Recombinant Legionella pneumophila subsp. pneumophila UPF0145 protein lpg0197 (lpg0197)

What is the lpg0197 protein and what organism does it originate from?

The lpg0197 protein is classified as a UPF0145 family protein originating from Legionella pneumophila subspecies pneumophila, specifically the Philadelphia 1 strain (ATCC 33152 / DSM 7513). This strain is historically significant as it was isolated during the first recognized outbreak of Legionnaires' disease. The protein's function is not fully characterized, but it belongs to a family of proteins of unknown function (UPF0145) . Legionella pneumophila is an environmental bacterium and the leading cause of Legionnaires' disease, which has significant public health implications worldwide .

What are the basic biochemical properties of recombinant lpg0197?

The recombinant lpg0197 protein has the following biochemical properties:

The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and repeated freezing and thawing is not recommended. For short-term storage, working aliquots can be kept at 4°C for up to one week .

How should I design experiments to investigate the function of lpg0197 in Legionella pathogenesis?

To investigate the potential role of lpg0197 in Legionella pathogenesis, a multifaceted experimental approach is recommended:

• Gene knockout studies: Generate lpg0197 deletion mutants in L. pneumophila and assess changes in virulence using established infection models (amoeba, macrophages, or mouse models).

• Complementation assays: Reintroduce the wild-type gene to confirm phenotypic changes are specifically due to lpg0197 deletion.

• Protein interaction studies: Perform pull-down assays, yeast two-hybrid screens, or co-immunoprecipitation to identify binding partners within host cells.

• Localization studies: Use fluorescently tagged lpg0197 to determine its subcellular localization during infection.

• Comparative genomics: Analyze the conservation of lpg0197 across different sequence types (STs) of L. pneumophila, particularly focusing on the five major disease-associated STs (1, 23, 37, 47, and 62) that are responsible for nearly half of all epidemiologically unrelated Legionnaires' disease cases in northwest Europe .

Design your experiments with appropriate controls, including investigation of other UPF0145 family proteins for comparison, and consider the clonal nature of pathogenic L. pneumophila strains that has been observed in recent epidemiological studies .

What expression systems are most suitable for producing recombinant lpg0197 for structural studies?

For structural studies of lpg0197, consider the following expression systems and optimization strategies:

• E. coli-based expression: The standard approach using BL21(DE3) or derivatives is effective for basic production of lpg0197 . For enhanced solubility, consider:

  • Using fusion tags (His, GST, MBP, SUMO)

  • Optimizing induction conditions (temperature, IPTG concentration, duration)

  • Codon optimization for E. coli

  • Utilizing specialized E. coli strains for disulfide bond formation if needed

• Insect cell expression: For proteins requiring eukaryotic post-translational modifications, baculovirus expression systems may yield more properly folded protein.

• Cell-free protein synthesis: For difficult-to-express proteins, cell-free systems can provide an alternative to traditional cell-based expression.

For crystallography or cryo-EM studies, protein purity should exceed 95%, which may require additional purification steps beyond what is used for standard biochemical applications. Given the relatively small size of lpg0197 (105 amino acids), NMR spectroscopy may be particularly suitable for structural analysis, requiring 15N and 13C isotope labeling in minimal media.

How can I assess the potential role of lpg0197 in the context of recently emerged L. pneumophila clones?

To investigate lpg0197's potential contribution to the emergence of disease-associated L. pneumophila clones:

• Comparative genomic analysis: Sequence lpg0197 across multiple clinical and environmental isolates, focusing on the five sequence types (ST1, ST23, ST37, ST47, and ST62) that cause nearly half of disease cases in northwest Europe .

• SNP analysis: Determine if lpg0197 contains any single-nucleotide polymorphisms (SNPs) that correlate with increased virulence or adaptation to man-made water systems. This is particularly relevant given the observation that disease-associated sequence types show remarkably low genetic diversity, with maximum pairwise SNPs ranging from only 19 (ST47) to 127 (ST1) .

• Transcriptional profiling: Compare lpg0197 expression levels across different strains under various environmental conditions to detect potential regulatory differences.

• Functional conservation testing: Express lpg0197 variants from different sequence types in a common genetic background to assess functional differences.

• Analysis of recombination events: Determine if lpg0197 is part of genomic regions that show evidence of recombination between disease-associated sequence types, as recombination has been identified as a potential contributor to increased disease propensity .

This systematic approach can help determine whether lpg0197 plays a role in the recently observed phenomenon of emergent, globally dispersed L. pneumophila clones with enhanced disease association .

How might lpg0197 interact with host cell factors during Legionella infection?

Understanding potential interactions between lpg0197 and host cell factors requires a systematic investigation based on the protein's predicted structural features and what is known about Legionella pathogenesis mechanisms:

• Structural prediction and domain analysis: While lpg0197 is a UPF0145 family protein with unknown function, structural analysis tools can identify potential functional domains or interaction motifs. Given that Legionella encodes multiple effector proteins that interact with host processes, lpg0197 may contain domains similar to known effectors.

• Yeast two-hybrid screening: Perform targeted screens against host protein libraries, particularly focusing on components involved in:

  • Endoplasmic reticulum interaction

  • Vesicular trafficking

  • Protein synthesis machinery

  • Immune response signaling

• Proximity labeling approaches: Use BioID or APEX2 fusion proteins to identify proximal interacting partners in living cells during infection.

• Comparison with other Legionella effectors: Consider potential functional redundancy with known effectors that modify eukaryotic elongation factors, such as Lgt1, which glucosylates eEF1A at serine 53 and inhibits protein synthesis .

• Investigation of potential post-translational modifications: Examine whether lpg0197 undergoes modifications in host cells or whether it modifies host proteins, similar to how Lgt1 functions as a glucosyltransferase targeting host eEF1A .

These approaches can help determine whether lpg0197 contributes to Legionella's ability to manipulate host cellular functions during infection cycles.

What is the evolutionary history of lpg0197 and how does it compare across Legionella species?

To understand the evolutionary context of lpg0197:

This evolutionary perspective can provide insights into whether lpg0197 contributes to the adaptation of L. pneumophila to new environmental niches, potentially including man-made water systems that have been hypothesized to play a role in the emergence of disease-associated clones .

How does lpg0197 expression change under different environmental conditions relevant to Legionella's lifecycle?

To characterize lpg0197 expression patterns:

• Transcriptomic analysis across lifecycle stages:

  • Exponential vs. stationary growth phases

  • Intracellular vs. extracellular environments

  • Growth in amoebae vs. macrophages

  • Planktonic vs. biofilm states

  • Various temperatures (environmental, human host)

• Promoter analysis and regulation:

  • Identify transcription factor binding sites

  • Construct reporter gene fusions to monitor expression

  • Evaluate the role of quorum sensing in regulation

  • Assess impact of nutrient availability

• Proteomic verification:

  • Confirm protein abundance correlates with transcript levels

  • Identify post-translational modifications under different conditions

  • Determine protein half-life and turnover rates

• Response to stressors:

  • Heat shock

  • Oxidative stress

  • Nutrient limitation

  • Disinfection treatments

  • Antibiotic exposure

Understanding these expression patterns could provide insights into whether lpg0197 plays a role in adaptation to specific environmental niches, particularly man-made water systems that have been suggested as a potential factor in the emergence and spread of disease-associated L. pneumophila clones .

What are the optimal storage and handling conditions for recombinant lpg0197 to maintain functional integrity?

Proper storage and handling of recombinant lpg0197 is crucial for experimental reproducibility:

• Reconstitution protocol:

  • Briefly centrifuge the vial before opening

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (with 50% recommended as optimal)

  • Aliquot to minimize freeze-thaw cycles

• Storage recommendations:

  • Long-term storage: -20°C to -80°C

  • Liquid form shelf life: approximately 6 months

  • Lyophilized form shelf life: approximately 12 months

  • Working aliquots: 4°C for up to one week

• Stability considerations:

  • Avoid repeated freeze-thaw cycles

  • Consider adding protease inhibitors for applications requiring extended handling

  • Maintain sterile conditions to prevent microbial contamination

• Quality control measures:

  • Periodically verify protein integrity by SDS-PAGE

  • Consider activity assays if function is known

  • Monitor for aggregation using dynamic light scattering

Following these guidelines will help ensure experimental consistency and reliable results when working with recombinant lpg0197.

What techniques are most appropriate for studying potential protein-protein interactions involving lpg0197?

To investigate protein-protein interactions involving lpg0197, consider the following methodologies:

• In vitro interaction methods:

  • Pull-down assays using tagged recombinant lpg0197

  • Surface plasmon resonance (SPR) for kinetic and affinity measurements

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Microscale thermophoresis (MST) for interactions in solution

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for identifying interaction interfaces

• Cell-based interaction methods:

  • Co-immunoprecipitation from infected host cells

  • Proximity labeling approaches (BioID, APEX2)

  • Fluorescence resonance energy transfer (FRET)

  • Split complementation assays (yeast two-hybrid, split-GFP)

  • Protein-fragment complementation assays (PCA)

• Structural biology approaches:

  • X-ray crystallography of protein complexes

  • Cryo-electron microscopy

  • NMR spectroscopy for studying dynamic interactions

• Computational prediction methods:

  • Molecular docking simulations

  • Protein-protein interaction interface prediction

  • Evolutionary coupling analysis

When designing these experiments, consider both bacterial and host interaction partners. For bacterial partners, focus on other proteins in the same operon or functionally related pathways. For host interactions, concentrate on cellular pathways known to be manipulated during Legionella infection, such as vesicular trafficking, immune signaling, or protein synthesis pathways .

How can I develop reliable antibodies against lpg0197 for detection in complex samples?

Developing antibodies against lpg0197 requires careful planning and validation:

• Antigen design strategies:

  • Full-length recombinant protein approach:

    • Express and purify the entire lpg0197 protein

    • Verify proper folding through circular dichroism or activity assays

  • Peptide-based approach:

    • Identify antigenic epitopes using prediction algorithms

    • Select peptides from surface-exposed regions

    • Consider conjugation to carrier proteins (KLH, BSA)

  • Combined approach:

    • Develop antibodies against both full protein and specific peptides

    • Compare specificity and sensitivity

• Antibody production options:

  • Polyclonal antibodies:

    • Faster development timeline

    • Recognize multiple epitopes

    • Generally higher sensitivity

  • Monoclonal antibodies:

    • Higher specificity

    • Consistent performance between batches

    • Better for distinguishing between closely related proteins

• Validation requirements:

  • Western blot against recombinant protein

  • Testing against native protein in Legionella lysates

  • Negative controls using deletion mutants

  • Cross-reactivity testing against related bacterial proteins

  • Immunoprecipitation efficiency assessment

  • Immunofluorescence localization validation

• Application-specific considerations:

  • For intracellular detection, test antibody performance in fixed cells

  • For tissue samples, validate on infected lung tissue sections

  • For ELISA development, determine optimal coating and detection conditions

A comprehensive validation approach ensures that antibodies against lpg0197 will be reliable tools for studying this protein in complex experimental systems.

What are the key knowledge gaps regarding lpg0197 that merit further investigation?

Despite advances in Legionella research, several critical knowledge gaps regarding lpg0197 remain to be addressed:

• Functional characterization: The fundamental biological function of lpg0197 remains unknown, as indicated by its UPF0145 family designation. Determining its enzymatic activity or structural role should be a priority.

• Role in virulence: Whether lpg0197 contributes to L. pneumophila pathogenesis has not been established. This is particularly relevant given the emergence of globally dispersed, disease-associated clones identified in recent epidemiological studies .

• Structural information: The three-dimensional structure of lpg0197 has not been determined, limiting insights into its potential functional mechanisms.

• Expression regulation: The conditions that regulate lpg0197 expression during different phases of the Legionella lifecycle remain to be characterized.

• Evolutionary significance: The conservation and potential adaptive evolution of lpg0197 across Legionella species and strains requires investigation, particularly in the context of recently emerged pathogenic clones that show global dissemination .

Addressing these knowledge gaps will contribute to a better understanding of Legionella biology and potentially reveal new targets for diagnostic or therapeutic development.

How might research on lpg0197 contribute to understanding the recent emergence of globally dispersed pathogenic Legionella clones?

Research on lpg0197 could provide valuable insights into the puzzling recent emergence and rapid global spread of specific L. pneumophila clones:

• Comparative functional genomics: By determining if lpg0197 variants confer fitness advantages or enhanced virulence in disease-associated sequence types, researchers could identify whether this protein contributes to the successful dissemination of these clones.

• Adaptation signatures: If lpg0197 shows evidence of recent adaptive evolution in disease-associated sequence types, this could support the hypothesis that these clones have adapted to new man-made environmental niches .

• Host interaction dynamics: Investigation of how lpg0197 interacts with host systems could reveal mechanisms that facilitate human infection and potential for human-to-human transmission, which has been hypothesized to explain the rapid geographic spread of certain sequence types .

• Environmental persistence factors: Understanding if lpg0197 contributes to survival in built environment water systems could help explain why certain sequence types have become predominant in clinical cases. This is particularly relevant given the hypothesis that adaptation to man-made water systems may be linked to the emergence of these successful clones .