Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni UPF0109 protein LIC_11556 (LIC_11556)

How should LIC_11556 recombinant protein be stored for optimal stability?

The shelf life of LIC_11556 recombinant protein depends on several factors including storage state, buffer ingredients, storage temperature, and the inherent stability of the protein. For optimal results:

• Store lyophilized form at -20°C/-80°C, where it typically maintains stability for up to 12 months

• Store liquid preparations at -20°C/-80°C for up to 6 months

• After reconstitution, create working aliquots stored at 4°C that should be used within one week

• Avoid repeated freeze-thaw cycles as these significantly decrease protein stability

When preparing aliquots for long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being the manufacturer's default recommendation) to prevent freeze-thaw damage .

What is the recommended protocol for reconstituting lyophilized LIC_11556?

For optimal reconstitution of lyophilized LIC_11556 protein:

• Briefly centrifuge the vial prior to opening to bring all contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being standard)

• Aliquot the solution into smaller volumes for storage to minimize freeze-thaw cycles

• Store aliquots at -20°C/-80°C for long-term storage

This method ensures maximum retention of protein activity and extends shelf life. The specific reconstitution volume should be calculated based on the protein amount and desired final concentration for your experimental applications.

How can I verify the purity and integrity of recombinant LIC_11556 before using it in experiments?

To verify the purity and integrity of recombinant LIC_11556:

• SDS-PAGE Analysis: Run the protein on a gel to confirm the expected molecular weight of approximately 8.7 kDa and assess purity (should be >85% as specified by suppliers)

• Western Blot Analysis: Use anti-His tag antibodies (if the protein contains a His tag) or specific antibodies against LIC_11556 to confirm identity

• Mass Spectrometry: For more precise molecular weight confirmation and to identify any post-translational modifications or truncations

• Functional Assays: Though the specific function of UPF0109 proteins is not fully characterized, comparative binding assays similar to those used for other leptospiral proteins can help verify functional integrity

A typical verification protocol should include at minimum SDS-PAGE analysis under both reducing and non-reducing conditions to assess both purity and potential oligomerization states.

What are the recommended control proteins when studying LIC_11556 in experimental settings?

When designing experiments involving LIC_11556, appropriate controls are essential for result validation:

• Positive Controls:

  • Other characterized leptospiral proteins like LipL32, which has well-documented immunoreactivity

  • Functionally characterized UPF0109 family proteins from related bacterial species

• Negative Controls:

  • Recombinant LipL36, which is downregulated during infection and shows minimal reactivity with patient sera

  • Unrelated bacterial proteins of similar size expressed in the same system

  • Tag-only proteins (if LIC_11556 is expressed with tags)

• Internal Controls:

  • Housekeeping proteins from Leptospira for comparison in expression studies

  • Recombinant LIC_10976, another UPF protein from the same organism, for comparative binding studies

The choice of controls should be tailored to the specific research question. For immunological studies, LipL32 serves as an excellent positive control due to its high immunoreactivity (94% sensitivity in convalescent phase sera), while LipL36 provides a good negative control .

What is known about the immunogenicity of LIC_11556 compared to other leptospiral proteins?

While specific immunogenicity data for LIC_11556 is limited in the available literature, we can make inferences by comparison with other leptospiral proteins:

The immunogenicity profile of leptospiral proteins varies significantly:

• LipL32 demonstrates high immunogenicity with 56% sensitivity in acute phase and 94% in convalescent phase of leptospirosis

• OmpL1, LipL41, and Hsp58 show lower sensitivities of 16%, 24%, and 18% during acute phase and 72%, 44%, and 32% during convalescence, respectively

• Lsa19 (LIC11122) shows significant recognition by antibodies from leptospirosis patients, while Lsa14 (LIC12287) shows lesser reactivity

• LIC_10713 (imelysin-like protein) demonstrates high immunogenicity with 90.9% sensitivity and 100% specificity in patient samples

How can I develop an ELISA-based detection system for LIC_11556?

Developing an ELISA system for LIC_11556 requires optimization of multiple parameters:

Protocol Development:

• Antigen Concentration Optimization:

  • Test multiple concentrations (5-100 ng/well) as different leptospiral proteins show varying optimal concentrations (e.g., rLipL32 performs best at 25 ng/well while rOmpL1 shows decreasing absorbance above 5 ng/well)

  • Create a dose-response curve to identify the optimal concentration

• Serum Dilution Optimization:

  • Test serial dilutions (typically 1:100 to 1:3,200) to determine optimal signal-to-noise ratio

  • Different classes of antibodies (IgG vs. IgM) may require different dilutions

• Buffer Composition:

  • PBS with 1-5% BSA or casein as blocking agent

  • Include 0.05% Tween-20 in wash buffers

• Control Selection:

  • Use sera from healthy individuals from endemic regions to establish specificity cutoffs (96% specificity is the standard used for other leptospiral proteins)

  • Include cross-reactivity controls from patients with dengue, hepatitis, Lyme disease, and syphilis

An example ELISA optimization matrix based on protocols used for other leptospiral proteins:

What computational approaches can be used to predict the structure and function of the uncharacterized LIC_11556 protein?

For UPF0109 family proteins like LIC_11556, several computational approaches can provide insights into structure and function:

• Homology Modeling:

  • Use structures of related UPF0109 proteins as templates

  • Software options include SWISS-MODEL, Phyre2, and I-TASSER

  • Validate models using PROCHECK or MolProbity

• Sequence-Based Function Prediction:

  • Conserved domain analysis using CDD, PFAM, or InterPro

  • Motif scanning for functional sites using PROSITE or ELM

  • GO term prediction using tools like DeepGOPlus

• Structural Bioinformatics:

  • Active site prediction using CASTp or POOL

  • Protein-protein interaction surface prediction using PredUs or SPPIDER

  • Molecular dynamics simulations to understand flexibility and potential binding modes

• Comparative Genomics:

  • Synteny analysis to identify conserved gene neighborhoods

  • Co-evolution analysis to identify potential interaction partners

  • Phylogenetic profiling to infer functional associations

The integrative approach combining these methods can provide testable hypotheses about LIC_11556 function. Since other leptospiral proteins like LruB (LIC_10713) have been characterized as adhesins that interact with extracellular matrix components , similar analyses for LIC_11556 may reveal potential binding properties.

How can I determine if LIC_11556 is surface-exposed in Leptospira interrogans?

Determining surface exposure of leptospiral proteins is critical for understanding their potential roles in host-pathogen interactions. For LIC_11556, several complementary approaches can be used:

• Proteinase K Accessibility Assay:

  • Treat intact bacteria with Proteinase K

  • Compare protein levels before and after treatment using Western blot

  • Surface-exposed proteins will be degraded (similar to methodology used for Lsa19 and Lsa14)

• Immunofluorescence Microscopy:

  • Generate antibodies against recombinant LIC_11556

  • Perform immunofluorescence on fixed but non-permeabilized bacteria

  • Compare with permeabilized controls

• Surface Biotinylation:

  • Label intact bacteria with a membrane-impermeable biotinylation reagent

  • Purify biotinylated proteins and detect LIC_11556 by Western blot

• Subcellular Fractionation:

  • Separate inner membrane, outer membrane, periplasmic, and cytoplasmic fractions

  • Identify LIC_11556 location by Western blot

  • Include known compartment markers as controls

The subcellular localization will provide clues about function - outer membrane or surface-exposed proteins often function in adhesion, immune evasion, or nutrient acquisition.

How can I investigate potential interactions between LIC_11556 and host extracellular matrix (ECM) components?

Many leptospiral proteins interact with host ECM components, which may be crucial for adhesion and colonization. To investigate whether LIC_11556 shares these properties:

• ELISA-Based Binding Assays:

  • Coat plates with purified ECM components (laminin, fibronectin, collagen types I and IV)

  • Add varying concentrations of recombinant LIC_11556

  • Detect binding using anti-LIC_11556 or anti-tag antibodies

  • Include dose-dependent saturation analysis to confirm specific binding

• Surface Plasmon Resonance (SPR):

  • Immobilize either LIC_11556 or ECM components on sensor chips

  • Measure real-time binding kinetics (ka, kd, KD)

  • Perform competition assays to determine binding specificity

• Pull-Down Assays:

  • Immobilize recombinant LIC_11556 on appropriate resin

  • Incubate with soluble ECM components or host cell lysates

  • Identify binding partners by Western blot or mass spectrometry

• Cell Adhesion Inhibition Assays:

  • Pre-incubate host cells with recombinant LIC_11556

  • Challenge with Leptospira

  • Quantify adhesion inhibition compared to controls

Based on studies with other leptospiral proteins like Lsa19, Lsa14, and LIC_10713, which demonstrated binding to laminin, fibronectin, and collagens in a dose-dependent manner , similar methodologies can be applied to investigate LIC_11556's potential interactions.

What approaches can be used to generate knockout or knockdown models of LIC_11556 to study its function in vivo?

Creating genetic manipulation models for LIC_11556 requires specialized techniques for Leptospira:

• Homologous Recombination:

  • Design construct with antibiotic resistance cassette flanked by LIC_11556 upstream and downstream regions

  • Transform into Leptospira using electroporation

  • Select on appropriate antibiotics

  • Confirm gene disruption by PCR and Western blot

• Transposon Mutagenesis:

  • Use random transposon insertion to generate a library of mutants

  • Screen for insertions in LIC_11556 by PCR

  • Phenotypically characterize the identified mutants

• CRISPR-Cas9 System:

  • Design sgRNAs targeting LIC_11556

  • Introduce Cas9 and sgRNA into Leptospira

  • Screen for successful editing events

  • Validate knockout by sequencing and Western blot

• Antisense RNA Approach:

  • Design antisense RNA constructs targeting LIC_11556 mRNA

  • Express in Leptospira under inducible promoter

  • Confirm knockdown by qRT-PCR and Western blot

• Phenotypic Analysis of Mutants:

  • Assess growth kinetics in various media

  • Evaluate virulence in hamster model (commonly used for Leptospira)

  • Test adhesion to ECM components and host cells

  • Compare immune response elicitation versus wild-type

Note that genetic manipulation of pathogenic Leptospira remains challenging. The biological significance of proteins like LIC_11556 ultimately requires these genetic approaches, as emphasized in studies of other leptospiral proteins like Lsa19 and Lsa14 .

How does LIC_11556 compare phylogenetically with other UPF proteins in Leptospira species?

Phylogenetic analysis of LIC_11556 within the context of other UPF proteins can provide evolutionary insights:

• Sequence Conservation Analysis:

  • Compare LIC_11556 with homologs in other Leptospira serovars and species

  • Calculate sequence identity and similarity percentages

  • Identify conserved residues potentially crucial for function

• Phylogenetic Tree Construction:

  • Align LIC_11556 with homologs using MUSCLE or CLUSTAL

  • Build trees using Maximum Likelihood or Bayesian methods

  • Root trees with homologs from distantly related bacteria

• Distribution Across Pathogenicity Groups:

  • Analyze presence/absence patterns across pathogenic, intermediate, and saprophytic Leptospira

  • Similar to analysis performed for LIC_10713, which was found predominantly in pathogenic species

• Synteny Analysis:

  • Examine conservation of genomic context around LIC_11556

  • Identify co-evolved genes that may function together

Understanding phylogenetic distribution could indicate whether LIC_11556 is associated with virulence. For instance, proteins like imelysin (LIC_10713) that are predominantly found in pathogenic Leptospira species may play roles in pathogenesis .

What experimental design would best determine if LIC_11556 has potential as a vaccine candidate?

Evaluating LIC_11556 as a potential vaccine candidate requires a systematic approach:

• Immunogenicity Assessment:

  • Immunize hamsters or guinea pigs with recombinant LIC_11556

  • Measure antibody titers using ELISA

  • Characterize antibody isotypes (IgG, IgM)

  • Assess T-cell responses (Th1/Th2 balance)

• Challenge Studies:

  • Immunize animals with recombinant LIC_11556 with appropriate adjuvant

  • Challenge with virulent L. interrogans

  • Monitor survival rates, bacterial burden, and histopathological changes

  • Compare with non-immunized controls and established vaccine candidates

• Cross-Protection Evaluation:

  • Challenge immunized animals with heterologous Leptospira serovars

  • Assess protection breadth compared to conventional vaccines

• Combination with Other Antigens:

  • Test LIC_11556 in combination with established immunogens like LigA

  • Evaluate for synergistic protection

• Immune Response Characterization:

  • Determine if protection is antibody-mediated or cell-mediated

  • Analyze cytokine profiles (similar to studies showing Th1 response for serovar Hardjo protection)

This approach mirrors successful studies with other leptospiral proteins, such as LigA, which demonstrated 100% survival in immunized hamsters challenged with L. interrogans serovar Pomona, while control animals developed severe nephritis or died .

What are the most promising research directions for further characterizing LIC_11556?

Based on the available data on LIC_11556 and related proteins, several promising research directions emerge:

• Structural Biology:

  • Determine the 3D structure using X-ray crystallography or cryo-EM

  • Map functional domains and potential binding sites

  • Compare with structures of functionally characterized UPF0109 proteins

• Host-Pathogen Interaction Studies:

  • Investigate interactions with host cells and ECM components

  • Determine if LIC_11556 plays a role in adhesion or immune evasion

  • Explore potential moonlighting functions (multiple biological roles)

• Expression Regulation:

  • Examine expression patterns during different growth phases

  • Determine if expression is upregulated during infection, like LIC_10713

  • Identify environmental triggers that modify expression

• Genetic Manipulation:

  • Generate knockout mutants to determine virulence effects

  • Perform complementation studies to confirm phenotypes

  • Create site-directed mutants to identify critical residues

• Diagnostic and Vaccine Potential:

  • Evaluate immunoreactivity with patient sera at different disease stages

  • Assess protective immunity in animal models

  • Test as part of multi-antigen vaccine formulations

These directions would address the significant knowledge gap regarding UPF0109 proteins in Leptospira and potentially reveal novel targets for diagnostic and therapeutic interventions against leptospirosis.

How can I design experiments to resolve contradictory data about LIC_11556 function?

When facing contradictory data about LIC_11556 function, a systematic approach is required:

• Replication with Standardized Methods:

  • Standardize protein preparation (expression system, purification method, storage)

  • Use consistent experimental conditions across laboratories

  • Implement blinded analysis to minimize bias

• Multi-Method Validation:

  • Employ orthogonal techniques to test the same hypothesis

  • For binding studies: combine ELISA, SPR, and cell-based assays

  • For localization: combine fractionation, immunofluorescence, and protease accessibility

• Strain and Serovar Comparison:

  • Test function across multiple L. interrogans strains

  • Compare results between different serovars

  • Consider host-specificity factors

• Context-Dependent Function Assessment:

  • Test under varying physiological conditions (pH, temperature, ion concentration)

  • Evaluate function during different growth phases

  • Compare in vitro versus in vivo results

• Collaborative Cross-Validation:

  • Establish multi-laboratory studies with standardized protocols

  • Share reagents (recombinant proteins, antibodies) between research groups

  • Create a consensus methodology for future studies