Syphilis, caused by the spirochetal bacterium Treponema pallidum, continues to play prominently as a sexually transmitted disease. Syphilis also is a paradigm of bacterial chronicity and immune evasion, but virtually nothing is known about how T. pallidum carries out these enigmatic processes. Unfortunately, there is a paucity of information on the functions of T. pallidum membrane proteins that likely contribute to the spirochete's complex parasitic strategy. T. pallidum is postulated to encode 45 membrane lipoproteins (4.3% of its genome). Membrane lipoproteins typically serve many important physiological roles and also have significance as virulence factors, modular components of ABC-type transporters, stabilizers of membrane integrity, protective immune targets, and proinflammatory agonists. However, the functions of the treponemal lipoproteins have remained largely undefined. In a departure from traditional T. pallidum research, we have been crystallizing the membrane lipoproteins of T. pallidum and inferring their functions from structural determinations. State-of-the-art biophysical and biochemical techniques are being applied to corroborate functions derived from structural data. From significant progress made over the past funding interval in solving the three-dimensional structures of five T. pallidum lipoproteins (Tp32, TP0319 [TmpC;PnrA], Tp34, TP0655 [PotD], and Tp0956), it is now well documented that our structural biology approach represents a successful discovery platform for exploring lipoprotein functions in the context of T. pallidum's atypical membrane biology (a key aspect of treponemal pathogenesis). Furthermore, from prior progress, we are now able to propose hypothesis-driven experiments that will allow us to place our lipoprotein functional assignments better in the context of T. pallidum physiology and membrane biology. Given this, the Specific Aims of this renewal proposal are (1) To assess the levels of expression and membrane surface localization in T. pallidum of lipoproteins whose functions are being determined;(2) To continue to clone, express in E. coli, purify, and crystallize recombinant T. pallidum lipoproteins, with emphasis on solving their three-dimensional structures. Structural data then will be used to formulate new testable hypotheses regarding potential protein function(s);and (3) To conduct follow-up biological, biochemical, and biophysical experiments that will complement functional predictions and place them in the context of T. pallidum biology and syphilis pathogenesis. Clarifying the functions of the membrane lipoproteins is essential for understanding many of the unusual aspects of T. pallidum membrane biology and its relationship to syphilis pathogenesis.

Public Health Relevance

Syphilis remains an important sexually transmitted disease in the United States. Efforts to understand the complex nature of syphilis pathogenesis have been hindered by the inability to culture the etiological agent, Treponema pallidum, in the laboratory. This project seeks to use molecular biology and structural biology to determine the structures and functions of key membrane (lipo) proteins of the organism. These membrane proteins likely play strategic roles in sustaining T. pallidum during human infection. As such, our studies have the potential to elucidate the molecular bases of many new aspects of syphilis pathogenesis and, consequently, new potential avenues to thwart infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI056305-10
Application #
8423767
Study Section
Special Emphasis Panel (ZRG1-IDM-R (02))
Program Officer
Hiltke, Thomas J
Project Start
2003-07-01
Project End
2014-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
10
Fiscal Year
2013
Total Cost
$361,609
Indirect Cost
$131,285
Name
University of Texas Sw Medical Center Dallas
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Brautigam, Chad A; Deka, Ranjit K; Liu, Wei Z et al. (2016) The Tp0684 (MglB-2) Lipoprotein of Treponema pallidum: A Glucose-Binding Protein with Divergent Topology. PLoS One 11:e0161022
Radolf, Justin D; Deka, Ranjit K; Anand, Arvind et al. (2016) Treponema pallidum, the syphilis spirochete: making a living as a stealth pathogen. Nat Rev Microbiol 14:744-759
Deka, Ranjit K; Brautigam, Chad A; Liu, Wei Z et al. (2016) Molecular insights into the enzymatic diversity of flavin-trafficking protein (Ftp; formerly ApbE) in flavoprotein biogenesis in the bacterial periplasm. Microbiologyopen 5:21-38
Brautigam, Chad A; Deka, Ranjit K; Liu, Wei Z et al. (2015) Insights into the potential function and membrane organization of the TP0435 (Tp17) lipoprotein from Treponema pallidum derived from structural and biophysical analyses. Protein Sci 24:11-9
Deka, Ranjit K; Brautigam, Chad A; Liu, Wei Z et al. (2015) Evidence for Posttranslational Protein Flavinylation in the Syphilis Spirochete Treponema pallidum: Structural and Biochemical Insights from the Catalytic Core of a Periplasmic Flavin-Trafficking Protein. MBio 6:e00519-15
Scheuermann, Thomas H; Brautigam, Chad A (2015) High-precision, automated integration of multiple isothermal titration calorimetric thermograms: new features of NITPIC. Methods 76:87-98
Brautigam, Chad A (2015) Fitting two- and three-site binding models to isothermal titration calorimetric data. Methods 76:124-36
Bashiri, Ghader; Baker, Edward N (2015) Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies. Protein Sci 24:1-10
Deka, Ranjit K; Brautigam, Chad A; Biddy, Brent A et al. (2013) Evidence for an ABC-type riboflavin transporter system in pathogenic spirochetes. MBio 4:e00615-12
Deka, Ranjit K; Brautigam, Chad A; Liu, Wei Z et al. (2013) The TP0796 lipoprotein of Treponema pallidum is a bimetal-dependent FAD pyrophosphatase with a potential role in flavin homeostasis. J Biol Chem 288:11106-21

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