Treponema p47 47.7kDa

What is Treponema p47 47.7kDa and what organism does it originate from?

Treponema p47 47.7kDa is a surface immunogen found in Treponema pallidum subspecies pallidum, the causative agent of syphilis. It is a non-glycosylated polypeptide chain with a molecular mass of 47.7kDa . Treponema pallidum is a gram-negative spirochaete bacterium with a corkscrew motion that allows it to move through viscous mediums such as mucus. The bacterium has one of the smallest bacterial genomes at 1.14 million base pairs, reflecting its adaptation through genome reduction to the rich environment of mammalian tissue .

How is the recombinant form of Treponema p47 47.7kDa produced?

Recombinant Treponema p47 is produced in Escherichia coli expression systems. The protein is typically fused to a histidine tag at the N-terminus to facilitate purification . The gene encoding the 47-kDa surface immunogen has been localized to a 1.3-kilobase DNA fragment of T. pallidum . Significant enhancement (20- to 100-fold) in expression can be achieved using a T7 RNA polymerase-dependent expression vector system, which allows for efficient production of the recombinant protein . When expressed in E. coli, the protein localizes to both the inner and outer membranes of the bacterial cells, indicating its membrane-associated nature .

What are the physicochemical properties of Treponema p47 47.7kDa?

Treponema p47 47.7kDa is a hydrophobic, integral membrane protein. This is evidenced by the absolute requirement of detergents for its solubilization from E. coli cell envelopes, including N-lauroylsarcosine, 3-[(3-chloramidopropyl)dimethylammonio]-1-propane sulfonate, N-octyl-beta-D-glucopyranoside, or Nonidet P-40 . Additionally, the recombinant protein partitions into the detergent phase during Triton X-114 solubilization, further confirming its hydrophobic nature . In commercial preparations, it appears as a sterile filtered white lyophilized powder and is typically formulated in 20mM sodium carbonate at pH 10 . For reconstitution, it is recommended to use sterile water at concentrations not less than 100µg/ml .

What is the role of Treponema p47 47.7kDa in T. pallidum pathogenesis?

Treponema p47 47.7kDa plays a crucial role in the immunopathogenesis of syphilis . It functions as a penicillin-binding protein (pbp) and is involved in the turnover of β-lactam antibiotics . Despite this function, T. pallidum remains sensitive to penicillin therapy, suggesting a complex relationship between this protein's activity and antibiotic resistance mechanisms . The protein is specific to pathogenic treponemes, as confirmed by Western blot analyses of the immunoaffinity-purified antigen, indicating its potential utility as a diagnostic marker for pathogenic Treponema species .

How can researchers distinguish between different molecular forms of Treponema p47?

Western blot analysis is the primary method for distinguishing between different molecular forms of Treponema p47. Using this technique, researchers have observed several derivatives of the recombinant 47-kDa protein possessing different molecular masses that are identical to those previously detected in native T. pallidum antigens using monoclonal antibodies . For optimal resolution, SDS-PAGE should be performed under reducing conditions, followed by immunoblotting with specific anti-Tp47 antibodies. Purity analysis can be conducted using SDS-PAGE, with recombinant preparations typically showing greater than 95.0% purity .

What storage and handling conditions are recommended for Treponema p47 47.7kDa?

While Treponema p47 exhibits stability at room temperature for up to 4 weeks, optimal long-term storage should be below -18°C . Freeze-thaw cycles should be prevented to maintain protein integrity and activity. For solubilization, it is recommended to reconstitute the lyophilized Treponema p47 in sterile 18MΩ-cm H₂O at concentrations not less than 100µg/ml, which can then be further diluted to other aqueous solutions as needed . When working with the protein, standard precautions for handling recombinant proteins should be followed, including the use of appropriate personal protective equipment.

How does Treponema p47 47.7kDa compare to penicillin-binding proteins in other Treponema species?

Phylogenetic analysis has revealed that Tp47 shows no substantial similarity to other β-lactamases in treponemes . This divergence suggests a unique evolutionary pathway for this protein in T. pallidum. The relatedness of Treponema denticola to other treponemes, including T. pallidum, highlights the importance of investigating the diversity of penicillin-binding protein (pbp) genes in Treponema species . Such studies can provide greater understanding of the potential for horizontal gene transfer of antibiotic resistance determinants, which could impact the treatment and control of syphilis .

What methodologies are effective for studying the membrane localization of Treponema p47 47.7kDa?

To study the membrane localization of Treponema p47, researchers typically employ Sarkosyl extraction of E. coli recombinant cell envelopes, which has shown that the 47-kDa protein localizes to both the inner and outer membranes of E. coli . Phase separation using Triton X-114 is another effective method, with the protein partitioning into the detergent phase, confirming its integral membrane protein nature . For more precise localization studies, immunoelectron microscopy using gold-labeled antibodies can be employed to visualize the distribution of the protein within the bacterial cell envelope. Additionally, membrane fractionation techniques combined with Western blotting can provide quantitative assessment of the protein's distribution across different membrane compartments.

What are the implications of Treponema p47 in antibiotic resistance studies?

Although T. pallidum remains sensitive to penicillin, clinically significant resistance to macrolides has emerged in many developing countries . Understanding the role of Tp47 in potential resistance mechanisms is crucial. Research methodologies should include minimum inhibitory concentration (MIC) determinations using various β-lactam antibiotics against recombinant strains expressing different levels of Tp47. Binding affinity studies using purified Tp47 and labeled antibiotics can provide insights into the molecular interactions. Additionally, site-directed mutagenesis of the Tp47 gene followed by functional analysis can identify key residues involved in antibiotic binding. The potential for horizontal gene transfer of resistance determinants between different Treponema species should also be investigated, particularly given the relatedness of various treponemes found in human dental plaque .

How prevalent are different Treponema species in health and disease conditions?

These findings indicate that even young adults with healthy periodontal tissues may harbor several oral Treponema species, and the presence of T. pectinovorum and/or T. vincentii in health-associated plaque may correlate with relative risk for disease on a site-specific basis .

What molecular techniques are most effective for detecting various Treponema species in clinical samples?

Species-specific nested PCR (nPCR) has proven to be an effective molecular technique for the detection of Treponema species in clinical samples . This approach provides greater sensitivity compared to direct microscopic observation, which likely underestimates cell numbers. The nested PCR methodology involves two rounds of amplification, with the first round using universal primers for Treponema species, followed by a second round using species-specific primers. This technique has been shown to be more sensitive than dot blot hybridization used in previous studies .

For researchers implementing this method, it is crucial to design primers targeting conserved regions of the 16S rRNA gene for the first round of amplification, followed by species-specific primers for the second round. Proper controls must be included to account for potential cross-reactivity between closely related species. Additionally, quantitative PCR (qPCR) can be employed for more precise estimation of bacterial loads in clinical samples, providing valuable data for correlation with disease severity.

How do phylogenetic relationships among Treponema species inform our understanding of potential horizontal gene transfer of antibiotic resistance?

Phylogenetic analysis of penicillin-binding protein (pbp) genes in treponemes reveals that genes encoding these proteins that show significant similarity to each other appear in separate clusters . This clustering pattern provides insights into the evolutionary relationships among different Treponema species and their respective pbp genes. The relatedness of Treponema denticola to other treponemes, including T. pallidum, coupled with the reported presence of natural mobile antibiotic determinants, highlights the importance of monitoring for potential horizontal gene transfer events .

For research in this area, whole genome sequencing followed by comparative genomic analysis can identify potential mobile genetic elements carrying resistance determinants. In vitro conjugation experiments between different Treponema species can assess the transferability of resistance genes. Additionally, monitoring the genetic diversity of clinical isolates over time and across geographic regions can provide early warning of emerging resistance patterns. Such studies are crucial for maintaining the effectiveness of current antibiotic regimens for treponematoses like syphilis .

What are the promising approaches for developing improved detection methods for Treponema p47 47.7kDa?

Development of novel detection methods for Treponema p47 47.7kDa should focus on enhancing sensitivity and specificity while reducing assay time. Potential approaches include:

• CRISPR-Cas-based detection systems targeting specific sequences in the Tp47 gene, which can provide rapid, point-of-care diagnostics with single-molecule sensitivity.

• Aptamer-based biosensors that can specifically bind to Tp47 protein, offering label-free detection with high sensitivity.

• Digital PCR methods for absolute quantification of Tp47 gene copies in clinical samples.

• Multiplexed assays that simultaneously detect multiple Treponema virulence factors, including Tp47, providing a more comprehensive assessment of pathogen load and virulence potential.

These advanced methods could significantly improve both research capabilities and clinical diagnostics for treponematoses.

What structural studies would advance our understanding of Treponema p47 47.7kDa function?

Despite its importance in T. pallidum pathogenesis, detailed structural information about Treponema p47 47.7kDa remains limited. Future research should focus on:

• X-ray crystallography or cryo-electron microscopy studies of purified Tp47 to determine its three-dimensional structure.

• Structure-function analyses through site-directed mutagenesis to identify critical residues involved in penicillin binding and membrane association.

• Protein-protein interaction studies to identify binding partners within the T. pallidum cell envelope.

• Molecular dynamics simulations to understand protein flexibility and potential conformational changes upon antibiotic binding.

These structural insights would provide a foundation for rational drug design targeting this important virulence factor.

How might Treponema p47 47.7kDa be utilized in vaccine development strategies?

As a surface-exposed immunogen specific to pathogenic treponemes, Tp47 represents a potential vaccine candidate. Future research in this direction should investigate:

• Immunogenicity studies of recombinant Tp47 in animal models, assessing both humoral and cell-mediated immune responses.

• Identification of immunodominant epitopes within the Tp47 protein that elicit protective antibodies.

• Development of multi-antigen vaccine formulations combining Tp47 with other T. pallidum surface antigens.

• Evaluation of different adjuvants and delivery systems to enhance immune responses to Tp47-based vaccines.

• Assessment of cross-protection against different pathogenic Treponema species or subspecies.

Such studies could potentially lead to novel preventive strategies for syphilis and related treponematoses, addressing a significant global health challenge.