The Major Outer Sheath Protein (Msp) of Treponema denticola Has a Bipartite Domain Architecture and Exists as Periplasmic and Outer Membrane-Spanning Conformers

Anand, Asha; Luthra, Amit; Edmond, Maxwell E.; LeDoyt, Morgan; Caimano, Melissa J.; Radolf, Justin D.

doi:10.1128/jb.00078-13

Cited by 30 publications

(74 citation statements)

References 79 publications

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“…Importantly, the size of the P66 pore is large enough to allow the influx of many nutrients and amino acids that are required for B. burgdorferi survival. Interestingly, although we found that P66 was not heat modifiable like many other bacterial porin proteins, this observation is consistent with the characteristics of ␤-barrel proteins BamA and Msp from the spirochetes T. pallidum and T. denticola, respectively, which also are not heat modifiable (24,84). Although we used recombinant protein in our studies and cannot rule out the possibility that the protein was not in its native confirmation, recombinant P66 formed pores in membrane vesicles, suggesting that the protein was, indeed, folded correctly.…”

Section: Discussionsupporting

confidence: 73%

Structural Modeling and Physicochemical Characterization Provide Evidence that P66 Forms a β-Barrel in the Borrelia burgdorferi Outer Membrane

et al. 2014

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The Borrelia burgdorferi outer membrane (OM) contains numerous surface-exposed lipoproteins but a relatively low density of integral OM proteins (OMPs). Few membrane-spanning OMPs of B. burgdorferi have been definitively identified, and none are well characterized structurally. Here, we provide evidence that the borrelial OMP P66, a known adhesin with pore-forming activity, forms a ␤-barrel in the B. burgdorferi OM. Multiple computer-based algorithms predict that P66 forms a ␤-barrel with either 22 or 24 transmembrane domains. According to our predicted P66 topology, a lysine residue (K487) known to be sensitive to trypsin cleavage is located within a surface-exposed loop. When we aligned the mature P66 amino acid sequences from B. burgdorferi and B. garinii, we found that K487 was present only in the B. burgdorferi P66 protein sequence. When intact cells from each strain were treated with trypsin, only B. burgdorferi P66 was trypsin sensitive, indicating that K487 is surface exposed, as predicted. Consistent with this observation, when we inserted a c-Myc tag adjacent to K487 and utilized surface localization immunofluorescence, we detected the loop containing K487 on the surface of B. burgdorferi. P66 was examined by both Triton X-114 phase partitioning and circular dichroism, confirming that the protein is amphiphilic and contains extensive (48%) ␤-sheets, respectively. Moreover, P66 also was able to incorporate into liposomes and form channels in large unilamellar vesicles. Finally, blue native PAGE (BN-PAGE) revealed that under nondenaturing conditions, P66 is found in large complexes of ϳ400 kDa and ϳ600 kDa. Outer surface lipoprotein A (OspA) and OspB both coimmunoprecipitate with P66, demonstrating that P66 associates with OspA and OspB in B. burgdorferi. The combined computer-based structural analyses and supporting physicochemical properties of P66 provide a working model to further examine the porin and integrin-binding activities of this OMP as they relate to B. burgdorferi physiology and Lyme disease pathogenesis.

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Section: Discussionsupporting

confidence: 73%

Structural Modeling and Physicochemical Characterization Provide Evidence that P66 Forms a β-Barrel in the Borrelia burgdorferi Outer Membrane

et al. 2014

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“…We also noted that TprC expressed in T. pallidum is stably tethered within the periplasm. Unexpectedly, using the Conserved Domain Database server, we discovered that TprC contains N-and C-terminal domains (TprC N and TprC C , respectively) corresponding to regions in the major outer sheath protein (MOSP) of the oral commensal Treponema denticola, the parental Tpr ortholog (30). Using a battery of biophysical methodologies, we determined that TprC C , but not TprC N , can fold to form an amphiphilic ␤-barrel with porin activity equivalent to that of the fulllength polypeptide.…”

Section: We Previously Identified Treponema Pallidum Repeat Proteins mentioning

confidence: 99%

Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I

Anand

LeDoyt

Karanian

et al. 2015

Journal of Biological Chemistry

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“…This strain is pathogenic, and its pathogenicity can be tested in animal models (46)(47)(48). Most importantly, more than two-thirds of T. pallidum genes can be found in the genome of ATCC 35405 (4), including the majority of known T. pallidum virulence factors (10,16,44,(49)(50)(51)(52)(53). These advantages make this strain an ideal model a Parallel to the growth curve measurements shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The TDE_0143 gene and the tap1 promoter-driven gentamicin resistance marker (P tap1 -aacC m ) were PCR amplified with primers P 13 /P 14 and P 15 / P 16 , respectively. The resulting fragments were fused together by PCR using primers P 13 /P 16 . The fused TDE_0143-P tap1 -aacC m fragment was cloned into a pGEM-T Easy vector and then released using SpeI and NotI.…”

Section: Methodsmentioning

confidence: 99%

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Evidence that TP_0144 of Treponema pallidum Is a Thiamine-Binding Protein

Bian

Wang

et al. 2015

J. Bacteriol.

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e Thiamine pyrophosphate (TPP), the biologically active form of thiamine (also known as vitamin B 1 ), is an essential cofactor for several important enzymes involved in carbohydrate metabolism, and therefore, it is required for all living organisms. We recently found that a thiamine-binding protein (TDE_0143) is essential for the survival of Treponema denticola, an important bacterial pathogen that is associated with human periodontitis. In this report, we provide experimental evidence showing that TP_0144, a homolog of TDE_0143 from the syphilis spirochete Treponema pallidum, is a thiamine-binding protein that has biochemical features and functions that are similar to those of TDE_0143. First, structural modeling analysis reveal that both TDE_0143 and TP_0144 contain a conserved TPP-binding site and share similar structures to the thiamine-binding protein of Escherichia coli. Second, biochemical analysis shows that these two proteins bind to TPP with similar dissociation constant (K d ) values (TDE_0143, K d of 36.50 nM; TP_0144, K d of 32.62 nM). Finally, heterologous expression of TP_0144 in a ⌬TDE_0143 strain, a previously constructed TDE_0143 mutant of T. denticola, fully restores its growth and TPP uptake when exogenous thiamine is limited. Collectively, these results indicate that TP_0144 is a thiamine-binding protein that is indispensable for T. pallidum to acquire exogenous thiamine, a key nutrient for bacterial survival. In addition, the studies shown in this report further underscore the feasibility of using T. denticola as a platform to study the biology and pathogenicity of T. pallidum and probably other uncultivable treponemal species as well. Spirochetes comprise a monophyletic phylum that exhibits distinct long and coiled cell shapes and a characteristic corkscrew-like motility (1). The phylum comprises nine genera. One of these genera is Treponema, which contains several important human pathogens such as Treponema pallidum, the causative agent of syphilis (2, 3). The genus of Treponema consists of more than 60 different species, and many of these species cannot be cultivated in vitro (e.g., T. pallidum can be grown experimentally only in rabbit testes) (4-7). The lack of reliable in vitro cultivation systems has substantially hindered our understanding of the biology of T. pallidum and syphilis pathogenesis (3). Identifying virulence determinants of T. pallidum still depends on expressing potential virulence genes in heterologous systems such as Escherichia coli (8-11). However, the physiological differences between the spirochetes and E. coli have limited the use of E. coli systems for functional investigations.Among the treponemal species, Treponema denticola, an oral spirochete associated with periodontitis, can be easily cultivated and is genetically manipulable (12-14). Due to the high physiological similarities between T. denticola and T. pallidum, the oral spirochete has been explored to study the physiology, biosynthetic pathways, and virulence determinants of T. pallidum (e.g., heterol...

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The Major Outer Sheath Protein (Msp) of Treponema denticola Has a Bipartite Domain Architecture and Exists as Periplasmic and Outer Membrane-Spanning Conformers

Cited by 30 publications

References 79 publications

Structural Modeling and Physicochemical Characterization Provide Evidence that P66 Forms a β-Barrel in the Borrelia burgdorferi Outer Membrane

Structural Modeling and Physicochemical Characterization Provide Evidence that P66 Forms a β-Barrel in the Borrelia burgdorferi Outer Membrane

Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I

Evidence that TP_0144 of Treponema pallidum Is a Thiamine-Binding Protein

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