Recombinant Legionella pneumophila subsp. pneumophila Protein grpE (grpE)

What is Legionella pneumophila and how does it cause disease?

Legionella pneumophila is a γ-proteobacterial species that naturally parasitizes free-living amoeba in freshwater environments . It is the most common cause of Legionnaires' disease, a severe respiratory infection, though the microorganism typically exists as a symbiont of free-living amoeba . L. pneumophila causes disease by hijacking the phagocytic process in both amoebae and human alveolar macrophages, subverting host cellular mechanisms to promote intracellular replication . The bacterium presents clinically as either severe pneumonia (Legionnaires' disease) or Pontiac fever, a self-limiting flu-like syndrome .

Importantly, L. pneumophila is not transmitted person-to-person; the U.S. Centers for Disease Control and Prevention (CDC) has recorded only one case of person-to-person transmission . Infection occurs when humans breathe in water vapor containing the bacteria in the immediate vicinity of that vapor, such as directly over a faucet . Symptoms include fever, chills, muscle aches, cough, shortness of breath, headaches, fatigue, loss of appetite, confusion, and sometimes diarrhea .

What is the role of grpE in L. pneumophila biology?

The grpE protein in L. pneumophila functions as a nucleotide exchange factor co-chaperone that works in concert with the DnaK-DnaJ chaperone system. While not explicitly mentioned in the search results, bacterial grpE proteins typically facilitate the release of ADP from DnaK (Hsp70), allowing ATP to bind and completing the chaperone cycle necessary for protein folding. In L. pneumophila, this system likely plays crucial roles in stress response, protein quality control, and potentially virulence, similar to other chaperone systems like HtpB that have been more extensively studied .

When comparing with other chaperone systems in L. pneumophila, we can note that the HtpB chaperonin is multifunctional, playing different roles depending on its location within the bacterial cell or in infected host cells . By analogy, grpE likely contributes to protein homeostasis under various stress conditions encountered during the L. pneumophila life cycle, both in aquatic environments and during host infection.

How does grpE relate to other chaperone systems in L. pneumophila?

L. pneumophila possesses several chaperone systems, including the well-studied HtpB chaperonin (a GroEL homolog) and likely the DnaK-DnaJ-grpE system. These systems function cooperatively but distinctly in protein quality control. The HtpB chaperonin has been shown to be multifunctional, with roles beyond protein folding . When expressed on the bacterial surface, HtpB can act as an invasion factor for non-phagocytic cells, while HtpB released in the host cell can alter organelle trafficking, actin microfilament organization, and cell signaling pathways .

The grpE protein would function primarily in the DnaK chaperone cycle, which typically handles different protein substrates than the GroEL/GroES (HtpB) system. While HtpB forms large oligomeric structures and can encapsulate substrate proteins, the DnaK-DnaJ-grpE system works on exposed hydrophobic segments of partially folded proteins. The two systems may function sequentially or in parallel during protein folding and stress response.

What expression systems are optimal for recombinant L. pneumophila grpE production?

Based on methodologies used for other L. pneumophila proteins, an E. coli expression system with pET vectors (such as pET15b or pET22b) would be suitable for recombinant grpE production . The protocol might follow a similar approach to that used for LceB expression:

• Clone the grpE gene from L. pneumophila into a pET vector with an N-terminal 6His-tag

• Transform the expression construct into E. coli BL21 DE3 Gold cells

• Grow cells in Lysogeny Broth (LB) at 37°C until an OD600 of 0.6

• Induce protein expression with IPTG (1 mM final concentration) for approximately 20 hours at 20°C

What purification strategies yield high-purity recombinant L. pneumophila grpE?

A multi-step purification protocol would likely yield high-purity recombinant grpE:

• Cell lysis using an Emulsiflex-C3 High Pressure Homogenizer with protease inhibitors (1 mM PMSF), 10 mM MgCl₂, and DNase

• Initial purification via nickel affinity chromatography using a wash buffer (50 mM Tris pH 7.5, 500-750 mM NaCl, 25 mM imidazole)

• Size exclusion chromatography to separate monomeric, dimeric, and potentially aggregated forms

• If necessary, ion exchange chromatography to remove contaminants with different charge properties

Analyzing purified grpE by SDS-PAGE under both reducing and non-reducing conditions would be important to verify its oligomeric state, as seen with HtpB protein which showed different forms under various conditions . Two-dimensional protein gel analysis might reveal post-translational modifications such as phosphorylation, which has been observed in related chaperones .

How can researchers assess the nucleotide exchange activity of purified grpE?

The nucleotide exchange activity of purified grpE can be assessed through several complementary approaches:

• ADP-ATP Exchange Assay: Measure the rate of exchange of radiolabeled ADP with ATP in the presence of DnaK and grpE using filter binding or HPLC methods

• Fluorescence-Based Assays: Use fluorescently labeled nucleotides (such as MANT-ADP) to monitor binding and release from DnaK in real-time upon addition of grpE

• Coupled Enzyme Assay: Measure ATPase activity of the DnaK-grpE system using a coupled enzyme system that links ATP hydrolysis to NADH oxidation, which can be monitored spectrophotometrically

• Thermal Stability Assessment: Determine if grpE enhances the thermal stability of DnaK in the presence of various nucleotides using differential scanning fluorimetry

This functional characterization is essential to confirm that recombinant grpE retains its native nucleotide exchange factor activity and to compare its kinetic parameters with grpE proteins from other bacterial species.

How might L. pneumophila grpE contribute to pathogenesis?

While direct evidence is limited in the search results, several mechanisms can be proposed for grpE's role in L. pneumophila pathogenesis based on studies of other chaperone systems:

• Stress Adaptation: grpE likely helps L. pneumophila adapt to temperature shifts and other stresses encountered during environmental-to-host transitions, similar to how HtpB functions as an essential stress protein

• Protein Quality Control During Infection: The DnaK-DnaJ-grpE system may ensure proper folding of virulence factors secreted through the Dot/Icm type IV secretion system

• Host-Pathogen Interactions: Like HtpB, which has been shown to act as an invasion factor and effector , grpE might have moonlighting functions during infection, potentially interacting with host proteins

• Survival in Macrophages: The chaperone system could contribute to bacterial survival in the harsh environment of macrophages, where the bacterium must evade host defense mechanisms

The confirmation of these hypotheses would require experimental approaches such as creating conditional grpE mutants (since complete deletion might be lethal as observed with htpB ) and assessing their ability to replicate in macrophages or amoebae.

What structural features of grpE are important for its co-chaperone function?

Bacterial grpE proteins typically form homodimers with distinct structural domains that mediate different aspects of their function:

• N-terminal Domain: Contains a long α-helical region that forms a coiled-coil structure important for dimerization and temperature sensing

• Central Domain: Mediates interactions with the ATPase domain of DnaK and facilitates nucleotide exchange

• C-terminal Domain: Contains a four-helix bundle structure that contributes to DnaK binding

A structural approach similar to that used for LceB characterization could be valuable for L. pneumophila grpE, combining AlphaFold modeling with X-ray crystallography to capture various conformational states. The temperature-sensing function of the N-terminal domain is particularly relevant for L. pneumophila, which must adapt to temperature shifts during environmental-to-host transitions.

How does L. pneumophila grpE compare with grpE proteins from other bacterial pathogens?

A comprehensive comparative analysis would examine:

• Sequence Conservation: Analysis of sequence homology between L. pneumophila grpE and other bacterial grpE proteins, particularly focusing on functional domains

• Structural Variations: Comparison of predicted or experimentally determined structures to identify unique features in L. pneumophila grpE

• Functional Complementation: Testing whether L. pneumophila grpE can complement grpE-deficient strains of other bacteria (similar to studies showing that HtpB could not complement GroEL deficiency in E. coli )

• Expression Patterns: Examining whether L. pneumophila grpE shows unique expression patterns during infection compared to other bacterial pathogens

This comparative approach might reveal adaptations specific to L. pneumophila's unique lifecycle, which involves transitions between aquatic environments, amoebae, and human macrophages.

What are common pitfalls in working with recombinant L. pneumophila grpE?

Researchers working with recombinant L. pneumophila grpE should be aware of several potential challenges:

• Protein Stability: The temperature-sensitive nature of grpE proteins may lead to conformational changes or aggregation during purification

• Oligomeric State Variability: As observed with HtpB, which showed multiple forms in SDS-PAGE analysis , grpE might exist in different oligomeric states depending on experimental conditions

• Co-purification of E. coli DnaK: When expressed in E. coli, grpE may form complexes with the host's DnaK, potentially contaminating the preparation

• Post-translational Modifications: Different patterns of post-translational modifications might occur in E. coli compared to native modifications in L. pneumophila, affecting functional studies

Strategies to address these challenges include careful buffer optimization, inclusion of stabilizing agents, thorough characterization of oligomeric states, and potentially exploring alternative expression hosts.

How can researchers investigate grpE-DnaK interactions specific to L. pneumophila?

To study the specific interactions between L. pneumophila grpE and DnaK:

• Co-expression and Co-purification: Express both proteins in E. coli with different tags to enable co-purification of stable complexes

• Surface Plasmon Resonance (SPR): Quantify binding kinetics and affinity between the purified proteins under various conditions

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Map interaction interfaces and conformational changes upon complex formation

• Mutational Analysis: Create point mutations in predicted interaction interfaces to validate their importance

• Cross-linking Studies: Use chemical cross-linking combined with mass spectrometry to identify interaction points between the proteins

These approaches would provide detailed insights into how L. pneumophila grpE and DnaK interact, potentially revealing adaptations specific to this pathogen's lifecycle.

How might grpE contribute to L. pneumophila adaptation to different hosts?

L. pneumophila has evolved to infect both amoebae and human macrophages, suggesting adaptations that enable survival in diverse host environments. The grpE co-chaperone might contribute to this adaptability through:

• Temperature Adaptation: grpE's thermosensing properties could help the bacterium adjust to temperature shifts between environmental water (cooler) and host cells (warmer)

• Stress Response Regulation: Different stress conditions in various hosts might be managed through modulated grpE-DnaK interactions

• Host-Specific Protein Folding Requirements: The chaperone system might be optimized for folding proteins needed in specific host environments

Research approaches to test these hypotheses could include comparing grpE expression and activity during growth in different host cells and examining the impact of grpE mutations on host-specific survival.

Could L. pneumophila grpE serve as a target for novel antimicrobial development?

The essential nature of chaperone systems makes grpE a potential target for antimicrobial development. A research strategy could include:

• Target Validation: Confirm whether grpE is essential for L. pneumophila survival, similar to attempts with htpB gene replacement that yielded negative results

• Small Molecule Screening: Develop high-throughput assays to identify compounds that specifically inhibit the nucleotide exchange activity of L. pneumophila grpE

• Structure-Based Drug Design: Use structural information to design inhibitors that target unique features of L. pneumophila grpE

• Specificity Assessment: Evaluate potential inhibitors for selectivity toward bacterial versus human homologs to minimize toxicity

• Efficacy Testing: Test promising compounds in cellular and animal models of L. pneumophila infection

This approach could potentially yield new therapeutic options for Legionnaires' disease, particularly important given the increasing public health concern of Legionella infections .