Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure

Blanco, David R.; Champion, Cheryl I.; Exner, Maurice M.; Shang, Ellen S.; Skare, Jon T.; Hancock, Robert E. W.; Miller, James N.; Lovett, Michael

doi:10.1128/jb.178.23.6685-6692.1996

Cited by 29 publications

(31 citation statements)

References 29 publications

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“…If the development of the humoral immune response between secondary and early latent syphilis in humans coincides with protective immunity, then the 11 proteins that exhibited reactivity only during early latency are of great interest. Four of the 11 proteins, including TP0163 (Mn 2ϩ /Mg 2ϩ ABC transport, periplasmic binding protein TroA), TP0216 (heat shock protein 70), TP0292 (conserved hypothetical protein), and TP1038 (bacterioferrin), have been previously identified as antigens (3,17,20). Five of the seven remaining novel antigens were also identified as antigens in our previous analysis using sera collected from rabbits (13).…”

Section: Resultsmentioning

confidence: 96%

Reactivity of Antibodies from Syphilis Patients to a Protein Array Representing theTreponema pallidumProteome

Brinkman¹,

McKevitt²,

McLoughlin³

et al. 2006

J Clin Microbiol

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To identify antigens important in the human immune response to syphilis, the serum antibody reactivity of syphilitic patients was examined with 908 of the 1,039 proteins in the proteome of Treponema pallidum subsp. pallidum using a protein array enzyme-linked immunosorbent assay. Thirty-four proteins exhibited significant reactivity when assayed with human sera from patients in the early latent stage of syphilis. A subset of antigens identified were further scrutinized for antibody reactivity at primary, secondary, and latent disease stages, and the results demonstrate that the humoral immune response to individual T. pallidum proteins develops at different rates during the time course of infection.

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Section: Resultsmentioning

confidence: 96%

Reactivity of Antibodies from Syphilis Patients to a Protein Array Representing theTreponema pallidumProteome

Brinkman¹,

McKevitt²,

McLoughlin³

et al. 2006

J Clin Microbiol

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“…In this regard, although T. pallidum possesses an outer membrane, the structure bears little resemblance to the outer membranes of Gram-negative bacteria; the treponemal outer membrane has a paucity of protein(s), lacks lipopolysaccharide, and seems to be devoid of channel-forming (e.g. porin) proteins (48 -51) despite early reports to the contrary (52,53).…”

Section: Table 2 Oligonucleotide Primers Used For Rt-pcr In This Studymentioning

confidence: 82%

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

Deka

Brautigam

Yang

et al. 2006

Journal of Biological Chemistry

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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 ...

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“…The rTromp2-containing sample and the control sample were tested for porin activity by the black lipid bilayer assay (1,4,5,16). Unlike native Tromp1 or rTromp1 (3,4), the addition of the sample containing rTromp2 to the model membrane system resulted in only a small number of insertional events (data not shown). A similar and greater amount of control sample showed no porin activity.…”

Section: Resultsmentioning

confidence: 99%

“…Based on the fact that most porin proteins have molecular masses between 28 and 48 kDa (25), we focused on the isolation of two outer membrane protein constituents of 28 and 31 kDa in size. We recently reported on the cloning of the gene encoding the 31-kDa rare outer membrane protein, designated Tromp1, and demonstrated that purified native Tromp1 and rTromp1 showed porin activity (3,4).…”

Section: Discussionmentioning

confidence: 99%

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Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2)

et al. 1997

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In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.

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Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure

Cited by 29 publications

References 29 publications

Reactivity of Antibodies from Syphilis Patients to a Protein Array Representing theTreponema pallidumProteome

Reactivity of Antibodies from Syphilis Patients to a Protein Array Representing theTreponema pallidumProteome

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

Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2)

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