Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Acylphosphatase (acyP)

How does acyP fit into the broader context of Leptospira interrogans metabolism?

While specific metabolic pathway analysis for acyP in L. interrogans is limited in the literature, its function can be contextualized within the bacterium's unique metabolic profile. L. interrogans requires CO2 for in vitro growth and employs a partial 3-hydroxypropionate pathway involving two acyl-CoA carboxylases for CO2 assimilation .

Acylphosphatase activity may intersect with these pathways by modulating the levels of acylphosphates, which are high-energy intermediates in various metabolic processes. The bacterium demonstrates both acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) activities, with a tri-subunit holoenzyme (LA_2736-LA_2735 and LA_3803) showing preference for acetyl-CoA and a bi-subunit holoenzyme (LA_2432-LA_2433) preferentially acting on propionyl-CoA .

When growing under host-like conditions (iron limitation and serum presence), L. interrogans exhibits significant changes in protein expression patterns, potentially including metabolic enzymes like acyP, as revealed by global proteome analysis using iTRAQ and LC-ESI-tandem mass spectrometry complemented with two-dimensional gel electrophoresis .

What are the optimal conditions for expressing recombinant L. interrogans acyP in E. coli?

Based on established protocols for recombinant L. interrogans proteins, the following methodology is recommended:

• Vector selection: Use expression vectors with strong, inducible promoters such as pET-based systems for laboratory-scale production.

• Host strain: BL21(DE3) or Rosetta-2 E. coli strains are recommended, particularly if the target gene contains rare codons .

• Expression conditions:

  • Culture temperature: 20°C for 16 hours post-induction (based on protocols for other leptospiral proteins)

  • IPTG concentration: 0.5 mM

  • Media: LB supplemented with appropriate antibiotics

• Protein extraction:

  • Cell lysis via sonication in appropriate buffer

  • Addition of benzonase (150 U/mL) to reduce viscosity from nucleic acids

  • Clarification by centrifugation at 16,000 g

• Purification strategy:

  • For His-tagged constructs, use immobilized metal affinity chromatography (IMAC)

  • Consider including 10-50% glycerol in storage buffers to enhance stability

Protocols may need optimization based on specific construct designs and experimental requirements.

What techniques are most effective for assessing acyP enzymatic activity in vitro?

The enzymatic activity of recombinant acyP can be assessed using multiple complementary approaches:

• Spectrophotometric assays:

  • Measure the release of inorganic phosphate from acylphosphate substrates

  • Monitor absorbance at 340 nm when coupled with auxiliary enzymes that utilize NADH

  • Typical substrates include benzoyl phosphate or acetyl phosphate

• Coupled enzyme assays:

  • Link acylphosphate hydrolysis to pyruvate kinase and lactate dehydrogenase reactions

  • Monitor NADH oxidation at 340 nm

• Direct measurement protocols:

  • Use malachite green assay to quantify released phosphate

  • Consider using isothermal titration calorimetry (ITC) for binding and kinetic studies

• Assessment of in vivo activity:

  • Compare wild-type L. interrogans with genetically modified strains using CRISPR/Cas9 and NHEJ from M. smegmatis for targeted genetic manipulation

For optimal results, maintain assay temperatures at 30°C (the growth temperature of Leptospira) and pH consistent with leptospiral physiology (pH 7.2-7.4).

How might acyP be involved in L. interrogans pathogenesis and host-pathogen interactions?

The potential role of acyP in L. interrogans pathogenesis can be examined through multiple perspectives:

• Metabolic adaptation during infection:

  • Transcriptomic studies show that L. interrogans significantly alters expressions of genes involved in carbohydrate and lipid metabolism, energy production, and signal transduction when interacting with host macrophages

  • acyP may contribute to these metabolic shifts, enabling adaptation to host environments

• Protective mechanisms against host defenses:

  • L. interrogans evades host immune responses through various mechanisms, including preventing macrophage cell death and escaping both pyroptosis and apoptosis

  • Metabolic enzymes like acyP may indirectly support these evasion strategies by maintaining energy homeostasis under stress conditions

• Potential moonlighting functions:

  • Some metabolic enzymes in pathogens exhibit secondary "moonlighting" functions in virulence

  • acyP's small size (95 amino acids) and potential localization suggest it might interact with host molecules or other bacterial components beyond its primary enzymatic function

• Comparative analysis with other pathogens:

  • In other bacterial pathogens, metabolic enzymes often play roles in virulence and adaptation

  • Similar functions may exist for acyP in L. interrogans

Investigation of these possibilities requires techniques such as:

• Gene knockdown using CRISPR interference (CRISPRi) systems adapted for Leptospira

• Protein-protein interaction studies to identify potential binding partners

• Immunolabeling to determine localization during infection

What potential exists for acyP as a therapeutic target or vaccine component?

Several lines of evidence suggest acyP could be evaluated as a therapeutic target or vaccine component:

• Target for antimicrobial development:

  • If essential for L. interrogans survival or virulence, acyP inhibitors could be developed

  • Structure-based drug design approaches could utilize the protein's known sequence and predicted structure

  • Small-molecule screening assays could identify potential inhibitors

• Vaccine development considerations:

  • Current leptospirosis vaccine development focuses on outer membrane proteins (OMPs) like LipL32, LipL41, and OmpL1

  • While acyP is not an OMP, it could be considered as part of a multi-component subunit vaccine if immunogenic

• Precedent from other bacterial systems:

  • Metabolic enzymes have been used successfully as vaccine components in other bacterial infections

  • Chimeric constructs combining epitopes from different proteins offer enhanced protection, as demonstrated with LigB domains

• Evidence from structural and immunological studies:

  • Extended low-resolution structures of leptospiral antigens have been used to design chimeric vaccines with improved protection

  • Similar approaches could be applied to acyP if structurally characterized

Research in this direction would require:

• Immunogenicity studies in animal models

• Protection assays following challenge with virulent L. interrogans

• Evaluation of cross-protection against different serovars

What are common challenges in purifying active recombinant acyP and how can they be addressed?

Researchers frequently encounter several challenges when purifying recombinant acyP:

• Protein solubility issues:

  • Challenge: Recombinant acyP may form inclusion bodies in E. coli

  • Solution: Express at lower temperatures (16-20°C), reduce inducer concentration, or use solubility-enhancing fusion tags like SUMO or MBP

• Protein stability concerns:

  • Challenge: acyP may show reduced stability after purification

  • Solution: Include 5-50% glycerol in storage buffers and store at -20°C/-80°C to maintain activity

• Enzymatic activity preservation:

  • Challenge: Loss of activity during purification steps

  • Solution: Minimize freeze-thaw cycles, as repeated freezing and thawing is not recommended

• Contamination with host proteins:

  • Challenge: Co-purification of E. coli proteins

  • Solution: Implement additional purification steps such as ion exchange or size exclusion chromatography after initial affinity purification

• Expression toxicity:

  • Challenge: Some leptospiral proteins show toxicity when expressed in E. coli, leading to impaired growth (as observed with LipL21, where growth rate was significantly reduced)

  • Solution: Use tightly regulated expression systems or consider alternative expression hosts

A systematic optimization approach is recommended, testing multiple expression conditions and purification strategies to identify optimal protocols for specific research applications.

How can researchers effectively analyze acyP interactions with other leptospiral proteins or host targets?

Multiple complementary approaches can be employed to study acyP interactions:

• In vitro protein-protein interaction assays:

  • Pull-down assays: Using tagged recombinant acyP to identify binding partners

  • Surface plasmon resonance (SPR): For quantifying binding kinetics and affinities

  • Isothermal titration calorimetry (ITC): For thermodynamic characterization of interactions

• Structural biology approaches:

  • X-ray crystallography: To determine atomic resolution structures of acyP alone or in complex with partners

  • Small-angle X-ray scattering (SAXS): For low-resolution structural information in solution

  • Cryo-electron microscopy: Particularly useful for larger complexes

• Cellular and in vivo approaches:

  • Bacterial two-hybrid systems: Adapted for identifying protein interactions

  • Immunoprecipitation from leptospiral lysates: To identify native interaction partners

  • Cell-surface immunoprecipitation (CSIP): A technique successfully used with leptospiral proteins

• Computational prediction methods:

  • Molecular docking simulations

  • Protein-protein interaction network analysis

  • Comparative analysis with homologous proteins

When analyzing potential host targets, researchers should consider:

• Using cell-based assays with relevant host cell types (e.g., macrophages, kidney cells)

• Comparing results across multiple mammalian species, as L. interrogans can infect various hosts with different outcomes

• Employing fluorescently labeled proteins to track localization during host-pathogen interactions

How should researchers interpret acyP enzymatic activity data in the context of L. interrogans metabolism?

When analyzing acyP enzymatic activity data, researchers should consider several contextual factors:

• Metabolic network integration:

  • Compare acyP activity with related metabolic enzymes, particularly those involved in the partial 3-hydroxypropionate pathway utilized by L. interrogans for CO2 assimilation

  • Consider that the specific activity of acetyl-CoA carboxylase (ACC) detected in crude cell extract was reported to be too low to account for bacterial growth in minimal medium, suggesting complex metabolic regulation

• Environmental influences:

  • Evaluate how enzymatic parameters (Km, Vmax, substrate specificity) change under conditions mimicking host environments

  • Consider that L. interrogans alters its metabolism during host-pathogen interactions, with significant changes in energy metabolism, oxygen tolerance, and signal transduction

• Comparative analysis framework:

  • Benchmark against enzymatic parameters from related species or other pathogenic bacteria

  • Consider acyP activity in the context of L. interrogans' unique metabolic requirements, including its dependence on CO2

• Statistical analysis recommendations:

  • Use appropriate statistical methods for enzyme kinetics data

  • Implement controls for variations in protein preparation and assay conditions

  • Follow the example of global proteome analysis studies that used stringent statistical frameworks with biological replicates (e.g., 75 independent cultures pooled for each condition)

What considerations are important when designing genetic manipulation studies involving acyP in L. interrogans?

Genetic manipulation of L. interrogans presents unique challenges that researchers should address:

• Available genetic tools:

  • CRISPR interference (CRISPRi) has been successfully applied for gene silencing in pathogenic Leptospira species, including L. interrogans

  • CRISPR/Cas9 systems with NHEJ repair mechanisms from M. smegmatis have been used to generate knockouts in L. interrogans

• Experimental design considerations:

  • Control selection: Include carefully chosen positive and negative controls

    • Example: When targeting LipL32 for knockout, researchers included controls with plasmids lacking sgRNA cassettes to demonstrate that double-strand breaks are not tolerated in L. interrogans

  • Phenotype verification: Confirm genetic modifications by multiple methods

    • Example: LipL32 knockouts were verified by both immunoblotting with anti-LipL32 antibodies and PCR amplification/sequencing of the target region

• Potential challenges:

  • Polar effects: Genetic modifications may affect neighboring genes

    • Example: In one LipL32 knockout study, a 345-bp deletion affected 42 bp of the upstream gene, although no growth defects were observed

  • Target essentiality: If acyP is essential, complete knockout may not be achievable

  • Technical limitations: Conjugation efficiencies can be heterogeneous, resulting in variable numbers of transformants

• Complementation strategies:

  • Include genetic complementation to confirm phenotypes are specifically due to acyP modification

  • Consider inducible expression systems for complementation of essential genes

• Phenotypic assessment protocols:

  • Evaluate growth under various conditions (standard media, minimal media, stress conditions)

  • Assess pathogenicity in appropriate animal models

  • Examine metabolic profiles using techniques such as metabolomics

What approaches would be most effective for elucidating the three-dimensional structure of L. interrogans acyP?

Multiple complementary structural biology techniques can be applied to determine acyP structure:

For functional interpretation, compare structural features with those of acylphosphatases from other organisms, identifying conserved catalytic residues and potential unique features of the leptospiral enzyme.

How can researchers effectively study the regulation of acyP expression in L. interrogans under different environmental conditions?

Understanding acyP regulation requires multiple experimental approaches examining both transcriptional and post-transcriptional control mechanisms:

• Transcriptional regulation analysis:

  • RT-qPCR: Measure acyP mRNA levels under various conditions

  • Transcriptome sequencing: Analyze global transcriptional responses, similar to studies examining L. interrogans responses to macrophage interaction

  • Promoter analysis: Identify regulatory elements and potential transcription factor binding sites

• Protocol for environmental condition testing:

  • Host-mimicking conditions:

    • Iron limitation: Culture in EMJH medium with iron chelators

    • Serum presence: Add 10% fetal bovine serum to culture media

    • Temperature shifts: Compare growth at 30°C (environmental) vs. 37°C (mammalian host)

  • Infection-relevant conditions:

    • Oxidative stress: Expose to sub-lethal H2O2 concentrations

    • pH variation: Test acyP expression across pH range 6.0-7.5

    • Nutrient limitation: Compare minimal vs. rich media formulations

• Protein expression quantification:

  • Western blotting: Using specific antibodies against acyP

  • Proteomics approaches:

    • iTRAQ labeling followed by LC-ESI-tandem mass spectrometry

    • Two-dimensional gel electrophoresis complemented with MALDI-TOF mass spectrometry

• Experimental design considerations:

  • Use biological replicates (pooled from multiple independent cultures)

  • Include technical replicates to control for variation in analytical methods

  • Implement appropriate statistical analysis for multi-condition comparisons

These approaches can reveal how acyP expression is modulated during the transition between environmental survival and host infection, providing insights into its potential role in leptospiral pathogenesis.