existing NMR structural data. In vitro binding experiments confirmed that recombinant DbpA proteins with mutations in K82, K163, or K170 did not bind decorin, which was due to an inability to interact with dermatan sulfate. Most importantly, we determined that the in vitro binding defect observed upon mutation of K82, K163, or K170 in DbpA also led to a defect during infection. The infectivity of B. burgdorferi expressing individual dbpA lysine point mutants was assessed in mice challenged via needle inoculation. Murine infection studies showed that strains expressing dbpA with mutations in K82, K163, and K170 were significantly attenuated and could not be cultured from any tissue. Proper expression and cellular localization of the mutated DbpA proteins were examined, and NMR spectroscopy determined that the mutant DbpA proteins were structurally similar to wild-type DbpA. Taken together, these data showed that lysines K82, K163, and K170 potentiate the binding of DbpA to dermatan sulfate and that an interaction(s) mediated by these lysines is essential for B. burgdorferi murine infection.
Assuntos
Adesinas Bacterianas/metabolismo , Borrelia burgdorferi/fisiologia , Doença de Lyme/microbiologia , Lisina/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Substituição de Aminoácidos , Animais , Borrelia burgdorferi/genética , Cristalografia por Raios X , Análise Mutacional de DNA , Lisina/química , Lisina/genética , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação ProteicaRESUMO
In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ54 (RpoN) directly activates transcription of another alternative σ factor, σ(S) (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ54-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ54-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ54-dependent gene regulation in bacteria.
Assuntos
Proteínas de Bactérias/fisiologia , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidade , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Polimerase Sigma 54/fisiologia , Proteínas Repressoras/fisiologia , Fator sigma/fisiologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Genes Reguladores , Doença de Lyme/genética , Doença de Lyme/microbiologia , Doença de Lyme/patologia , Camundongos , Dados de Sequência Molecular , Organismos Geneticamente Modificados , Ligação Proteica , RNA Polimerase Sigma 54/genética , RNA Polimerase Sigma 54/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Fator sigma/genética , Fator sigma/metabolismo , Transdução de Sinais/genética , Virulência/genéticaRESUMO
Syphilis, caused by the bacterial spirochete Treponema pallidum, remains a prominent sexually transmitted infection worldwide. Despite sequencing of the genome of this obligate human pathogen 15 years ago, the functions of a large number of the gene products of T. pallidum are still unknown, particularly with respect to those of the organism's periplasmic lipoproteins. To better understand their functions, a structural biology approach has been pursued. To this end, the soluble portion of the T. pallidum TP0435 lipoprotein (also known as Tp17) was cloned, hyper-expressed in Escherichia coli and purified to apparent homogeneity. The protein crystals obtained from this preparation diffracted to 2.4 Å resolution and had the symmetry of space group R3. In the hexagonal setting, the unit-cell parameters were a = b = 85.7, c = 85.4 Å.
Assuntos
Proteínas de Bactérias/química , Lipoproteínas/química , Treponema pallidum/química , Proteínas de Bactérias/isolamento & purificação , Cristalização , Cristalografia por Raios X , Lipoproteínas/isolamento & purificaçãoRESUMO
The mechanisms of energy generation and carbon-source utilization in the syphilis spirochete Treponema pallidum have remained enigmatic despite complete genomic sequence information. Whereas the bacterium harbors enzymes for glycolysis, the apparatus for more efficient use of glucose catabolites, namely the citric-acid cycle, is apparently not present. Yet, the organism's energy needs likely exceed the modest output from glycolysis alone. Recently, building on our structure-function studies of T. pallidum lipoproteins, we proposed a "flavin-centric" metabolic lifestyle for the organism that partially resolves this conundrum. As a part of the hypothesis, we have proposed that T. pallidum contains an acetogenic energy-conservation pathway that catabolizes D-lactate, yielding acetate, reducing equivalents for the generation and maintenance of chemiosmotic potential, and ATP. We already have confirmed the D-lactate dehydrogenase activity in T. pallidum necessary for this pathway to operate. In the current study, we focused on another enzyme ostensibly involved in treponemal acetogenesis, phosphotransacetylase (Pta). This enzyme is putatively identified as TP0094 and, in this study, we determined a high-resolution (1.95 Å) X-ray crystal structure of the protein, finding that its fold comports with other known Pta enzymes. Further studies on its solution behavior and enzyme activity confirmed that it has the properties of a Pta. These results are consistent with the proposed acetogenesis pathway in T. pallidum, and we propose that the protein be referred to henceforth as TpPta.
Assuntos
Sífilis , Treponema pallidum , Humanos , Treponema pallidum/genética , Fosfato Acetiltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Sífilis/microbiologia , Treponema/genéticaRESUMO
Metal ion homeostasis is a critical function of many integral and peripheral membrane proteins. The genome of the etiologic agent of syphilis, Treponema pallidum, is compact and devoid of many metabolic enzyme genes. Nevertheless, it harbors genes coding for homologs of several enzymes that typically require either iron or zinc. The product of the tp0971 gene of T. pallidum, designated Tp34, is a periplasmic lipoprotein that is thought to be tethered to the inner membrane of this organism. Previous work on a water-soluble (nonacylated) recombinant version of Tp34 established that this protein binds to Zn(2+), which, like other transition metal ions, stabilizes the dimeric form of the protein. In this study, we employed analytical ultracentrifugation to establish that four transition metal ions (Ni(2+), Co(2+), Cu(2+), and Zn(2+)) readily induce the dimerization of Tp34; Cu(2+) (50% effective concentration [EC(50)] = 1.7 µM) and Zn(2+) (EC(50) = 6.2 µM) were the most efficacious of these ions. Mutations of the crystallographically identified metal-binding residues hindered the ability of Tp34 to dimerize. X-ray crystallography performed on crystals of Tp34 that had been incubated with metal ions indicated that the binding site could accommodate the metals examined. The findings presented herein, coupled with bioinformatic analyses of related proteins, point to Tp34's likely role in metal ion homeostasis in T. pallidum.
Assuntos
Lipoproteínas/química , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Metais/metabolismo , Treponema pallidum/metabolismo , Sítios de Ligação/genética , Transporte Biológico , Cobalto/química , Cobalto/metabolismo , Biologia Computacional , Cobre/química , Cobre/metabolismo , Cristalografia por Raios X , Dimerização , Genes Bacterianos , Lipoproteínas/genética , Metais/química , Família Multigênica , Mutação , Níquel/química , Níquel/metabolismo , Ligação Proteica , Multimerização Proteica , Treponema pallidum/genética , Zinco/química , Zinco/metabolismoRESUMO
Antibiotic resistance is a challenge for the control of bacterial infections. In an effort to explore unconventional avenues for antibacterial drug development, we focused on the FMN-transferase activity of the enzyme Ftp from the syphilis spirochete, Treponema pallidum (Ftp_Tp). This enzyme, which is only found in prokaryotes and trypanosomatids, post-translationally modifies proteins in the periplasm, covalently linking FMN (from FAD) to proteins that typically are important for establishing an essential electrochemical gradient across the cytoplasmic membrane. As such, Ftp inhibitors potentially represent a new class of antimicrobials. Previously, we showed that AMP is both a product of the Ftp_tp-catalyzed reaction and an inhibitor of the enzyme. As a preliminary step in exploiting this property to develop a novel Ftp_Tp inhibitor, we have used structural and solution studies to examine the inhibitory and enzyme-binding properties of several adenine-based nucleosides, with particular focus on the 2-position of the purine ring. Implications for future drug design are discussed.
Assuntos
Farmacorresistência Bacteriana , Mononucleotídeo de Flavina , Transferases , Treponema pallidum , Antibacterianos/farmacologia , Flavina-Adenina Dinucleotídeo/química , Treponema pallidum/efeitos dos fármacos , Treponema pallidum/enzimologiaRESUMO
Determination of the stoichiometry of macromolecular assemblies is fundamental to an understanding of how they function. Many different biophysical methodologies may be used to determine stoichiometry. In the past, both sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation have been employed to determine component stoichiometries. Recently, a method of globally analyzing multisignal sedimentation velocity data was introduced by Schuck and coworkers. This global analysis removes some of the experimental inconveniences and inaccuracies that could occur in the previously used strategies. This method uses spectral differences between the macromolecular components to decompose the well-known c(s) distribution into component distributions c(k)(s); that is, each component k has its own c(k)(s) distribution. Integration of these distributions allows the calculation of the populations of each component in cosedimenting complexes, yielding their stoichiometry. In our laboratories, we have used this method extensively to determine the component stoichiometries of several protein-protein complexes involved in cytoskeletal remodeling, sugar metabolism, and host-pathogen interactions. The overall method is described in detail in this work, as are experimental examples and caveats.
Assuntos
Complexos Multiproteicos/química , Ultracentrifugação/métodos , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Humanos , Lactoferrina/química , Ligação Proteica , Complexo Piruvato Desidrogenase/químicaRESUMO
A longstanding conundrum in Treponema pallidum biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. For decades, it has been assumed that the bacterium relies solely on glucose catabolism (via glycolysis) for generation of its ATP. However, the organism's robust motility, believed to be essential for human tissue invasion and dissemination, would require abundant ATP likely not provided by the parsimony of glycolysis. As such, additional ATP generation, either via a chemiosmotic gradient, substrate-level phosphorylation, or both, likely exists in T. pallidum Along these lines, we have hypothesized that T. pallidum exploits an acetogenic energy conservation pathway that relies on the redox chemistry of flavins. Central to this hypothesis is the apparent existence in T. pallidum of an acetogenic pathway for the conversion of d-lactate to acetate. Herein we have characterized the structural, biophysical, and biochemical properties of the first enzyme (d-lactate dehydrogenase [d-LDH]; TP0037) predicted in this pathway. Binding and enzymatic studies showed that recombinant TP0037 consumed d-lactate and NAD+ to produce pyruvate and NADH. The crystal structure of TP0037 revealed a fold similar to that of other d-acid dehydrogenases; residues in the cofactor-binding and active sites were homologous to those of other known d-LDHs. The crystal structure and solution biophysical experiments revealed the protein's propensity to dimerize, akin to other d-LDHs. This study is the first to elucidate the enzymatic properties of T. pallidum's d-LDH, thereby providing new compelling evidence for a flavin-dependent acetogenic energy conservation (ATP-generating) pathway in T. pallidumIMPORTANCE Because T. pallidum lacks a Krebs cycle and the capability for oxidative phosphorylation, historically it has been difficult to reconcile how the syphilis spirochete generates sufficient ATP to fulfill its energy needs, particularly for its robust motility, solely from glycolysis. We have postulated the existence in T. pallidum of a flavin-dependent acetogenic energy conservation pathway that would generate additional ATP for T. pallidum bioenergetics. In the proposed acetogenic pathway, first d-lactate would be converted to pyruvate. Pyruvate would then be metabolized to acetate in three additional steps, with ATP being generated via substrate-level phosphorylation. This study provides structural, biochemical, and biophysical evidence for the first T. pallidum enzyme in the pathway (TP0037; d-lactate dehydrogenase) requisite for the conversion of d-lactate to pyruvate. The findings represent the first experimental evidence to support a role for an acetogenic energy conservation pathway that would contribute to nonglycolytic ATP production in T. pallidum.
Assuntos
Acetatos/metabolismo , Metabolismo Energético , Lactato Desidrogenases/metabolismo , Ácido Láctico/metabolismo , Redes e Vias Metabólicas , Treponema pallidum/enzimologia , Trifosfato de Adenosina/metabolismo , Ácido Pirúvico/metabolismoRESUMO
Tp0655 of Treponema pallidum, the causative agent of syphilis, is predicted to be a 40 kDa membrane lipoprotein. Previous sequence analysis of Tp0655 noted its homology to polyamine-binding proteins of the bacterial PotD family, which serve as periplasmic ligand-binding proteins of ATP-binding-cassette (ABC) transport systems. Here, the 1.8 A crystal structure of Tp0655 demonstrated structural homology to Escherichia coli PotD and PotF. The latter two proteins preferentially bind spermidine and putrescine, respectively. All of these proteins contain two domains that sandwich the ligand between them. The ligand-binding site of Tp0655 can be occupied by 2-(N-morpholino)ethanesulfanoic acid, a component of the crystallization medium. To discern the polyamine binding preferences of Tp0655, the protein was subjected to isothermal titration calorimetric experiments. The titrations established that Tp0655 binds polyamines avidly, with a marked preference for putrescine (Kd=10 nM) over spermidine (Kd=430 nM), but the related compounds cadaverine and spermine did not bind. Structural comparisons and structure-based sequence analyses provide insights into how polyamine-binding proteins recognize their ligands. In particular, these comparisons allow the derivation of rules that may be used to predict the function of other members of the PotD family. The sequential, structural, and functional homology of Tp0655 to PotD and PotF prompt the conclusion that the former likely is the polyamine-binding component of an ABC-type polyamine transport system in T. pallidum. We thus rename Tp0655 as TpPotD. The ramifications of TpPotD as a polyamine-binding protein to the parasitic strategy of T. pallidum are discussed.
Assuntos
Proteínas Periplásmicas de Ligação/química , Poliaminas/metabolismo , Treponema pallidum/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Proteínas Periplásmicas de Ligação/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Previously, we determined the crystal structure of apo-TpMglB-2, a d-glucose-binding component of a putative ABC transporter from the syphilis spirochete Treponema pallidum. The protein had an unusual topology for this class of proteins, raising the question of whether the d-glucose-binding mode would be different in TpMglB-2. Here, we present the crystal structures of a variant of TpMglB-2 with and without d-glucose bound. The structures demonstrate that, despite its aberrant topology, the protein undergoes conformational changes and binds d-glucose similarly to other Mgl-type proteins, likely facilitating d-glucose uptake in T. pallidum.
Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte de Monossacarídeos/química , Treponema pallidum/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Glucose/metabolismo , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/metabolismo , Conformação ProteicaRESUMO
Biophysical and biochemical studies on the lipoproteins and other periplasmic proteins from the spirochetal species Treponema pallidum have yielded numerous insights into the functioning of the organism's peculiar membrane organization, its nutritional requirements, and intermediary metabolism. However, not all T. pallidum proteins have proven to be amenable to biophysical studies. One such recalcitrant protein is Tp0309, a putative polar-amino-acid-binding protein of an ABC transporter system. To gain further information on its possible function, a homolog of the protein from the related species T. vincentii was used as a surrogate. This protein, Tv2483, was crystallized, resulting in the determination of its crystal structure at a resolution of 1.75 Å. The protein has a typical fold for a ligand-binding protein, and a single molecule of l-arginine was bound between its two lobes. Differential scanning fluorimetry and isothermal titration calorimetry experiments confirmed that l-arginine bound to the protein with unusually high selectivity. However, further comparison to Tp0309 showed differences in key amino-acid-binding residues may impart an alternate specificity for the T. pallidum protein.
Assuntos
Arginina/metabolismo , Lipoproteínas/metabolismo , Treponema pallidum/química , Sequência de Aminoácidos , Arginina/química , Sítios de Ligação , Calorimetria , Ligantes , Lipoproteínas/química , Lipoproteínas/isolamento & purificação , Modelos Moleculares , Alinhamento de SequênciaRESUMO
The spirochete Treponema pallidum is the causative agent of syphilis, a sexually transmitted infection of major global importance. Other closely related subspecies of Treponema also are the etiological agents of the endemic treponematoses, such as yaws, pinta, and bejel. The inability of T. pallidum and its close relatives to be cultured in vitro has prompted efforts to characterize T. pallidum's proteins structurally and biophysically, particularly those potentially relevant to treponemal membrane biology, with the goal of possibly revealing the functions of those proteins. This report describes the structure of the treponemal protein Tp0737; this polypeptide has a fold characteristic of a class of periplasmic ligand-binding proteins associated with ABC-type transporters. Although no ligand for the protein was observed in electron-density maps, and thus the nature of the native ligand remains obscure, the structural data described herein provide a foundation for further efforts to elucidate the ligand and thus the function of this protein in T. pallidum.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas Periplásmicas/química , Treponema pallidum/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cristalografia por Raios X , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade , Treponema pallidum/genética , Treponema pallidum/metabolismoRESUMO
Treponema pallidum, the bacterium that causes syphilis, is an obligate human parasite. As such, it must acquire energy, in the form of carbon sources, from the host. There is ample evidence that the principal source of energy for this spirochete is D-glucose acquired from its environment, likely via an ABC transporter. Further, there is genetic evidence of a D-glucose chemotaxis system in T. pallidum. Both of these processes may be dependent on a single lipidated chemoreceptor: Tp0684, also called TpMglB-2 for its sequence homology to MglB of Escherichia coli. To broaden our understanding of this potentially vital protein, we determined a 2.05-Å X-ray crystal structure of a soluble form of the recombinant protein. Like its namesake, TpMglB-2 adopts a bilobed fold that is similar to that of the ligand-binding proteins (LBPs) of other ABC transporters. However, the protein has an unusual, circularly permuted topology. This feature prompted a series of biophysical studies that examined whether the protein's topological distinctiveness affected its putative chemoreceptor functions. Differential scanning fluorimetry and isothermal titration calorimetry were used to confirm that the protein bound D-glucose in a cleft between its two lobes. Additionally, analytical ultracentrifugation was employed to reveal that D-glucose binding is accompanied by a significant conformational change. TpMglB-2 thus appears to be fully functional in vitro, and given the probable central importance of the protein to T. pallidum's physiology, our results have implications for the viability and pathogenicity of this obligate human pathogen.
Assuntos
Proteínas de Bactérias/metabolismo , Lipoproteínas/metabolismo , Treponema pallidum/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glucose/metabolismo , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Conformação Proteica , Homologia de Sequência , Sífilis/metabolismo , Sífilis/microbiologia , Treponema pallidum/genética , Treponema pallidum/fisiologiaRESUMO
We recently reported a flavin-trafficking protein (Ftp) in the syphilis spirochete Treponema pallidum (Ftp_Tp) as the first bacterial metal-dependent FAD pyrophosphatase that hydrolyzes FAD into AMP and FMN in the periplasm. Orthologs of Ftp_Tp in other bacteria (formerly ApbE) appear to lack this hydrolytic activity; rather, they flavinylate the redox subunit, NqrC, via their metal-dependent FMN transferase activity. However, nothing has been known about the nature or mechanism of metal-dependent Ftp catalysis in either Nqr- or Rnf-redox-containing bacteria. In the current study, we identified a bimetal center in the crystal structure of Escherichia coli Ftp (Ftp_Ec) and show via mutagenesis that a single amino acid substitution converts it from an FAD-binding protein to a Mg(2+)-dependent FAD pyrophosphatase (Ftp_Tp-like). Furthermore, in the presence of protein substrates, both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. A high-resolution structure of the Ftp-mediated flavinylated protein of Shewanella oneidensis NqrC identified an essential lysine in phosphoester-threonyl-FMN bond formation in the posttranslationally modified flavoproteins. Together, these discoveries broaden our understanding of the physiological capabilities of the bacterial periplasm, and they also clarify a possible mechanism by which flavoproteins are generated.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Flavoproteínas/metabolismo , Periplasma/enzimologia , Processamento de Proteína Pós-Traducional , Pirofosfatases/metabolismo , Shewanella/enzimologia , Monofosfato de Adenosina/metabolismo , Cristalografia por Raios X , Escherichia coli/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/biossíntese , Mutagênese Sítio-Dirigida , Oxirredução , Periplasma/metabolismo , Transporte Proteico , Pirofosfatases/genética , Shewanella/metabolismoRESUMO
The past two decades have seen a worldwide resurgence in infections caused by Treponema pallidum subsp. pallidum, the syphilis spirochete. The well-recognized capacity of the syphilis spirochete for early dissemination and immune evasion has earned it the designation 'the stealth pathogen'. Despite the many hurdles to studying syphilis pathogenesis, most notably the inability to culture and to genetically manipulate T. pallidum, in recent years, considerable progress has been made in elucidating the structural, physiological, and regulatory facets of T. pallidum pathogenicity. In this Review, we integrate this eclectic body of information to garner fresh insights into the highly successful parasitic lifestyles of the syphilis spirochete and related pathogenic treponemes.
Assuntos
Evasão da Resposta Imune , Sífilis/microbiologia , Treponema pallidum/imunologia , Treponema pallidum/patogenicidade , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Genômica , Humanos , Alinhamento de Sequência , Sífilis/imunologia , Sífilis/transmissão , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Treponema pallidum/genética , Treponema pallidum/fisiologiaRESUMO
UNLABELLED: The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete's periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg(2+)-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg(2+)-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp's dual activities, thereby underscoring the role of Mg(2+) in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm. IMPORTANCE: Treponema pallidum, the syphilis spirochete, exploits its periplasmic lipoproteins for a number of essential physiologic processes. One of these, flavin-trafficking protein (Ftp), not only exploits its catalytic center to mediate posttranslational flavinylation of proteins (to create flavoproteins) but also likely maintains the periplasmic flavin pool via its unique ability to hydrolyze FAD. This functional diversity within a single lipoprotein is quite remarkable and reflects the enzymatic versatility of the treponemal lipoproteins, as well as molecular parsimony in an organism with a limited genome. Ftp-mediated protein flavinylation in the periplasm also likely is a key aspect of a predicted flavin-dependent Rnf-based redox homeostasis system at the cytoplasmic membrane of T. pallidum. In addition to its importance in T. pallidum physiology, Ftp homologs exist in other bacteria, thereby expanding our understanding of the bacterial periplasm as a metabolically active subcellular compartment for flavoprotein biogenesis as well as flavin homeostasis.