Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Bifunctional protein FolD (folD)

What is the functional role of FolD in Leptospira interrogans metabolism?

FolD (5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase) is a bifunctional enzyme involved in one-carbon metabolism, which is critical for nucleotide synthesis, methionine regeneration, and other biosynthetic pathways. In bacterial pathogens like L. interrogans, FolD catalyzes two sequential reactions:

• Conversion of 5,10-methylene-tetrahydrofolate to 5,10-methenyl-tetrahydrofolate (dehydrogenase activity)

• Conversion of 5,10-methenyl-tetrahydrofolate to 10-formyl-tetrahydrofolate (cyclohydrolase activity)

These reactions are essential for purine biosynthesis and cellular survival, particularly in nutrient-limited environments encountered during infection .

How does FolD expression change in different growth conditions?

Proteomic analyses of L. interrogans have shown that proteins involved in metabolism and energy production undergo significant expression changes in response to environmental conditions that mimic in vivo infection. While specific data on FolD regulation is limited, similar metabolic enzymes show altered expression under iron-limited conditions with serum factors present (−Fe/FBS media) . The average fold changes in protein expression under these conditions can range from −5.863 to 2.731, suggesting that FolD may also respond to host environmental cues.

How might FolD contribute to L. interrogans pathogenesis?

While FolD has not been specifically identified as a virulence factor in the available literature, several lines of evidence suggest it may play a role in pathogenesis:

• Essential metabolic enzymes often contribute to pathogen survival during infection

• Proteins involved in energy metabolism show significant regulatory changes under in vivo-like conditions

• Other metabolic enzymes with dual roles in both metabolism and virulence have been identified in L. interrogans

Comparative proteomic studies have identified several proteins upregulated under in vivo-like conditions that exhibit sequence similarity to proteins with key functional roles in other pathogens, including outer surface lipoproteins, catalase, and glycosyl hydrolase . FolD could potentially serve a similar dual role.

What structural similarities exist between L. interrogans FolD and FolD proteins from other pathogens?

In silico analysis of FolD would likely reveal conserved domains characteristic of the bifunctional enzyme while potentially identifying unique structural elements that could be exploited for selective targeting.

What experimental approaches can differentiate the two catalytic activities of FolD?

The bifunctional nature of FolD presents a methodological challenge for researchers. Based on approaches used with other bifunctional enzymes in L. interrogans, the following methods could be applied:

• Spectrophotometric assays monitoring NADP+/NADPH conversion for the dehydrogenase activity

• HPLC-based detection of substrate/product conversion for both activities

• Site-directed mutagenesis targeting residues specific to each active site

• Design of activity-specific inhibitors to selectively block one function while preserving the other

What expression systems are optimal for producing recombinant L. interrogans FolD?

Based on successful expression of other L. interrogans proteins, the following approach is recommended:

• Expression vector: pET or pGEX systems with N-terminal His6 or GST tags

• Host strain: E. coli BL21(DE3) or Rosetta for rare codon optimization

• Culture conditions: LB medium, induction with 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Induction temperature: 16-18°C for 16-18 hours to maximize soluble protein yield

Protein purification can follow established protocols used for other L. interrogans proteins, typically involving:

• Initial capture using Ni-NTA or glutathione affinity chromatography

• Secondary purification via ion exchange or size exclusion chromatography

• Buffer optimization to maintain enzyme stability and activity

How can researchers determine the stability characteristics of L. interrogans FolD?

Protein stability assessment should follow approaches used for other L. interrogans enzymes, such as:

• Thermal stability analysis using differential scanning fluorimetry (DSF)

• Chemical denaturation with urea or guanidine hydrochloride

• pH stability profiling across physiologically relevant ranges (pH 5.0-8.0)

Notably, some L. interrogans enzymes like FNR demonstrate remarkable stability, retaining 38% activity even in 6M urea . Researchers should investigate whether FolD exhibits similar stability characteristics which could contribute to pathogen persistence under harsh host conditions.

What strategies can optimize the functional analysis of recombinant FolD?

Based on approaches used for other L. interrogans enzymes:

• Optimize reaction conditions (pH, temperature, ionic strength) - for example, the cytochrome c reductase activity of L. interrogans FNR exhibits an optimum at pH 6.5

• Identify potential electron donors/acceptors specific to L. interrogans metabolism

• Investigate substrate specificity using natural and synthetic tetrahydrofolate derivatives

• Develop coupled enzyme assays to monitor both dehydrogenase and cyclohydrolase activities simultaneously

Could FolD serve as a potential drug target against L. interrogans?

Several factors support FolD as a potential therapeutic target:

• FolD catalyzes essential steps in folate metabolism, which is critical for bacterial survival

• The bifunctional nature offers multiple sites for potential inhibitor binding

• Successful targeting of folate metabolism in other pathogens (e.g., via sulfonamides and trimethoprim)

Challenge considerations include:

• Potential structural similarities with human FolD requiring selective targeting

• The need for inhibitors that can penetrate the unique outer membrane structure of spirochetes

• Requirement for drug delivery to tissues where L. interrogans persists during chronic infection

How might FolD expression change during different stages of leptospirosis?

While specific data on FolD expression during infection is not provided in the search results, global proteome analyses of L. interrogans under in vivo-like conditions reveal significant regulation of metabolic enzymes . Proteins involved in energy production and metabolism show altered expression under these conditions, suggesting FolD may similarly be regulated during different infection stages.

A comprehensive investigation would require:

• Temporal transcriptomic analysis during infection in animal models

• Tissue-specific proteomics from infected organs (kidney, liver, lungs)

• Comparison of acute vs. chronic infection phases

Can FolD interact with host factors during infection?

L. interrogans proteins have demonstrated interactions with host molecules, particularly in adhesion processes. Pathogenic L. interrogans binds more efficiently to host cells than to extracellular matrix (ECM) components . While FolD has not been specifically identified as a host-interacting protein, researchers could investigate potential interactions using:

• Pull-down assays with host proteins

• Surface plasmon resonance to quantify binding affinities

• Cell culture infection models comparing wild-type and folD-depleted strains

What analytical methods would best characterize the dual functionality of FolD?

Based on approaches used for other L. interrogans enzymes with multiple functions:

• X-ray crystallography with and without substrates/inhibitors bound

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map flexible regions

• Site-directed mutagenesis of predicted catalytic residues

• Isothermal titration calorimetry (ITC) to determine binding parameters

How might molecular dynamics simulations inform understanding of FolD function?

Computational approaches could provide insights into:

• Conformational changes during catalysis transitioning between dehydrogenase and cyclohydrolase functions

• Substrate channeling between active sites

• Effects of pH and temperature on enzyme dynamics

• Identification of allosteric sites for potential inhibitor development

Simulations could be validated experimentally through enzyme kinetics studies and structural biology approaches.

What proteomic approaches could identify potential FolD interaction partners?

Global proteome analyses of L. interrogans have successfully identified proteins with altered expression under different conditions . Similar approaches could identify FolD interaction partners:

• Co-immunoprecipitation followed by mass spectrometry

• Chemical cross-linking coupled with mass spectrometry (XL-MS)

• Bacterial two-hybrid screening

• Label-free quantitative proteomics comparing wild-type and folD-knockout strains

Potential interaction partners might include other enzymes in folate metabolism pathways, regulatory proteins, or cellular structures that may localize FolD activity within the bacterial cell.