Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni Lipoprotein-releasing system ATP-binding protein LolD (lolD)

What is LolD and what is its role in Leptospira interrogans?

LolD is a critical component of the ABC transporter complex LolCDE involved in the translocation of mature outer membrane-directed lipoproteins from the inner membrane to the periplasmic chaperone, LolA. In L. interrogans serogroup Icterohaemorrhagiae serovar copenhageni (strain Fiocruz L1-130), LolD is responsible for the formation of the LolA-lipoprotein complex in an ATP-dependent manner. This 237-amino acid protein belongs to the ABC transporter superfamily, specifically the Lipoprotein translocase (TC 3.A.1.125) family, and has a molecular mass of approximately 26.9 kDa .

The translocation process is essential for the proper localization of lipoproteins, which are major components of the leptospiral outer membrane (OM) and play crucial roles in leptospiral physiology and pathogenesis . Given that L. interrogans has 21 ABC efflux systems, including lipoprotein-releasing systems among its 220 transport proteins, LolD represents an important element of bacterial membrane organization .

How does the lipoprotein transport system function in Leptospira?

The lipoprotein transport pathway in Leptospira involves multiple coordinated steps:

• Initial protein export: Proteins with amino-terminal signal peptides, including lipoproteins, are exported across the inner membrane by the Sec translocase complex, which has orthologs of all essential components present in Leptospira .

• Lipoprotein processing: Upon reaching the periplasm, lipoproteins undergo processing by a series of three enzymes:

  • Lipoprotein diacylglyceryl transferase (Lgt): Attaches a diacyl group containing two fatty acids to the sulfhydryl residue of cysteine via a thioester linkage

  • Lipoprotein signal peptidase (Lsp): Removes the signal peptide, making cysteine the N-terminal amino acid

  • Lipoprotein N-acyl transferase (Lnt): Adds a third fatty acid to the amino residue of cysteine via an amide bond

• Lipoprotein sorting and transport: The LolCDE complex (including LolD) extracts lipoproteins from the inner membrane and transfers them to the periplasmic chaperone LolA in an ATP-dependent process, where LolD is specifically responsible for ATP hydrolysis that powers this transfer .

What experimental approaches are used to express recombinant LolD protein?

To express recombinant LolD from L. interrogans, researchers typically employ the following methodological approach:

• Gene cloning: The lolD gene (without its signal peptide) is amplified from L. interrogans genomic DNA using PCR with specific primers containing appropriate restriction sites. This approach is similar to that used for other leptospiral protein studies .

• Expression vector construction: The amplified gene is cloned into expression vectors such as pET-28a(+), which adds a His-tag for purification. Alternative approaches include expression as a fusion with glutathione-S-transferase (GST) as demonstrated for other leptospiral proteins .

• Expression conditions: The recombinant protein is typically expressed in E. coli BL21(DE3) cells induced with IPTG. Optimization of expression conditions may include testing different temperatures (typically 18-37°C), IPTG concentrations, and induction times .

• Protein purification: The recombinant protein can be purified using affinity chromatography (Ni-NTA for His-tagged proteins or glutathione sepharose for GST fusion proteins), followed by optional additional purification steps such as ion exchange or size exclusion chromatography .

The expression and purification protocol should yield approximately 10 mg of purified protein per liter of culture, comparable to the yields reported for other recombinant leptospiral proteins .

How can researchers assess the ATP-binding and hydrolysis activity of recombinant LolD?

To characterize the ATP-binding and hydrolysis activity of recombinant LolD, researchers can employ several complementary approaches:

• Colorimetric ATPase assays: Malachite green-based assays can quantify phosphate release during ATP hydrolysis. Typically, purified recombinant LolD (1-5 μg) is incubated with ATP (0.1-5 mM) in a buffer containing magnesium (essential for activity) at 37°C. The reaction is stopped at various time points using acidic malachite green solution, and absorbance is measured at 620-650 nm .

• Isothermal titration calorimetry (ITC): This technique can determine the binding affinity (Kd), stoichiometry, and thermodynamic parameters of ATP binding to LolD. Typically, the protein solution (20-50 μM) is placed in the sample cell, and ATP solution (0.5-1 mM) is added in small aliquots while measuring heat changes .

• ATP-binding assays using fluorescent ATP analogs: ATP analogs such as TNP-ATP (2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate) exhibit increased fluorescence when bound to proteins, allowing for direct measurement of binding kinetics.

• Site-directed mutagenesis: Mutating key residues in the Walker A motif (GSGKS in LolD ) can confirm their role in ATP binding and hydrolysis. Mutations typically target the conserved lysine residue, which is essential for ATP binding in ABC transporters.

For example, experiments with the recombinant protein LIC13086 from L. interrogans used circular dichroism spectroscopy to assess structural changes upon adding divalent cations like Ca²⁺ and Mg²⁺, which could be adapted to study how metal ions affect LolD's ATP-binding capability .

What strategies can be employed to study LolD interactions with other components of the LolCDE complex?

Investigating LolD interactions within the LolCDE complex requires specialized approaches:

• Bacterial two-hybrid (B2H) system: The coding sequences of LolD, LolC, and LolE can be cloned into B2H vectors to detect protein-protein interactions in a bacterial host. This approach has been used to study protein interactions in Leptospira .

• Co-immunoprecipitation (Co-IP): Anti-LolD antibodies can be used to pull down the entire LolCDE complex from L. interrogans membrane preparations, followed by Western blot analysis using antibodies against LolC and LolE to confirm interactions.

• Surface plasmon resonance (SPR): Purified recombinant LolD can be immobilized on a sensor chip, and solutions containing purified LolC and/or LolE are flowed over the surface to measure binding kinetics and affinity. This technique has been used to study the interaction of leptospiral proteins with host components .

• Isothermal titration calorimetry (ITC): As with ATP binding, ITC can be used to measure the thermodynamic parameters of LolD binding to other Lol components.

• Chemical cross-linking and mass spectrometry: This approach can identify interaction interfaces between LolD and other components of the complex. Purified proteins or membrane preparations are treated with cross-linking reagents, followed by proteolytic digestion and mass spectrometric analysis .

• Structural studies: X-ray crystallography or cryo-EM can provide detailed structural information about the LolCDE complex. Researchers could adapt methods similar to those used for structural characterization of other leptospiral proteins .

How can CRISPR interference (CRISPRi) be adapted to study lolD function in Leptospira interrogans?

CRISPRi has recently been adapted for use in Leptospira and can be employed to study LolD function through the following methodological approach:

• sgRNA design: Design single guide RNAs (sgRNAs) targeting the lolD gene. Typically, sgRNAs targeting the non-template strand near the 5' end of the gene provide the best silencing efficiency. Multiple sgRNAs should be designed and tested to identify the most effective one .

• CRISPRi system construction: Clone the sgRNA into a CRISPRi vector containing a catalytically inactive Cas9 (dCas9) under the control of a suitable promoter. The system developed for silencing LipL32 and LigA/LigB in L. interrogans can be adapted for LolD .

• Transformation: Introduce the CRISPRi constructs into L. interrogans through conjugation with E. coli carrying the construct, as described for other leptospiral genes .

• Verification of gene knockdown: Confirm reduced LolD expression using:

  • RT-qPCR to quantify lolD mRNA levels

  • Western blot with anti-LolD antibodies to assess protein levels

  • Functional assays measuring lipoprotein translocation efficiency

• Phenotypic analysis: Examine changes in:

  • Growth rate and morphology

  • Lipoprotein localization using fractionation and immunoblotting

  • Membrane integrity using dye exclusion assays

  • Virulence using animal models

Prior work has shown that CRISPRi can achieve significant knockdown of target genes in L. interrogans without adverse effects on bacterial viability or LPS structure, making it suitable for studying essential genes like lolD .

What approaches can be used to investigate LolD's contribution to L. interrogans virulence and pathogenesis?

To investigate LolD's role in virulence, researchers can employ several complementary approaches:

• Gene knockdown and overexpression: Use CRISPRi to knockdown lolD expression or introduce a plasmid for controlled overexpression, then assess effects on:

  • Survival in host cells (macrophages, epithelial cells)

  • Resistance to complement-mediated killing

  • Adhesion to extracellular matrix components

  • Biofilm formation capability

• Lipoprotein profiling: Compare the outer membrane proteome between wild-type and LolD-depleted strains using mass spectrometry-based proteomics to identify lipoproteins that depend on LolD for proper localization .

• Animal infection models: Assess the virulence of LolD-depleted strains in hamster or mouse models of leptospirosis, examining:

  • LD50 values

  • Organ colonization (particularly kidneys and liver)

  • Histopathological changes

  • Bacterial load in blood and tissues using qPCR targeting the lipL32 gene

• Transcriptome analysis: Compare gene expression patterns between wild-type and LolD-depleted strains during infection or under stress conditions using RNA-seq .

• Immune response analysis: Evaluate how LolD depletion affects the interaction with host immune components, particularly:

  • TLR2 and TLR4 signaling

  • Complement system evasion

  • NOD1 and NOD2 recognition

  • Inflammasome activation

• Environmental stress resistance: Test how LolD depletion affects survival under conditions mimicking host environments (pH fluctuations, oxidative stress, temperature shifts) .

How does LolD expression vary under different environmental conditions relevant to L. interrogans pathogenesis?

To investigate LolD expression regulation under different environmental conditions, researchers can use the following methodological approaches:

• Quantitative RT-PCR: Design primers specific to the lolD gene to quantify transcript levels under different conditions, normalizing to housekeeping genes like 16S rRNA or rpoB . Conditions to test include:

  • Growth in standard EMJH medium versus serum-supplemented media

  • Iron limitation (a key host environment signal)

  • Different temperatures (28°C for environmental survival versus 37°C for mammalian host)

  • pH variations (pH 6.0-7.5)

  • Oxidative stress conditions (H₂O₂ exposure)

• Protein quantification: Use Western blotting with anti-LolD antibodies or targeted proteomics (selected reaction monitoring) to measure LolD protein levels under different conditions .

• Reporter gene assays: Fuse the lolD promoter region to a reporter gene like GFP or luciferase to monitor promoter activity under different conditions in real-time.

• Global proteome analysis: Compare LolD expression in the context of the global proteome under different conditions using iTRAQ or similar quantitative proteomics techniques, as has been done for other leptospiral proteins .

• Transcriptome analysis: Perform RNA-seq under different environmental conditions to identify transcriptional networks that regulate lolD expression along with other virulence-associated genes .

Prior studies have shown that virulent leptospires undergo complex changes in protein expression profiles in response to shifts to in vivo-like environments, with proteins involved in motility, outer membrane organization, and virulence being differentially regulated .

What structural bioinformatics approaches can help predict functional domains and potential inhibitor binding sites in LolD?

To identify functional domains and potential inhibitor binding sites in LolD, researchers can employ the following computational approaches:

• Primary sequence analysis: Analyze the LolD sequence (MQFVNSGISVTDLRKTFGNSEIIKGVTLDIEDGDYVSLTGKSGSGKSTLLYMISSLDPPSSGTIKIDKKDIYRMNEEEIHEFRNKRMGFIFQFHYLLPEFTAIENVLMPARKAGLLKEYQSYAEHLLEEFDLKDRMNYRINRLSGGQAQRVAIARALVMNPKYIFADEPTGALDSTNTKVVMNILEKVNRETKTTILVVTHDPDFASKTKRQIHLVDGRVVSLKEFEAIKKSAKTAR) to identify:

  • Walker A motif (GSGKS): Essential for ATP binding

  • Walker B motif: Required for ATP hydrolysis

  • ABC signature motif: Characteristic of ABC transporters

  • Q-loop and H-loop: Involved in coordinating ATP hydrolysis with substrate transport

• Homology modeling: Create a 3D structural model of LolD based on crystal structures of related ABC transporter NBDs (Nucleotide Binding Domains) using software like SWISS-MODEL, Phyre2, or I-TASSER. These models can reveal:

  • The spatial arrangement of ATP-binding residues

  • Potential dimer interface residues

  • Interaction surfaces with other Lol components

• Molecular dynamics simulations: Simulate the dynamics of LolD, particularly:

  • Conformational changes during ATP binding and hydrolysis

  • Stability of the protein under different conditions

  • Effects of mutations on protein structure

• Virtual screening: Identify potential small molecule inhibitors by:

  • Structure-based virtual screening against the ATP-binding pocket

  • Pharmacophore-based screening targeting key functional motifs

  • Fragment-based approaches to identify scaffolds for further optimization

• Protein-protein docking: Model interactions between LolD and other components of the Lol system (LolC, LolE, LolA) to identify key interaction interfaces that could be targeted by inhibitors.

• Pocket analysis: Identify druggable pockets beyond the ATP-binding site using tools like fpocket, SiteMap, or DoGSiteScorer, focusing on regions unique to bacterial LolD compared to human ABC transporters.

This methodological approach has been successful in identifying potential inhibitors against other bacterial ABC transporters, which share functional similarities with LolD.

What methods can be used to investigate the impact of LolD inhibition on outer membrane composition and virulence of L. interrogans?

To assess how LolD inhibition affects outer membrane composition and virulence, researchers can employ the following approach:

• Selective inhibition of LolD:

  • Use CRISPRi to create a conditional knockdown of lolD

  • Design specific inhibitors targeting the ATP-binding site based on structural predictions

  • Create temperature-sensitive mutants by site-directed mutagenesis

• Outer membrane composition analysis:

  • Perform membrane fractionation to isolate the outer membrane

  • Analyze protein content using 2D gel electrophoresis followed by mass spectrometry

  • Quantify specific lipoproteins (e.g., LipL32, LipL41, LipL48) using immunoblotting

  • Examine LPS composition and structure using silver staining and mass spectrometry

• Functional membrane assays:

  • Assess membrane integrity using fluorescent dyes (e.g., propidium iodide)

  • Measure sensitivity to detergents and antimicrobial peptides

  • Evaluate resistance to complement-mediated killing

• Virulence factor expression and function:

  • Analyze expression of known virulence factors such as LigA/LigB, LipL32, and LRR proteins

  • Assess the ability of cells to bind to extracellular matrix components (laminin, E-cadherin)

  • Measure plasminogen binding and activation, which contributes to tissue invasion

• In vivo virulence assessment:

  • Compare the LD50 of wild-type and LolD-depleted strains in animal models

  • Quantify bacterial loads in tissues using qPCR or culture methods

  • Examine histopathological changes in infected tissues

  • Measure inflammatory markers and immune responses

This methodological approach can reveal how LolD-dependent lipoprotein transport affects multiple aspects of leptospiral pathogenesis, from membrane composition to host interaction and virulence.

Leptospira interrogans and Leptospirosis Research Timeline