Recombinant Legionella pneumophila subsp. pneumophila GTPase Der (der)

What is GTPase Der in Legionella pneumophila?

GTPase Der (also known as GTP-binding protein EngA) is a key protein encoded by the Legionella pneumophila genome. It belongs to the GTPase superfamily, which functions as molecular switches that cycle between GTP-bound (active) and GDP-bound (inactive) states. Der contains two GTPase domains and is involved in important cellular processes. The full-length Der protein from L. pneumophila strain Corby consists of 462 amino acids with a UniProt accession number of A5IC36 . Der plays roles in ribosome assembly and bacterial physiology, making it an important subject for research on L. pneumophila virulence mechanisms.

How does recombinant Der differ from native Der in L. pneumophila?

Recombinant Der is an exogenously expressed version of the native protein produced in host systems like mammalian cells . While the amino acid sequence remains identical to the native protein, several differences may exist:

• Post-translational modifications may differ depending on the expression system

• Recombinant Der typically contains affinity tags for purification purposes

• The protein may have different folding kinetics or stability characteristics

• Expression levels are typically higher than physiological conditions

• The recombinant protein is isolated from its natural bacterial environment and interaction partners

When using recombinant Der for research, these differences should be considered when interpreting results in relation to native protein function.

What is the role of GTPases in Legionella pneumophila pathogenesis?

GTPases play critical roles in L. pneumophila pathogenesis through several mechanisms:

• Interaction with host cell GTPases: L. pneumophila secretes effector proteins that modulate host GTPases like Ran and Rab1 to facilitate infection

• Subversion of host trafficking: Bacterial effectors target GTPases involved in vesicular trafficking to create a replicative niche called the Legionella-containing vacuole (LCV)

• Modification of host GTPase cycling: L. pneumophila effectors can manipulate the GTP/GDP cycle of host GTPases through processes like AMPylation and de-AMPylation

• Bacterial GTPases like Der contribute to ribosome biogenesis and bacterial fitness during infection

For example, the effector SidM recruits and AMPylates host Rab1 GTPase, locking it in an active state to promote fusion of secretory vesicles with LCVs, while another effector, SidD, later de-AMPylates Rab1 to allow its inactivation and removal from the vacuole membrane .

What are the optimal conditions for storing and reconstituting recombinant Der protein?

Proper storage and reconstitution of recombinant Der protein is critical for maintaining its biological activity:

For reconstitution, follow these methodological steps:

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

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

• Add glycerol to a final concentration of 5-50% (50% is recommended) for long-term storage

• Prepare small aliquots to avoid repeated freeze-thaw cycles

These conditions ensure optimal protein stability and activity for downstream applications.

What experimental approaches can be used to study Der GTPase activity in vitro?

Several methodological approaches are suitable for characterizing Der GTPase activity:

• GTP Hydrolysis Assays:

  • Colorimetric phosphate release assays using malachite green

  • HPLC-based assays to quantify GDP formation

  • Radiometric assays using [γ-32P]GTP to track GTP hydrolysis

• Nucleotide Binding Studies:

  • Fluorescence-based assays using MANT-GTP or BODIPY-GTP

  • Isothermal titration calorimetry (ITC) to determine binding affinities

  • Filter binding assays with radioactive nucleotides

• Structural Analyses:

  • X-ray crystallography of Der in different nucleotide-bound states

  • Hydrogen-deuterium exchange mass spectrometry to monitor conformational changes

  • Cryo-EM to visualize larger complexes involving Der

• Protein Interaction Studies:

  • Pull-down assays to identify interaction partners

  • Surface plasmon resonance to determine binding kinetics

  • FRET-based assays to monitor protein-protein interactions in real-time

When designing these experiments, researchers should consider the dual GTPase domains of Der and potentially study them independently to understand their distinct functions.

How can recombinant Der protein be used to study L. pneumophila-host interactions?

Recombinant Der can be utilized in multiple experimental strategies to elucidate L. pneumophila-host interactions:

• Binding Partner Identification:

  • Affinity chromatography using immobilized Der to pull down potential host targets

  • Yeast two-hybrid screening with Der as bait

  • Proximity labeling approaches (BioID or APEX) to identify proteins in close proximity to Der

• Functional Assays:

  • Microinjection of recombinant Der into host cells to observe phenotypic changes

  • Transfection of Der expression constructs into mammalian cells

  • Reconstitution of Der-dependent processes in cell-free systems

• Structural Vaccinology Approaches:

  • Using recombinant Der as an antigen to generate antibodies

  • Evaluating Der as a potential vaccine target

  • Structure-based design of Der inhibitors as potential therapeutics

• Cell Biology Applications:

  • Fluorescently labeled Der to track localization during infection

  • Der-specific antibodies for immunofluorescence studies

  • Construction of Der mutants to dissect protein domains and functions

These approaches leverage recombinant protein technology to understand the specific roles of Der in pathogenesis .

How do the GTPase domains of Der coordinate with each other, and what are the functional implications?

The Der protein contains two distinct GTPase domains that exhibit coordinated activity with significant functional implications:

• Domain Architecture and Coordination:

  • N-terminal GTPase domain (residues approximately 1-170) and C-terminal GTPase domain (residues approximately 170-300) based on sequence analysis

  • The domains likely communicate through conformational changes upon GTP binding/hydrolysis

  • Sequential or synchronized GTP hydrolysis may regulate Der's cellular functions

• Nucleotide Binding Preferences:

  • Based on sequence analysis, the N-terminal domain contains the motif "PNVGKSTLFN" while the C-terminal domain contains "PNVGKSTLIN"

  • These subtle differences in the G1 motif (P-loop) may confer different nucleotide binding affinities

  • The differential binding properties could create a sequential activation mechanism

• Functional Implications:

  • Dual GTPase domains may allow integration of different cellular signals

  • The two domains could recognize distinct interaction partners

  • Mutational analysis of individual domains could reveal their specific contributions to pathogenesis

• Evolutionary Considerations:

  • The two-domain structure may have arisen through gene duplication events

  • Comparing Der structure with other bacterial GTPases might reveal evolutionary adaptations specific to Legionella's intracellular lifestyle

A comprehensive understanding of this coordination would provide insights into bacterial GTPase function and potentially identify novel therapeutic targets.

What role does Der play in regulating L. pneumophila's secretion system or effector proteins?

While direct evidence for Der's role in regulating the Type IV secretion system is limited, several hypotheses can be proposed based on GTPase functions in related systems:

• Potential Regulatory Mechanisms:

  • Der may function as a checkpoint in the assembly or activation of secretion machinery

  • It could coordinate the timing of effector protein secretion during different infection stages

  • Der might regulate the translation of secretion system components or effector proteins through its role in ribosome assembly

• Indirect Influence on Effector Function:

  • L. pneumophila secretes over 300 different effector proteins during infection

  • Der could indirectly impact the production or function of GTPase-modulating effectors like LegG1 or PieG

  • This would create a bacterial regulatory network connecting Der to host GTPase manipulation

• Methodological Approaches to Test These Hypotheses:

  • Der depletion or overexpression studies to observe effects on effector secretion

  • Proteomics analysis to identify Der-dependent changes in the secretome

  • Genetic interaction studies between Der and secretion system components

  • Temporal analysis of Der activity throughout the infection cycle

Understanding this relationship would provide insights into the hierarchical regulation of L. pneumophila virulence mechanisms.

How do different L. pneumophila strains vary in Der sequence and function, and what are the implications for host interaction?

Strain variation in Der may contribute to differences in L. pneumophila virulence and host adaptation:

• Comparative Genomic Analysis:

  • Sequence alignment of Der from different strains (e.g., Philadelphia-1, Paris, Corby) reveals potential variations

  • The Corby strain Der sequence is well characterized with UniProt accession A5IC36

  • Critical residues for GTP binding (G1-G5 motifs) are likely conserved, while other regions may show greater variability

• Functional Diversity:

  • Strain-specific variations in Der could alter GTPase activity, binding partner selectivity, or subcellular localization

  • Similar to the RCC1 repeat effectors that have divergently evolved between strains to target different components of host GTPase cycles

  • Some strains might have optimized Der function for specific host environments (amoebae vs. human macrophages)

• Evolutionary Implications:

  • Der variation could reflect adaptations to different ecological niches

  • Like the pieG gene that evolved through fusion of legG1 and lpg1975 , Der may undergo evolutionary processes that affect its function

  • Horizontal gene transfer could contribute to Der diversity among strains

• Research Methodology:

  • Complementation studies with Der variants from different strains

  • Comparative biochemical characterization of Der proteins

  • Infection models using diverse host cells to assess strain-specific Der functions

This comparative approach could identify strain-specific virulence mechanisms and host adaptation strategies.

What are common challenges in expressing and purifying recombinant Der, and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant Der protein:

• Solubility Issues:

  • Challenge: GTPases often form inclusion bodies when overexpressed

  • Solution: Optimize expression conditions (lower temperature, reduced IPTG concentration)

  • Alternative: Use solubility tags like MBP, SUMO, or GST

  • Recommendation: Express in mammalian cells as indicated in product information

• Maintaining Nucleotide-Bound State:

  • Challenge: GTPases may lose bound nucleotides during purification

  • Solution: Include appropriate nucleotides in all buffers

  • Methodology: Use affinity chromatography followed by size exclusion in the presence of GTP/GDP

• Preserving Activity:

  • Challenge: Loss of GTPase activity during purification or storage

  • Solution: Add glycerol (5-50%) to storage buffer as recommended

  • Methodology: Aliquot and store at -80°C, avoid repeated freeze-thaw cycles

• Protein Purity Considerations:

  • Challenge: Contaminating proteins or nucleic acids

  • Solution: Multiple purification steps (typical purity should be >85% by SDS-PAGE)

  • Methodology: Consider ion exchange chromatography as a polishing step

• Tag Removal Issues:

  • Challenge: Inefficient tag cleavage or protein aggregation after tag removal

  • Solution: Optimize protease digestion conditions

  • Alternative: If function is not affected, consider using the tagged protein for experiments

These methodological considerations ensure obtaining functional recombinant Der protein suitable for downstream applications.

How can researchers distinguish between direct and indirect effects of Der GTPase in infection models?

Distinguishing direct from indirect effects requires careful experimental design:

• Genetic Approaches:

  • Generate catalytically inactive Der mutants (e.g., mutations in G1/G3 motifs)

  • Create domain-specific Der mutants to dissect individual functions

  • Deploy conditional expression systems to control Der levels temporally

• Biochemical Strategies:

  • Perform direct binding assays with purified components

  • Use proximity labeling approaches (BioID, APEX) to identify direct interaction partners

  • Conduct in vitro reconstitution of Der-dependent processes

• Temporal Analysis:

  • Use pulse-chase experiments to track the sequence of events following Der activation

  • Implement rapid depletion systems (e.g., auxin-inducible degron) to observe immediate effects

  • Perform time-resolved proteomics or transcriptomics following Der perturbation

• Spatial Resolution Approaches:

  • Utilize subcellular fractionation to determine where Der acts

  • Implement optogenetic tools to activate or inactivate Der in specific cellular locations

  • Use super-resolution microscopy to visualize Der localization relative to other components

• Control Experiments:

  • Include Der-independent controls in all experiments

  • Compare effects of Der perturbation with perturbation of known downstream factors

  • Use multiple methodological approaches to confirm direct interactions

These strategies help build a comprehensive understanding of Der's direct mechanistic roles versus its downstream effects.

What are the most effective methods for studying interactions between bacterial GTPases like Der and host cellular processes?

Several sophisticated methodologies can effectively probe bacterial-host GTPase interactions:

• Advanced Microscopy Techniques:

  • Live-cell imaging with fluorescently tagged Der and host GTPases

  • FRET/BRET approaches to detect direct interactions

  • Single-molecule tracking to observe dynamics of interactions

  • CLEM (Correlative Light and Electron Microscopy) for ultrastructural context

• Proteomics-Based Approaches:

  • BioID or APEX proximity labeling to identify proteins in Der's vicinity

  • Cross-linking mass spectrometry (XL-MS) to map interaction interfaces

  • Thermal proteome profiling to detect Der-induced stability changes in host proteins

  • Phosphoproteomics to identify signaling changes induced by Der

• Functional Genomics:

  • CRISPR screens to identify host factors required for Der function

  • Transcriptomics to characterize host responses to Der expression

  • Genetic interaction mapping to position Der in host pathways

• Biochemical Reconstitution:

  • Cell-free systems to reconstitute Der-dependent processes

  • Liposome-based assays to study membrane interactions

  • Single-molecule biochemistry to examine Der-host protein interactions

• Structural Biology Approaches:

  • Cryo-EM of Der-host protein complexes

  • HDX-MS to map conformational changes during interactions

  • NMR studies of dynamic interactions in solution

These methodologies, used in combination, can provide comprehensive insights into the complex interplay between bacterial GTPases and host cellular mechanisms during L. pneumophila infection.

How might Der GTPase be targeted for therapeutic development against Legionella infections?

Der GTPase presents several promising avenues for therapeutic development:

• Small Molecule Inhibitor Approaches:

  • Structure-based design of inhibitors targeting the GTP binding pockets

  • Allosteric inhibitors that prevent conformational changes between domains

  • Compounds that disrupt Der interaction with essential bacterial partners

  • Advantage: Potential for high specificity given differences from human GTPases

• Peptide-Based Strategies:

  • Develop peptides that mimic Der binding interfaces

  • Cell-penetrating peptides that interfere with Der function within bacteria

  • Peptide aptamers selected for high-affinity binding to Der

• Novel Antibiotic Development:

  • Since Der is involved in ribosome assembly, it represents a novel antibiotic target

  • Der inhibitors could be combined with existing antibiotics for synergistic effects

  • Screening chemical libraries against recombinant Der activity

• Vaccine Development:

  • Evaluating Der or its domains as potential vaccine antigens

  • Identifying immunogenic epitopes within Der sequence

  • Testing whether anti-Der antibodies can neutralize infection

• Methodological Research Pathway:

  • High-throughput screening of compound libraries against Der activity

  • Structure-activity relationship studies of lead compounds

  • Animal model testing of Der-targeting therapeutics

  • Resistance development assessment

These approaches could lead to novel treatments for Legionnaires' disease, particularly important for cases resistant to conventional antibiotics.

What can comparative studies between L. pneumophila Der and other bacterial GTPases reveal about pathogenesis mechanisms?

Comparative studies offer valuable insights into evolutionary adaptations and functional specialization:

• Evolutionary Analysis:

  • Phylogenetic comparison of Der across pathogenic and non-pathogenic bacteria

  • Identification of Legionella-specific adaptations in Der sequence and structure

  • Analysis of selective pressure on different Der domains

• Functional Comparison:

  • Contrasting Der with similar GTPases from other intracellular pathogens (e.g., Mycobacterium, Salmonella)

  • Comparing Der with GTPases from extracellular pathogens to identify niche-specific adaptations

  • Examining differences between Der and essential GTPases in non-pathogenic bacteria

• Methodological Approaches:

  • Complementation studies across species

  • Domain-swapping experiments to identify functionally important regions

  • Comparative biochemical characterization of GTPase activities

  • Cross-species interaction studies with host factors

• Potential Revelations:

  • Discovery of pathogen-specific mechanisms of host manipulation

  • Identification of conserved bacterial GTPase functions essential for virulence

  • Understanding how L. pneumophila has specialized Der function for its unique intracellular lifestyle

This comparative approach could uncover fundamental principles of bacterial pathogenesis while identifying Legionella-specific virulence mechanisms.

How does Der interact with the network of effector proteins that manipulate host GTPases during infection?

Understanding Der's position within L. pneumophila's complex effector network represents a frontier in research:

• Potential Regulatory Relationships:

  • Der may influence the expression, secretion, or activity of effectors like SidM and SidD that manipulate host Rab1

  • It could coordinate with effectors targeting the Ran GTPase cycle (LegG1, PpgA, PieG)

  • Der might serve as a timing mechanism for deploying different effectors during infection stages

• Integration of GTPase Networks:

  • L. pneumophila effectors target multiple host GTPases, including Ran and Rab1

  • Der could function as a bacterial sensor that integrates signals from these manipulated host pathways

  • This integration might allow L. pneumophila to adapt its virulence strategy based on host responses

• Methodological Research Approaches:

  • Interactome mapping between Der and effector proteins

  • Temporal analysis of Der activity in relation to effector deployment

  • Systems biology modeling of the GTPase networks during infection

  • Genetic interaction studies between Der and effector genes

• Functional Significance:

  • Coordinated GTPase manipulation may be critical for establishing the Legionella-containing vacuole

  • Understanding this network could reveal vulnerability points for therapeutic intervention

  • This knowledge may apply to other intracellular pathogens with similar strategies

Deciphering this network would provide a comprehensive view of how L. pneumophila orchestrates host cell takeover through GTPase manipulation.