T.pallidum p47

What is the structural characterization of T. pallidum p47?

Tp47 exhibits a unique protein structure that distinguishes it from other penicillin-binding proteins (PBPs). The crystal structure at 1.95 Å resolution reveals that Tp47 is predominantly composed of beta-sheet structures, which contrasts with the typical alpha/beta-fold observed in other PBPs . The protein comprises four distinct domains: two complex beta-sheet-containing N-terminal domains and two C-terminal domains that adopt immunoglobulin-like folds . This unique structural arrangement contributes to its distinct functional properties.

Despite containing three hypothetical consensus motifs (SVTK, TEN, and KTG) that typically form active centers in other PBPs, these motifs do not come together to form a conventional PBP active site in Tp47 . This structural deviation from established PBP patterns suggests that Tp47 likely represents a novel class of PBPs with potentially unique mechanisms of action.

What are the physical and biochemical properties of recombinant Tp47?

Recombinant Tp47 derived from the full-length TPN47 gene is commonly engineered with a 6xHis tag at the C-terminus for purification purposes . The protein has a molecular weight of approximately 47 kDa and an isoelectric point (pI) of 5.86 . When produced as a recombinant protein, Tp47 is typically purified using proprietary chromatographic techniques to achieve greater than 95% purity as determined by SDS-PAGE analysis .

For research applications, recombinant Tp47 is generally formulated in a solution containing PBS and 25mM potassium carbonate . While the protein remains stable at 4°C for approximately one week, long-term storage recommendations suggest temperatures below -18°C to maintain optimal activity . Freeze-thaw cycles should be avoided to prevent protein degradation and loss of functional properties.

How does Tp47 function as a penicillin-binding protein?

Unlike conventional penicillin-binding proteins, Tp47 displays unusual properties that challenge traditional classifications. Notably, Tp47 exhibits beta-lactamase activity with a catalytic rate constant (k(cat)) for penicillin of 271 ± 6 s⁻¹ . This enzymatic activity is significant as it represents a potential mechanism for interaction with beta-lactam antibiotics.

Interestingly, mutations of key amino acids within the hypothetical consensus motifs (SVTK, TEN, and KTG) fail to abolish the penicillin binding activity of Tp47 . This finding, combined with the structural observations that these motifs do not form a typical PBP active site, suggests that Tp47 employs a non-classical mechanism for penicillin binding. The beta-lactamase activity of Tp47 has historically complicated efforts to identify its active site through co-crystallization and mass spectrometric techniques .

What are the optimal conditions for producing and purifying recombinant Tp47?

Recombinant Tp47 is typically produced using Escherichia coli expression systems . The production process involves cloning the full-length TPN47 gene into an appropriate expression vector with a C-terminal 6xHis tag to facilitate purification . Following expression, the protein can be isolated using proprietary chromatographic techniques, including nickel affinity chromatography targeting the His-tag.

For optimal results, researchers should consider the following methodological approach:

• Express the recombinant protein in an E. coli strain optimized for protein expression

• Induce expression under controlled temperature and IPTG concentration

• Lyse cells under native conditions to preserve protein structure

• Purify using affinity chromatography followed by size exclusion chromatography

• Formulate in PBS with 25mM potassium carbonate for stability

• Verify purity using SDS-PAGE (target >95% purity)

• Store aliquots at temperatures below -18°C to prevent freeze-thaw cycles

What immunological methods are available for detecting and studying Tp47?

Monoclonal antibodies specific for Tp47 provide valuable tools for research applications. These antibodies typically do not stain the entire bacterial cell in immunocytochemistry/immunofluorescence (ICC/IF) applications, but specifically target the p47 protein . Purified preparations of monoclonal antibodies against Tp47 generally consist of >90% pure mouse monoclonal antibody, which has been purified from ascites fluid by protein A chromatography .

For experimental applications, the following working dilutions are recommended:

• ICC/IF: 1:10-1:50 dilution range

• ELISA: 1:20-1:200 dilution range

Researchers should optimize these dilutions based on their specific experimental conditions and detection systems. Additionally, commercial chimeric antigens combining p15, p17, and p47 are available for capture assays and serological studies . These chimeric constructs offer enhanced sensitivity for detecting anti-treponemal antibodies in research settings.

How can researchers effectively study Tp47's role in angiogenesis?

To investigate Tp47's pro-angiogenic effects, researchers can employ several complementary methodologies as demonstrated in recent studies:

• In vitro tube formation assay: This technique involves culturing human umbilical vein endothelial cells (HUVECs) with recombinant Tp47 and assessing tubule formation, which represents an early stage of angiogenesis .

• Three-dimensional angiogenesis analysis: This advanced method evaluates the formation of angiogenic sprouts in a three-dimensional matrix, providing more physiologically relevant data on vessel formation processes .

• Zebrafish embryo model: This in vivo approach involves injecting Tp47 into zebrafish embryos and quantifying the number of subintestinal vessel branch points, offering a whole-organism perspective on angiogenic activity .

• ROS measurement techniques: Since Tp47-induced angiogenesis involves reactive oxygen species (ROS), researchers should incorporate methods to measure intracellular and mitochondrial ROS levels, such as fluorescent probes specific for different ROS species .

• Pharmacological intervention: Using specific inhibitors such as the ROS inhibitor NAC can help establish mechanistic relationships between Tp47, ROS production, and angiogenesis .

• Autophagy flux assessment: Methods to measure autophagy-related proteins (P62, Beclin 1, LC3-II/LC3-I ratio) should be incorporated to evaluate the role of autophagy in Tp47-induced angiogenesis .

How does Tp47 contribute to pathological angiogenesis in syphilis?

Pathological angiogenesis represents a significant manifestation of syphilis, and Tp47 appears to play a crucial role in this process. Mechanistically, Tp47 promotes angiogenesis through a complex signaling cascade involving reactive oxygen species (ROS) and autophagy .

The pathway proceeds as follows:

• Tp47 enhances the production of mitochondrial ROS and increases the expression of NADPH oxidase-related proteins Nox2 and Nox4

• This leads to elevated intracellular ROS levels in a dose-dependent manner

• The increased ROS production subsequently promotes autophagy, as evidenced by increased expression of autophagy-related proteins P62 and Beclin 1, as well as an elevated LC3-II/LC3-I ratio

• The ROS-induced autophagy ultimately drives angiogenesis

Experimental evidence strongly supports this mechanism, as treatment with the ROS inhibitor NAC effectively prevents Tp47-induced tube formation and angiogenic sprout formation . Similarly, the autophagy inhibitor BafA1 significantly inhibits these angiogenic processes . These findings illuminate a critical pathway by which T. pallidum infection may contribute to the vascular manifestations observed in syphilis.

What is the role of Tp47 in activating inflammatory responses?

Tp47 plays a significant role in activating inflammatory responses through the NOD-like receptor family protein 3 (NLRP3) inflammasome pathway. Recent research has demonstrated that Tp47 promotes the expression of NLRP3, caspase-1, and IL-1β mRNA in macrophages . This activation appears to be metabolically regulated through glycolysis.

Specifically, Tp47 enhances glycolysis and the glycolytic capacity of macrophages, promoting the production of glycolytic metabolites including citrate, phosphoenolpyruvate, and lactate . This metabolic shift appears to be mediated by M2 pyruvate kinase (PKM2), as evidenced by experimental interventions:

• Treatment with the PKM2 inhibitor shikonin downregulates Tp47-promoted expression of NLRP3, caspase-1, and IL-1β mRNA in macrophages

• Shikonin also suppresses the Tp47-enhanced glycolysis and glycolytic capacity

• Silencing PKM2 with siRNA similarly inhibits Tp47-promoted inflammasome activation and enhanced glycolysis

These findings suggest that Tp47 activates NLRP3 inflammasomes via PKM2-dependent glycolysis, providing insight into how T. pallidum may trigger inflammatory responses during infection.

How does Tp47 interact with host endothelial cells?

Tp47 interacts with host endothelial cells to induce significant functional and metabolic changes that contribute to pathogenesis. In human umbilical vein endothelial cells (HUVECs), Tp47 promotes tubule formation and the development of angiogenic sprouts in a dose-dependent manner . This interaction appears to be primarily mediated through the generation of reactive oxygen species (ROS).

The interaction proceeds through several identifiable stages:

• Initial binding of Tp47 to endothelial cell surface receptors

• Induction of NADPH oxidase activity, particularly involving Nox2 and Nox4 proteins

• Enhanced production of mitochondrial ROS, which contributes to increased intracellular ROS levels

• ROS-mediated stimulation of autophagy pathways

• Autophagy-dependent promotion of angiogenic responses

The interaction between Tp47 and endothelial cells represents a critical aspect of T. pallidum pathogenesis, potentially contributing to the vascular manifestations observed in different stages of syphilis. The ability of Tp47 to induce these effects even in the absence of intact bacteria suggests it may function as a significant virulence factor during infection.

How can structural insights into Tp47 inform novel antimicrobial strategies?

The unique structural characteristics of Tp47 present opportunities for developing targeted antimicrobial strategies. Unlike traditional PBPs, Tp47 exhibits a predominantly beta-sheet structure with four distinct domains and unconventional arrangement of the PBP signature motifs (SVTK, TEN, and KTG) . These structural peculiarities, combined with its beta-lactamase activity (k(cat) = 271 ± 6 s⁻¹), position Tp47 as a potentially novel therapeutic target .

Future drug discovery efforts might focus on:

• Structure-based design of inhibitors targeting the unique beta-sheet domains of Tp47

• Development of compounds that specifically inhibit Tp47's beta-lactamase activity

• Creation of molecules that disrupt the interaction between Tp47 and host cell receptors

• Design of agents that prevent Tp47-induced ROS production and subsequent angiogenesis

Since T. pallidum remains exquisitely sensitive to penicillin despite decades of use, understanding how Tp47 interacts with beta-lactams may provide crucial insights for developing new antibiotics that maintain effectiveness against evolving bacterial pathogens.

What potential exists for Tp47 as a diagnostic or vaccine target?

Tp47 represents a promising target for both diagnostic and vaccine development efforts. As a membrane-associated lipoprotein with strong immunogenicity, Tp47 elicits significant antibody responses during infection. Current diagnostic approaches already utilize chimeric antigens incorporating Tp47 epitopes alongside other T. pallidum antigens (p15, p17) for enhanced sensitivity in serological assays .

For diagnostic applications, researchers should consider:

• Developing multiplexed assays that incorporate Tp47 alongside other treponemal antigens

• Exploring point-of-care diagnostic platforms utilizing recombinant Tp47

• Investigating whether Tp47 antibody profiles can differentiate between active and past infection

For vaccine development, Tp47's roles in angiogenesis, NLRP3 inflammasome activation, and glycolysis modulation suggest it may be a critical virulence factor . A vaccine targeting Tp47 might interfere with these pathogenic processes. Researchers should explore both passive immunization strategies using anti-Tp47 antibodies and active immunization approaches utilizing recombinant protein or epitope-based designs.

How might targeting Tp47-induced metabolic changes provide therapeutic opportunities?

Recent research reveals that Tp47 significantly alters host cell metabolism, particularly through enhancing glycolysis and modulating ROS production. These metabolic changes appear central to Tp47's roles in angiogenesis and inflammasome activation . This creates potential opportunities for metabolism-focused therapeutic interventions.

Promising therapeutic targets identified in recent studies include:

• NADPH oxidase pathway: Inhibitors of NADPH oxidase, such as DPI and apocynin, reduce Tp47-induced mitochondrial and intracellular ROS production . Further development of specific inhibitors targeting Nox2 and Nox4 might provide therapeutic benefit.

• PKM2-dependent glycolysis: The PKM2 inhibitor shikonin downregulates Tp47-promoted inflammasome activation and glycolytic enhancement . This suggests PKM2 inhibition as a potential therapeutic strategy.

• Autophagy modulation: Since Tp47-induced angiogenesis involves autophagy, as demonstrated by the inhibitory effect of BafA1 on tube formation , autophagy inhibitors may offer therapeutic potential.

• ROS scavenging approaches: The ROS inhibitor NAC effectively prevents Tp47-induced angiogenic processes , suggesting that antioxidant strategies might counteract certain pathogenic effects of T. pallidum infection.

These metabolic intervention strategies represent a novel approach to addressing T. pallidum pathogenesis, potentially complementing traditional antibiotic therapies. Future research should focus on developing specific inhibitors targeting these pathways and evaluating their efficacy in relevant model systems.

What are the relative advantages of different model systems for studying Tp47 functions?

Various experimental models offer distinct advantages for investigating Tp47 functions, as summarized in the following comparative table:

Selecting the appropriate model system depends on the specific research question, with many studies benefiting from a multi-model approach that combines the strengths of different systems.