Recombinant Legionella pneumophila subsp. pneumophila 30S ribosomal protein S14 (rpsN)

What is Legionella pneumophila and why is it significant in research?

Legionella pneumophila is a Gram-negative, nonencapsulated, aerobic bacillus belonging to the genus Legionella. It is the primary human pathogenic bacterium in this group and the causative agent of Legionnaires' disease (legionellosis). The bacterium inhabits freshwater ecosystems in biofilm or planktonic forms, primarily establishing parasitic relationships with protozoa, which provide nutrition sources and protection from adverse environmental conditions . L. pneumophila is scientifically significant because it transitions between different environments during its lifecycle, including environmental water sources, protozoan hosts, and human macrophages. Bacteria released from protozoa demonstrate enhanced infectious properties, including increased motility and improved survival within human monocytes compared to bacteria grown on artificial media . Among the 58 identified Legionella species, L. pneumophila serogroup 1 is responsible for over 85% of Legionnaires' disease cases worldwide, making it a primary focus for pathogenesis and vaccine development research .

What is the 30S ribosomal protein S14 (rpsN) and what functions does it serve?

The 30S ribosomal protein S14, encoded by the rpsN gene, is a component of the small (30S) subunit of the bacterial ribosome in Legionella pneumophila. This protein plays several critical roles in bacterial physiology and pathogenesis:

• Ribosome stabilization under stress conditions: rpsN contributes to maintaining ribosomal integrity when the bacterium encounters environmental stressors

• Participation in translation processes as a structural component of the ribosome

• Potential surface exposure, making it a candidate target for immune recognition and vaccine development

• Interaction with other ribosomal components to maintain proper ribosomal architecture

The protein has been identified as increasingly important in L. pneumophila research, particularly in studies focused on stress response mechanisms and potential vaccine development approaches due to its immunogenic properties.

What expression systems are used for recombinant L. pneumophila rpsN production?

Recombinant L. pneumophila rpsN can be produced using several expression systems, each with distinct advantages and limitations :

The choice of expression system should be determined by the intended application, with mammalian cell expression often preferred for immunological and vaccine-related studies as mentioned in product specifications .

What are the optimal reconstitution and storage conditions for recombinant rpsN?

Based on the product information, the optimal conditions for reconstituting and storing lyophilized recombinant L. pneumophila rpsN include :

Reconstitution protocol:

• Centrifuge the vial briefly before 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% (with 50% being the default recommendation)

• Aliquot for long-term storage to avoid repeated freeze-thaw cycles

Storage conditions:

• Short-term storage (up to one week): 4°C

• Long-term storage: -20°C/-80°C in glycerol-containing buffer

• Shelf life: 6 months for liquid form at -20°C/-80°C; 12 months for lyophilized form

• Critical note: "Repeated freezing and thawing is not recommended"

For optimal stability, it's advisable to prepare single-use aliquots sized appropriately for experimental needs to minimize the damaging effects of freeze-thaw cycles on protein structure and function.

How can researchers verify the purity and functionality of recombinant rpsN?

Verifying the purity and functionality of recombinant L. pneumophila rpsN requires a multi-method approach:

Purity assessment:

• SDS-PAGE analysis: The expected purity should be >85% as stated in the product specifications

• Western blotting with anti-rpsN antibodies or antibodies against purification tags

• Mass spectrometry for precise identification and to detect potential contaminants

Functional validation approaches:

• RNA binding assays to confirm the protein's ability to interact with ribosomal RNA

• Structural integrity assessment using circular dichroism spectroscopy

• Immunological activity testing if being studied as a vaccine candidate

• Ribosome reconstitution assays to evaluate integration into ribosomal subunits

Researchers should always validate new lots of recombinant protein before use in critical experiments, especially when studying immunological responses where contaminants could significantly affect results.

How is recombinant rpsN being utilized in vaccine development research?

Recombinant L. pneumophila rpsN is being explored as a potential component in vaccine development strategies due to several advantageous properties:

• Surface exposure and accessibility: rpsN is being investigated as an antigenic target due to its surface exposure on the bacterial cell and demonstrated immunogenicity

• Ribosomal proteins like rpsN (S14) and rpsJ (S10) are validated targets for antibody generation and potential vaccine candidates

• As stated directly in the research literature: "Recombinant ribosomal proteins (e.g., S10, S14) are explored as antigenic targets due to their surface exposure and immunogenicity"

Research approaches include:

• Subunit vaccine formulations combining purified recombinant rpsN with appropriate adjuvants

• Comparative studies evaluating rpsN alongside other ribosomal proteins like S10, S6, and S7

• Evaluation of both humoral and cell-mediated immune responses to recombinant rpsN

• Investigation of cross-protective potential against different L. pneumophila strains

It's important to note that current applications are restricted to research purposes only, as emphasized in product documentation: "All of our products can only be used for research purposes. These vaccine ingredients CANNOT be used directly on humans or animals" .

What experimental models are appropriate for studying rpsN-related phenomena?

Several experimental models are suitable for investigating L. pneumophila rpsN, depending on the specific research questions:

In vitro cellular models:

• Human alveolar macrophage cell lines (THP-1, U937): U937-derived macrophages have been used successfully for studying L. pneumophila proteins

• CHO cells: Employed for ectopic expression of Legionella proteins to study their effects on host cells

Functional models:

• Protein-coated beads: As described in the research literature, "HtpB-coated beads recruited mitochondria in CHO cells and U937-derived macrophages"—similar approaches can be applied with rpsN-coated beads

• Transfected cell lines expressing recombinant proteins: "Ectopic expression of HtpB in the cytoplasm of transfected CHO cells" provides a model applicable to rpsN studies

Comparative analysis models:

• Side-by-side comparison with other ribosomal proteins, as shown in this table from published research:

These diverse experimental models allow researchers to explore various aspects of rpsN biology, from basic structural studies to complex host-pathogen interactions and potential vaccine applications.

What methodological approaches are recommended for immunological studies with rpsN?

When conducting immunological studies with recombinant L. pneumophila rpsN, the following methodological approaches are recommended:

Sample preparation considerations:

• Endotoxin testing and removal is essential, especially for proteins expressed in bacterial systems

• Protein concentration determination using validated methods (BCA or Bradford assays)

• Quality control testing to ensure batch-to-batch consistency

Experimental controls:

• Include denatured rpsN as a control for conformation-dependent responses

• Use protein tag-only controls if the recombinant protein contains purification tags

• Include other L. pneumophila ribosomal proteins (e.g., rpsJ/S10) to assess specificity of responses

• Incorporate both positive controls (known immunogens) and negative controls (irrelevant proteins)

Analytical techniques:

• ELISA for antibody titer determination

• ELISpot for enumeration of antigen-specific antibody-secreting cells

• Flow cytometry with intracellular cytokine staining to characterize T-cell responses

• Western blotting to confirm antibody specificity

For protein-coated bead studies similar to those described for HtpB , researchers should employ fluorescence microscopy and flow cytometry to assess cellular interactions and responses to rpsN-coated beads, with appropriate controls including beads coated with irrelevant proteins.

How does L. pneumophila rpsN compare to other ribosomal proteins as research targets?

Legionella pneumophila ribosomal proteins represent an interesting group of potential research targets with distinct characteristics and applications:

Comparative analysis of L. pneumophila ribosomal proteins:

• rpsN (S14) is specifically studied for its role in "ribosome stabilization under stress" and is particularly valuable for stress response research

• rpsJ (S10) has been validated for "vaccine candidate validation" and is notable for antibiotic binding sites

• rpsG (S7) is emphasized for structural studies and antigen design approaches

• rpsF (S6) has applications in pathogenesis models due to its role in translational fidelity

Key factors influencing the selection of rpsN versus other ribosomal proteins include:

• Research focus: Stress response (rpsN) versus core translation (rpsG, rpsJ)

• Surface accessibility: rpsN and rpsJ are noted for surface exposure, enhancing their potential as immunological targets

• Technical considerations: Expression, purification, and stability characteristics vary among ribosomal proteins

When selecting a ribosomal protein target, researchers should consider these comparative aspects in relation to their specific research questions and technical capabilities.

What is known about rpsN's potential role in L. pneumophila pathogenesis?

While direct evidence for rpsN's role in pathogenesis requires further research, several connections can be made based on current knowledge:

Potential pathogenesis mechanisms involving rpsN:

• Stress adaptation: rpsN's role in "ribosome stabilization under stress" is particularly relevant for L. pneumophila, which must adapt to diverse environments including environmental water, protozoan hosts, and human macrophages

• Potential moonlighting functions: Similar to other bacterial proteins, rpsN may have non-ribosomal functions when expressed on the bacterial surface

• Translation regulation during host invasion: As part of the translation machinery, rpsN supports the production of virulence factors during different phases of infection

Contextual relevance to L. pneumophila life cycle:

• Search result describes how L. pneumophila undergoes a differentiation program when transitioning between environments, with "metabolic as well as morphogenetic changes"

• The translation machinery, including rpsN, would be essential for expressing the proteins responsible for enhanced virulence traits observed when bacteria transition from environmental to host settings

Research approaches to investigate rpsN in pathogenesis:

• Generation of rpsN mutants to assess effects on stress tolerance and virulence

• Localization studies to determine if rpsN is indeed surface-exposed during infection

• Comparative proteomics to examine rpsN expression patterns during different stages of infection

What challenges exist in functional studies of recombinant ribosomal proteins?

Researchers working with recombinant L. pneumophila rpsN face several significant challenges:

Structural and functional integrity:

• Ribosomal proteins naturally function as part of a complex with rRNA and other proteins

• Recombinant expression removes these proteins from their natural context, potentially affecting folding

• Solution: Consider co-purification with binding partners or include stabilizing agents in buffers

Nucleic acid contamination:

• Ribosomal proteins naturally bind RNA, leading to potential nucleic acid contamination

• This can affect downstream applications, particularly in immunological studies

• Solution: Include RNase treatment during purification and monitor A260/A280 ratio

Expression system selection challenges:

• Different expression systems (E. coli, yeast, baculovirus, mammalian cells) produce proteins with varying characteristics

• Post-translational modifications may differ between expression systems

• Solution: Compare expression products from multiple systems for critical applications

Validation of functional activity:

• Without the complete ribosomal context, assessing true functional activity is challenging

• Standardized functional assays specific to rpsN are not well-established

• Solution: Develop application-specific assays that reflect the intended research use

These challenges highlight the importance of rigorous experimental design and appropriate controls when working with recombinant ribosomal proteins like rpsN from L. pneumophila.

What emerging applications exist for recombinant L. pneumophila rpsN?

Several promising research directions for recombinant L. pneumophila rpsN are emerging:

Vaccine development advancements:

• Multi-component vaccine approaches combining rpsN with other immunogenic proteins

• Novel adjuvant formulations to enhance rpsN-specific immune responses

• Development of chimeric proteins incorporating rpsN epitopes with other bacterial antigens

Diagnostic applications:

• Development of rpsN-based serological assays for improved L. pneumophila detection

• Creation of rapid diagnostic tests targeting rpsN antibodies in patient samples

• Potential use in environmental monitoring systems

Structural biology opportunities:

• Cryo-electron microscopy studies of L. pneumophila ribosomes focusing on rpsN positioning

• Comparative structural analysis across different Legionella species and strains

• Investigation of rpsN structural changes during environmental stress adaptation

Antimicrobial development:

• Exploration of rpsN as a target for novel antimicrobial compounds

• Investigation of small molecules that could disrupt rpsN function or integration

• Development of antibody-antibiotic conjugates targeting surface-exposed rpsN

These emerging applications highlight the diverse potential of recombinant L. pneumophila rpsN beyond its current research applications, suggesting valuable avenues for future investigation.