Recombinant Leptospira biflexa serovar Patoc Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is the functional role of sucC in Leptospira biflexa compared to pathogenic Leptospira species?

Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) functions as a critical enzyme in the tricarboxylic acid (TCA) cycle, catalyzing the reversible conversion of succinyl-CoA to succinate with concomitant ADP phosphorylation to ATP. In saprophytic Leptospira like L. biflexa, the TCA cycle plays a fundamental role in environmental adaptation, potentially differing from pathogenic species that have evolved specialized metabolic strategies for host infection. While pathogenic Leptospira species such as L. interrogans utilize virulence factors for host interaction and immune evasion, saprophytic L. biflexa relies more heavily on metabolic versatility for environmental survival, potentially influencing sucC expression patterns and regulation .

How can L. biflexa be used as a surrogate host for investigating leptospiral proteins?

L. biflexa serovar Patoc serves as an excellent surrogate host for expressing and studying proteins from pathogenic Leptospira species due to its genetic tractability and non-pathogenic nature. Research demonstrates that L. biflexa can be effectively transformed with plasmids containing genes from pathogenic Leptospira under appropriate promoters. For example, studies have successfully expressed the pathogen-specific LIC11711 gene in L. biflexa using the strong lipL32 promoter (P32), resulting in protein overexpression approximately 600-fold higher than native levels in pathogenic strains . This approach allows researchers to study protein function in a safe, genetically manipulable system while maintaining a leptospiral cellular environment.

What genetic tools are available for expressing recombinant proteins in L. biflexa?

Several genetic tools have been validated for recombinant protein expression in L. biflexa:

These tools enable precise control of gene expression and stable maintenance of recombinant constructs in L. biflexa, making it a valuable model system for studying proteins like sucC in a native-like environment .

What expression systems would yield optimal activity for recombinant L. biflexa sucC?

For optimal expression of recombinant sucC from L. biflexa, researchers should consider both heterologous and homologous expression systems. Based on documented approaches for other leptospiral proteins, the following strategies are recommended:

• Homologous expression in L. biflexa:

  • Utilize the P32 (lipL32) promoter, which has demonstrated nearly 600-fold increase in target gene transcription compared to native expression levels

  • Employ the pMaOri vector system with spectinomycin selection (40 μg/mL)

  • Optimize codon usage for L. biflexa if expressing genes from other species

• Heterologous expression in E. coli:

  • Consider fusion tags (His, MBP, or SUMO) to improve solubility and facilitate purification

  • Express at lower temperatures (16-25°C) to enhance proper protein folding

  • Co-express with chaperones if misfolding occurs

The optimal choice would depend on downstream applications - homologous expression in L. biflexa would better preserve native folding and potential interaction partners, while E. coli systems typically yield higher protein quantities for structural and biochemical studies .

How can heterologous expression be used to investigate structure-function relationships in sucC?

Heterologous expression provides powerful tools for investigating structure-function relationships in sucC through domain mapping, site-directed mutagenesis, and comparative studies. Taking inspiration from successful approaches with other leptospiral proteins, researchers could:

• Generate truncation constructs similar to those used for LigB (Table 1), targeting specific domains to determine their functional contributions:

• Employ site-directed mutagenesis of conserved residues predicted to be involved in substrate binding or catalysis

• Create chimeric proteins combining domains from pathogenic and saprophytic Leptospira sucC to identify specificity determinants

• Utilize heterologous expression to produce protein for structural studies (X-ray crystallography or cryo-EM)

This systematic approach would map critical regions responsible for sucC's enzymatic function and potential unique adaptations in saprophytic versus pathogenic species .

What role might sucC play in L. biflexa's environmental adaptation compared to pathogenic leptospires?

The sucC enzyme likely serves as a critical metabolic node in L. biflexa's adaptation to diverse environmental conditions. Unlike pathogenic Leptospira, which have evolved specialized mechanisms for host infection and immune evasion, saprophytic L. biflexa must efficiently utilize available nutrients in soil and water environments.

Research indicates fundamental differences between saprophytic and pathogenic Leptospira - "Saprophytic leptospires do not have the ability to infect the target host and cannot cause leptospirosis. In contrast, pathogenic species have a number of surface proteins that are unique to these strains and can interact with the host" . These differences likely extend to metabolic enzymes like sucC, which may be optimized for:

Comparative studies examining sucC expression under various growth conditions between pathogenic and saprophytic species would elucidate these potential metabolic adaptations and their contribution to L. biflexa's environmental fitness .

What transformation protocol is most effective for expressing recombinant proteins in L. biflexa?

For effective transformation of L. biflexa with constructs expressing recombinant proteins like sucC, the following optimized protocol is recommended based on successful approaches documented in the literature:

• Culture preparation:

  • Grow L. biflexa in EMJH medium at 30°C until reaching exponential phase (OD420nm of 0.3-0.5)

  • Harvest cells by centrifugation (3500× g, room temperature)

  • Wash cell pellet with equivalent volume of sterile water

  • Resuspend to concentration of approximately 10^10 cells/mL in sterile water

• Electroporation:

  • Mix 100 μL of bacterial suspension with plasmid DNA (typically 1-5 μg)

  • Transfer mixture to 0.2 mm electroporation cuvette (pre-chilled at -20°C)

  • Perform electroporation with parameters: 1.8 kV, 25 μF, and 200 Ω

  • Immediately add 1 mL of fresh liquid EMJH medium

• Recovery and selection:

  • Incubate transformed cells at 30°C with shaking (150 rpm)

  • Plate on solid EMJH medium containing appropriate antibiotic (e.g., 40 μg/mL spectinomycin)

  • Incubate at 30°C until colonies become visible (typically 7-10 days)

  • Verify transformation by PCR using plasmid-specific primers

This protocol has been successfully used to transform L. biflexa with constructs expressing various proteins, including LIC11711 under the strong P32 promoter .

How can binding and functional assays be adapted to characterize sucC activity?

To characterize sucC activity and interactions, researchers can adapt established assays used for other leptospiral proteins. Based on methodologies described in the literature, the following approaches are recommended:

• Enzymatic activity assays:

  • Spectrophotometric measurement of ADP/ATP formation using coupled enzyme systems

  • Monitoring CoA release using thiol-reactive fluorescent probes

  • Isothermal titration calorimetry to determine substrate binding constants

• Comparative growth assays:

  • Construct sucC knockout or conditional expression strains

  • Assess growth under various carbon sources and environmental conditions

  • Compare metabolic flexibility between wild-type and mutant strains

These methodological approaches provide comprehensive characterization of sucC function and its role in L. biflexa metabolism .

What purification strategy would yield highest activity for recombinant sucC?

For optimal purification of active recombinant sucC from L. biflexa, a multi-step approach focused on preserving enzymatic activity is recommended:

• Expression optimization:

  • Use strong promoters (P32) for high-level expression in L. biflexa

  • Consider adding purification tags (His, FLAG) for affinity purification

  • Maintain physiological pH and temperature during cell growth

• Cell lysis and initial clarification:

  • Gentle lysis using non-ionic detergents or controlled sonication

  • Include protease inhibitor cocktail to prevent degradation

  • Maintain low temperature (4°C) throughout purification process

  • Clarify lysate by centrifugation (≥15,000 × g for 30 minutes)

• Multi-step chromatographic purification:

  • Initial capture: Affinity chromatography (if tagged) or ion exchange

  • Intermediate purification: Hydrophobic interaction chromatography

  • Polishing step: Size exclusion chromatography

  • Include stabilizing factors (glycerol, reducing agents) in all buffers

• Activity preservation:

  • Add substrate analogs or cofactors to maintain active conformation

  • Avoid freeze-thaw cycles (use small aliquots for storage)

  • Store with glycerol (10-20%) at -80°C for long-term stability

This systematic approach, informed by successful purification strategies for other leptospiral proteins, maximizes the likelihood of obtaining high-activity recombinant sucC suitable for enzymatic and structural studies .

How should researchers design experiments to compare sucC function between pathogenic and saprophytic Leptospira species?

For robust comparative analysis of sucC function between pathogenic and saprophytic Leptospira species, researchers should implement a multi-faceted experimental design:

• Comparative genomics and transcriptomics:

  • Sequence analysis of sucC genes and regulatory regions across multiple Leptospira species

  • RNA-Seq under identical growth conditions to compare expression patterns

  • Promoter analysis to identify potential regulatory differences

• Heterologous expression studies:

  • Express sucC from different Leptospira species in a common host (E. coli or L. biflexa)

  • Use standardized purification protocols to allow direct comparison

  • Perform detailed enzyme kinetics (Km, Vmax, substrate specificity)

• Functional complementation:

  • Create sucC knockout strains in L. biflexa

  • Complement with sucC genes from pathogenic species

  • Assess growth characteristics under various nutrient conditions

• Metabolic flux analysis:

  • Use 13C-labeled substrates to track carbon flow through central metabolism

  • Compare flux distribution between pathogenic and saprophytic species

  • Identify potential metabolic rewiring around the sucC reaction

This comprehensive approach would reveal meaningful differences in sucC function that may contribute to the distinct ecological niches occupied by pathogenic versus saprophytic Leptospira species .

What controls should be included when studying heterologously expressed sucC in L. biflexa?

• Vector-only control:

  • L. biflexa transformed with empty vector (e.g., pMaOri without insert)

  • Establishes baseline for growth, metabolism, and protein expression

• Expression verification controls:

  • RT-qPCR to confirm transcript levels (studies show P32 promoter can increase expression ~600-fold)

  • Western blotting using tag-specific or sucC-specific antibodies

  • Include housekeeping gene control (e.g., DnaK) to normalize expression levels

• Functional controls:

  • Wild-type L. biflexa strain (untransformed)

  • sucC knockout strain (if available)

  • L. biflexa expressing native sucC under the same promoter

• Specificity controls:

  • Expression of unrelated metabolic enzyme under identical conditions

  • Expression of catalytically inactive sucC mutant

• Experimental condition controls:

  • Growth media composition standardization

  • Consistent growth phase for all analyses

  • Identical purification conditions for all protein variants

These controls, adapted from successful approaches with other Leptospira proteins, ensure that observed phenotypes are specifically attributable to sucC function rather than transformation effects or experimental variables .

How should researchers interpret discrepancies in sucC function between in vitro and in vivo studies?

When encountering discrepancies between in vitro biochemical data and in vivo functional studies of sucC, researchers should consider several potential explanations and analytical approaches:

• Physiological context differences:

  • In vitro systems lack the complex metabolic networks present in vivo

  • Substrate concentrations in vitro often differ from physiological levels

  • Cofactor availability may vary between experimental systems

• Protein-protein interactions:

  • sucC likely functions as part of a multi-enzyme complex in vivo

  • Regulatory proteins may modulate activity in cellular contexts

  • These interactions are typically absent in purified protein studies

• Post-translational modifications:

  • Potential modifications affecting activity may occur in vivo but not in vitro

  • Differences in modification patterns between expression systems

• Methodological considerations:

  • Similar to studies with LIC11711 protein, where cellular localization in L. biflexa differed from recombinant protein behavior

  • Buffer conditions influencing observed enzymatic parameters

• Recommended analytical approaches:

  • Integrate multiple experimental methodologies

  • Perform studies in increasingly complex systems (purified protein → cell extracts → intact cells)

  • Use genetic approaches (knockout/complementation) to validate biochemical findings

  • Apply systems biology tools to place discrepancies in broader metabolic context

This comprehensive analytical framework helps reconcile contradictory observations and develop a more complete understanding of sucC function in L. biflexa metabolism .

What statistical approaches are appropriate for analyzing comparative sucC activity data?

For robust statistical analysis of comparative sucC activity data, researchers should employ:

• Experimental design considerations:

  • Minimum of three biological replicates per condition

  • Technical triplicates for each biological replicate

  • Randomization of sample processing order to minimize systematic errors

• Appropriate statistical tests:

  • For comparing two conditions: Student's t-test (parametric) or Mann-Whitney U test (non-parametric)

  • For multiple conditions: One-way ANOVA with appropriate post-hoc tests (Tukey's, Dunnett's)

  • For multi-factorial experiments: Two-way ANOVA to assess interaction effects

• Data normalization strategies:

  • Normalize enzyme activity to protein concentration

  • Consider internal standards for batch-to-batch comparison

  • Use relative activity ratios when comparing across different experimental setups

• Advanced analytical approaches:

  • For complex datasets: Principal component analysis to identify patterns

  • For time-course experiments: Repeated measures ANOVA or mixed-effects models

  • For concentration-dependent data: Non-linear regression for enzyme kinetics parameters

From the literature, successful statistical approaches include the Mann-Whitney U test for comparing binding of components to different Leptospira strains, with p-values below 0.05 considered statistically significant . Similar approaches would be appropriate for sucC activity comparisons between wild-type and recombinant strains.

What are the most promising research directions for understanding sucC's role in Leptospira metabolism?

The most promising research directions for elucidating sucC's role in Leptospira metabolism include:

• Comparative metabolomics:

  • Profiling metabolite levels in wild-type vs. sucC-modified strains

  • Identifying metabolic bottlenecks and regulatory nodes connected to sucC function

  • Comparing metabolic networks between saprophytic and pathogenic species

• Systems biology approaches:

  • Construction of genome-scale metabolic models incorporating experimentally determined sucC parameters

  • Flux balance analysis to predict metabolic adaptations under different conditions

  • Integration of transcriptomic and proteomic data with metabolic models

• Structural biology:

  • Determination of sucC crystal structure from L. biflexa

  • Comparative structural analysis with homologs from other bacterial species

  • Structure-guided design of specific inhibitors or activity modulators

• Environmental adaptation studies:

  • Investigation of sucC regulation under various environmental stresses

  • Determination of sucC's role in survival under nutrient limitation

  • Assessment of metabolic flexibility conferred by sucC in different ecological niches

These research directions build upon the successful experimental frameworks established for other Leptospira proteins, such as the heterologous expression and functional characterization of LIC11711 in L. biflexa, adapting them to investigate the central metabolic role of sucC .

How might insights from sucC research contribute to broader understanding of Leptospira biology?

Insights from sucC research have significant implications for broader understanding of Leptospira biology across several dimensions:

• Metabolic adaptation and evolution:

  • Comparing sucC function between saprophytic and pathogenic species provides insights into metabolic adaptations during evolution toward pathogenicity

  • Understanding how central carbon metabolism has been rewired during Leptospira speciation

  • Identifying metabolic signatures that distinguish environmental from host-adapted lifestyles

• Environmental persistence mechanisms:

  • sucC's role in energy generation may illuminate how L. biflexa survives in nutrient-poor environments

  • Potential connections between metabolic efficiency and long-term environmental persistence

  • Insights into seasonal variations in Leptospira prevalence in environmental reservoirs

• Pathogenesis and host adaptation:

  • Similar to findings with LIC11711, where exposure to saprophytic Leptospira influenced pathogenic outcomes , understanding metabolic differences may reveal how pathogenic species adapted to host environments

  • Potential metabolic targets for therapeutic intervention

  • Connections between metabolism and virulence factor expression

• Biotechnological applications:

  • Development of L. biflexa as a metabolic engineering platform

  • Potential bioremediation applications exploiting Leptospira's metabolic versatility

  • Biosynthetic production of valuable compounds through engineered metabolic pathways