Ranjit K. Deka

Treponema pallidum TroA is a periplasmic zinc-binding protein with a helical backbone

The crystal structure of recombinant TroA, a zinc-binding protein component of an ATP-binding cassette transport system in Treponema pallidum, was determined at a resolution of 1.8 Å. The organization of the protein is largely similar to other periplasmic ligand-binding proteins (PLBP), in that two independent globular domains interact with each other to create a zinc-binding cleft between them. The structure has one bound zinc pentavalently coordinated to residues from both domains. Unlike previous PLBP structures that have an interdomain hinge composed of β-strands, the N- and C-domains of TroA are linked by a single long backbone helix. This unique backbone helical conformation was possibly adopted to limit the hinge motion associated with ligand exchange.

BosR (BB0647) Controls the RpoN-RpoS Regulatory Pathway and Virulence Expression in Borrelia burgdorferi by a Novel DNA-Binding Mechanism

In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ54 (RpoN) directly activates transcription of another alternative σ factor, σS (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.

The PnrA (Tp0319; TmpC) lipoprotein represents a new family of bacterial purine nucleoside receptor encoded within an ATP-binding cassette (ABC)-like operon in Treponema pallidum

Treponema pallidum, the bacterial agent of syphilis, cannot be cultivated in vitro. This constraint has severely impeded the study of the membrane biology of this complex human pathogen. A structure-to-function approach thus was adopted as a means of discerning the likely function of Tp0319, a 35-kDa cytoplasmic membrane-associated lipoprotein of T. pallidum formerly designated as TmpC. A 1.7-Å crystal structure showed that recombinant Tp0319 (rTp0319) consists of two α/β domains, linked by three crossovers, with a deep cleft between them akin to ATP-binding cassette (ABC) receptors. In the cleft, a molecule of inosine was bound. Isothermal titration calorimetry demonstrated that rTp0319 specifically binds purine nucleosides (dissociation constant (Kd) ∼10-7 m). This predilection for purine nucleosides by rTp0319 is consistent with its likely role as a receptor component of a cytoplasmic membrane-associated transporter system. Reverse transcription-PCR analysis of RNA isolated from rabbit tissue-extracted T. pallidum additionally showed that tp0319 is transcriptionally linked to four other downstream open reading frames, thereby supporting the existence of an ABC-like operon (tp0319-0323). We herein thus re-name tp0319 as purine nucleoside receptor A (pnrA), with its operonic partners tp0320-0323 designated as pnrB-E, respectively. Our study not only infers that PnrA transports purine nucleosides essential for the survival of T. pallidum within its obligate human host, but to our knowledge, this is the first description of an ABC-type nucleoside transport system in any bacterium. PnrA has been grouped with a functionally uncharacterized protein family (HBG016869), thereby implying that other members of the family may have similar nucleoside-binding function(s).

Crystal Structure of the 47-kDa Lipoprotein of Treponema pallidum Reveals a Novel Penicillin-binding Protein

Syphilis is a complex sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. T. pallidum has remained exquisitely sensitive to penicillin, but the mode of action and lethal targets for β-lactams are still unknown. We previously identified the T. pallidum 47-kDa lipoprotein (Tp47) as a penicillin-binding protein (PBP). Tp47 contains three hypothetical consensus motifs (SVTK, TEN, and KTG) that typically form the active center of other PBPs. Yet, in this study, mutations of key amino acids within these motifs failed to abolish the penicillin binding activity of Tp47. The crystal structure of Tp47 at a resolution of 1.95 Å revealed a fold different from any other known PBP; Tp47 is predominantly β-sheet, in contrast to the α/β-fold common to other PBPs. It comprises four distinct domains: two complex β-sheet-containing N-terminal domains and two C-terminal domains that adopt immunoglobulin-like folds. The three hypothetical PBP signature motifs do not come together to form a typical PBP active site. Furthermore, Tp47 is unusual in that it displays β-lactamase activity (k cat for penicillin = 271 ± 6 s−1), a feature that hindered attempts to identify the active site in Tp47 by co-crystallization and mass spectrometric techniques. Taken together, Tp47 does not fit the classical structural and mechanistic paradigms for PBPs, and thus Tp47 appears to represent a new class of PBP.

Structural Evidence That the 32-Kilodalton Lipoprotein (Tp32) of Treponema pallidum Is an l-Methionine-binding Protein

A structure-to-function approach was undertaken to gain insights into the potential function of the 32-kDa membrane lipoprotein (Tp32) of Treponema pallidum, the syphilis bacterium. The crystal structure of rTp32 (determined at a resolution of 1.85 Å) shows that the organization of rTp32 is similar to other periplasmic ligand-binding proteins (PLBPs), in that it consists of two α/β domains, linked by two crossovers, with a binding pocket between them. In the pocket, a molecule of l-methionine was detected in the electron density map. Residues from both domains interact with the ligand. One of the crossover regions is comprised of a 310-helix, a feature not typical in other ligand-binding proteins. Sequence comparison shows strong similarity to other hypothetical methionine-binding proteins. Together, the data support the notion that rTp32 is a component of a periplasmic methionine uptake transporter system in T. pallidum.

Determination of protein complex stoichiometry through multisignal sedimentation velocity experiments.

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.

Crystal Structure of the Tp34 (TP0971) Lipoprotein of Treponema pallidum IMPLICATIONS OF ITS METAL-BOUND STATE AND AFFINITY FOR HUMAN LACTOFERRIN

The Tp34 (TP0971) membrane lipoprotein of Treponema pallidum, an obligate human pathogen and the agent of syphilis, was previously reported to have lactoferrin binding properties. Given the non-cultivatable nature of T. pallidum, a structure-to-function approach was pursued to clarify further potential relationships between the Tp34 structural and biochemical properties and its propensity to bind human lactoferrin. The crystal structure of a nonacylated, recombinant form of Tp34 (rTp34), solved to a resolution of 1.9Å, revealed two metaloccupied binding sites within a dimer; the identity of the ion most likely was zinc. Residues from both of the monomers contributed to the interfacial metal-binding sites; a novel feature was that the δ-sulfur of methionine coordinated the zinc ion. Analytical ultracentrifugation showed that, in solution, rTp34 formed a metal-stabilized dimer and that rTp34 bound human lactoferrin with a stoichiometry of 2:1. Isothermal titration calorimetry further revealed that rTp34 bound human lactoferrin at high (submicromolar) affinity. Finally, membrane topology studies revealed that native Tp34 is not located on the outer surface (outer membrane) of T. pallidum but, rather, is periplasmic. How propensity of Tp34 to bind zinc and the iron-sequestering lactoferrin may relate overall to the biology of T. pallidum infection in humans is discussed.

Evidence for an ABC-Type Riboflavin Transporter System in Pathogenic Spirochetes

ABSTRACT Bacterial transporter proteins are involved in the translocation of many essential nutrients and metabolites. However, many of these key bacterial transport systems remain to be identified, including those involved in the transport of riboflavin (vitamin B2). Pathogenic spirochetes lack riboflavin biosynthetic pathways, implying reliance on obtaining riboflavin from their hosts. Using structural and functional characterizations of possible ligand-binding components, we have identified an ABC-type riboflavin transport system within pathogenic spirochetes. The putative lipoprotein ligand-binding components of these systems from three different spirochetes were cloned, hyperexpressed in Escherichia coli, and purified to homogeneity. Solutions of all three of the purified recombinant proteins were bright yellow. UV-visible spectra demonstrated that these proteins were likely flavoproteins; electrospray ionization mass spectrometry and thin-layer chromatography confirmed that they contained riboflavin. A 1.3-Å crystal structure of the protein (TP0298) encoded by Treponema pallidum, the syphilis spirochete, demonstrated that the protein’s fold is similar to the ligand-binding components of ABC-type transporters. The structure also revealed other salient details of the riboflavin binding site. Comparative bioinformatics analyses of spirochetal genomes, coupled with experimental validation, facilitated the discovery of this new ABC-type riboflavin transport system(s). We denote the ligand-binding component as riboflavin uptake transporter A (RfuA). Taken together, it appears that pathogenic spirochetes have evolved an ABC-type transport system (RfuABCD) for survival in their host environments, particularly that of the human host. IMPORTANCE Syphilis remains a public health problem, but very little is known about the causative bacterium. This is because Treponema pallidum still cannot be cultured in the laboratory. Rather, T. pallidum must be cultivated in laboratory rabbits, a restriction that poses many insurmountable experimental obstacles. Approaches to learn more about the structure and function of T. pallidum’s cell envelope, which is both the physical and functional interface between T. pallidum and its human host, are severely limited. One approach for elucidating T. pallidum’s cell envelope has been to determine the three-dimensional structures of its membrane lipoproteins, molecules that serve many critical survival functions. Herein, we describe a previously unknown transport system that T. pallidum uses to import riboflavin, an essential nutrient for the organism’s survival. Moreover, we found that this transport system is present in other pathogenic spirochetes. This is the first description of this new type of bacterial riboflavin transport system. Syphilis remains a public health problem, but very little is known about the causative bacterium. This is because Treponema pallidum still cannot be cultured in the laboratory. Rather, T. pallidum must be cultivated in laboratory rabbits, a restriction that poses many insurmountable experimental obstacles. Approaches to learn more about the structure and function of T. pallidum’s cell envelope, which is both the physical and functional interface between T. pallidum and its human host, are severely limited. One approach for elucidating T. pallidum’s cell envelope has been to determine the three-dimensional structures of its membrane lipoproteins, molecules that serve many critical survival functions. Herein, we describe a previously unknown transport system that T. pallidum uses to import riboflavin, an essential nutrient for the organism’s survival. Moreover, we found that this transport system is present in other pathogenic spirochetes. This is the first description of this new type of bacterial riboflavin transport system.

Identification of Lysine Residues in the Borrelia burgdorferi DbpA Adhesin Required for Murine Infection

ABSTRACT 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.

The TP0796 Lipoprotein of Treponema pallidum Is a Bimetal-dependent FAD Pyrophosphatase with a Potential Role in Flavin Homeostasis.

Background: The TP0796 lipoprotein of Treponema pallidum belongs to the poorly characterized ApbE superfamily. Results: TP0796 hydrolyzed FAD into FMN and AMP, consistent with the general enzymatic mechanism of an FAD pyrophosphatase. Conclusion: This novel metal-dependent enzyme probably plays an essential role in flavin homeostasis in T. pallidum. Significance: This is the first description of a metal-dependent FAD pyrophosphatase in bacteria. 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 Mg2+-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 Mg2+ 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.