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

Abstract

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 membraneassociated lipoprotein of T. pallidum formerly designated as TmpC. A 1.7-Å crystal structure showed that recombinant Tp0319 (rTp0319) consists of two ␣/␤ 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 (K d ) ϳ10 ؊7 M). This predilection for purine nucleosides by rTp0319 is consistent with its likely role as a receptor component of a cytoplasmic membraneassociated 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).Treponema pallidum, the bacterial agent of syphilis, cannot be cultivated continuously in vitro (1). Historically, this constraint has severely hampered progress in understanding many features of this enigmatic pathogen. More specifically, key aspects of the membrane biology of T. pallidum, particularly as it pertains to the interaction of the pathogen with its human host, remain poorly defined (2-4). The initial discovery of membrane lipoproteins in T. pallidum (5, 6) spawned new avenues of investigation into treponemal membrane biology, inasmuch as in other bacteria, lipoproteins have importance as virulence factors, modular components of ATP-binding cassette (ABC) 3 transporters, receptors, protective immune targets, and proinflammatory agonists that elicit innate immune responses (7-14). In fact, genome analysis predicts that T. pallidum devotes as much as 3% of its genetic coding capacity to lipoproteins (15). However, to date, with one exception (9, 16), comparative sequence analyses have not been fruitful for predicting the functions of treponemal lipoproteins, and the inability to cultivate (and thus genetically manipulate) the organism has precluded using classical gene inactivation approaches (17) for investigating the functions of treponemal ...

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

“…The supernatant was applied to a 4-ml Ni 2ϩ -agarose column (Qiagen), immobilizing the tagged protein. The elution of the TP0796 followed a previously developed methodology (8). The eluted protein was further purified by size exclusion chromatography on a Superdex 200 (16/60) column (GE Healthcare) equilibrated with buffer A (20 mM Hepes, pH 7.5, 0.1 M NaCl, 2 mM ␤-octyl glucoside).…”

“…The plasmid was transformed into E. coli BL21 (DE3), and the recombinant protein was hyperproduced as described above. Both liganded and ligand-free ApbE_Ec were prepared as described for PnrA (8). The concentration was determined at 280 nm using an extinction coefficient of 46,410 M Ϫ1 cm…”

“…The mechanism(s) by which the parasite acquires and utilizes these essential nutrients can potentially help explain the peculiar membrane biology of T. pallidum, elucidate key aspects of its parasitic strategy, and prompt new avenues of investigation for potentially novel antimicrobial drug targets. To this end, we have found that many of the likely transport systems of T. pallidum are predicated on the organism's membrane lipoproteins (5)(6)(7)(8)(9)(10)(11)(12)(13), and, as a result, the organism devotes a large percentage of its genome to encoding these lipoproteins (3,14,15). Unfortunately, many of the putative lipoproteins are hypothetical (i.e.…”

“…FAD Binding Activity-Ligand-free ApbE_Ec was prepared by an on-column denaturation/renaturation method (8). To determine the FAD binding affinity to the ligand-free protein, changes in fluorescence (⌬F) upon binding of different concentrations of FAD were measured (as described above), and the binding affinity was calculated using the GraphPad software.…”

Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold ‘em, knowing when to fold ‘em

“…Thus, SBPs are vital to the specificity and directionality of ABC import systems. SBPs have been classified into at least nine subfamilies, reflecting the broad range of substrates transported [60][61][62] and make a vital contribution to the ability of prokaryotes to acquire diverse substrates from their environments. These include sugars, siderophores, divalent metal ions, anions (such as phosphate and sulphate), amino acids, oligopeptides and nucleosides.…”