Recombinant Legionella pneumophila subsp. pneumophila Proline--tRNA ligase (proS), partial

What is the role of Proline--tRNA ligase (proS) in Legionella pneumophila?

Proline--tRNA ligase (ProS) is an essential aminoacyl-tRNA synthetase responsible for charging tRNA^Pro with proline, a critical step in protein synthesis. In L. pneumophila, this enzyme plays a fundamental role in bacterial survival and virulence expression. The enzyme catalyzes the attachment of proline to its cognate tRNA, generating proline-charged tRNA^Pro that is essential for translation of proline codons during protein synthesis . Additionally, evidence suggests that proline-charged tRNA levels serve as regulatory signals for certain virulence genes, similar to mechanisms observed in related bacteria such as Salmonella .

How does proline metabolism connect to L. pneumophila pathogenesis?

Proline metabolism is intricately linked to L. pneumophila pathogenesis through several mechanisms:

• Intracellular infection induces proline biosynthetic genes in L. pneumophila

• Proline-charged tRNA^Pro levels influence expression of specific virulence determinants

• Proline limitation affects bacterial stress responses and virulence gene expression

L. pneumophila, which resides within phagosomes, induces proline biosynthetic genes when inside macrophages . This suggests that proline availability is an important environmental cue during intracellular infection. The bacterium's ability to sense and respond to changes in proline availability may be a key adaptation for survival in host cells.

What experimental systems are used to study recombinant ProS from L. pneumophila?

Standard experimental systems for studying recombinant ProS include:

For genetic manipulation of L. pneumophila, established methods include allelic exchange using plasmids with counter-selection markers such as SacB, which confers sensitivity to sucrose. This allows for construction of unmarked deletions in L. pneumophila while providing flexibility to examine recombination and repair mechanisms .

How can researchers distinguish between ProS effects on translation versus potential regulatory functions?

Distinguishing between ProS effects on translation versus regulatory functions requires multifaceted experimental approaches:

• Conditional expression systems: Utilizing inducible promoters to modulate proS expression levels can help separate essential translation functions from regulatory effects.

• Site-directed mutagenesis: Creating variants with reduced aminoacylation activity but preserved structural integrity can separate enzymatic from potential moonlighting functions.

• tRNA charging assays: Measuring proline-charged tRNA^Pro levels using acid-urea gel electrophoresis or northern blot techniques under different conditions . This approach was effective in demonstrating that proline limitation and hyperosmotic stress decrease the levels of proline-charged tRNA in Salmonella .

• Ribosome profiling: Analyzing ribosome occupancy at proline codons during infection can reveal translation defects versus regulatory effects.

• Protein-protein interaction studies: Identifying ProS interaction partners beyond tRNA^Pro through co-immunoprecipitation, bacterial two-hybrid, or pull-down assays.

What are the methodological challenges in purifying active recombinant L. pneumophila ProS?

Purifying active recombinant L. pneumophila ProS presents several methodological challenges:

• Protein solubility: ProS enzymes can form inclusion bodies in heterologous expression systems. Solution: Optimize expression conditions (temperature, inducer concentration) or use solubility tags such as MBP or SUMO.

• Structural integrity: Preserving the proper folding of multi-domain ProS structure is critical. Approach: Consider using mild purification conditions and including stabilizing agents in buffers.

• Co-purification of tRNAs: Endogenous tRNAs can co-purify with ProS, affecting activity assays. Method: Include high-salt washes (500-800 mM NaCl) during purification to remove bound nucleic acids.

• Activity preservation: ProS activity depends on proper coordination of zinc ions in the active site. Solution: Include low concentrations of zinc (10-50 μM ZnCl₂) in purification buffers.

• Homogeneity assessment: Ensuring preparation homogeneity requires multiple analytical techniques including size-exclusion chromatography, dynamic light scattering, and native PAGE.

Following successful purification, activity can be assessed through aminoacylation assays that monitor the attachment of radiolabeled proline to tRNA^Pro or through ATP-PPi exchange assays.

How can ProS or tRNA^Pro be leveraged as genetic tools in L. pneumophila?

ProS and tRNA^Pro can serve as powerful genetic tools in L. pneumophila research:

• Selectable markers: The essential nature of proS makes it suitable as a selection marker in gene deletion studies.

• Conditional expression: tRNA^Pro suppressor variants can be used for conditional gene expression systems.

• Reporter systems: Leveraging proline codon-rich leader sequences (similar to the mgtP system in Salmonella ) to create reporter constructs for studying environmental responses.

• Gene delivery: The RecA-independent recombination mechanism in L. pneumophila, which utilizes oligonucleotides with homologous DNA stretches (≥21 nucleotides), offers an efficient approach for genetic manipulation . This system requires the primosomal protein PriA and DNA Pol I, making it distinct from other described oligo recombination mechanisms .

• Synthetic biology applications: Manipulating tRNA^Pro charging levels to control expression of specific proline codon-enriched genes.

For efficient genetic manipulation, L. pneumophila researchers can utilize phage recombination coupled with site-specific Flp recombination to construct unmarked deletions while examining the role of single-stranded exonucleases in the process .

What controls and validations are essential when studying ProS function in L. pneumophila?

When studying ProS function in L. pneumophila, the following controls and validations are essential:

Additionally, researchers should consider the stringent response pathway when examining ProS function, as RelA affects ppGpp levels during the stationary phase in L. pneumophila and influences various phenotypes including pigment production and flagellum gene expression .

How does ProS activity relate to L. pneumophila virulence mechanisms?

ProS activity connects to L. pneumophila virulence mechanisms through several pathways:

• Regulation of virulence gene expression: Similar to systems discovered in Salmonella, changes in proline-charged tRNA^Pro levels likely influence expression of specific L. pneumophila virulence factors .

• Stress adaptation: ProS function under stress conditions (including hyperosmotic stress and nutrient limitation) may prepare the bacterium for host cell environments.

• Translation of virulence factors: Many L. pneumophila effector proteins contain proline-rich regions that require efficient ProS activity for proper synthesis.

• Intracellular survival: L. pneumophila induces proline biosynthetic genes during macrophage infection , indicating the importance of proline metabolism during intracellular growth.

• Interaction with bacterial secretion systems: L. pneumophila's Type IV secretion system translocates numerous effector proteins into host cells , and proper synthesis of these effectors depends on functional translation machinery, including ProS.

One particularly relevant mechanism is how L. pneumophila effector glucosyltransferases (lgt's) selectively modify eukaryotic elongation factor 1A, with the modification being enhanced 70-590 fold in the presence of aminoacyl-tRNAs . This demonstrates the critical role of the translation machinery in pathogenesis.

What methodologies can assess the impact of ProS inhibition on L. pneumophila infection?

Researchers can employ several methodologies to assess the impact of ProS inhibition on L. pneumophila infection:

• Chemical inhibition studies: Using ProS-specific inhibitors to assess effects on bacterial survival and virulence.

• Conditional depletion systems: Employing inducible degradation tags or expression systems to modulate ProS levels during infection.

• Host cell infection models: Quantifying bacterial replication in human macrophage cell lines (such as HL-60) or amoebae (such as Acanthamoeba castellanii) following ProS manipulation.

• Transcriptomic analysis: RNA-seq to identify genes differentially expressed upon ProS inhibition during infection.

• Animal infection models: Assessing virulence in guinea pig or mouse models of Legionnaires' disease.

• Microscopy techniques: Examining changes in intracellular trafficking and vacuole formation through fluorescence microscopy.

• Competition assays: Mixed infections with wild-type and ProS-deficient strains to detect fitness defects in vivo.

How does L. pneumophila ProS compare to homologs in other bacterial pathogens?

Comparative analysis of L. pneumophila ProS with homologs in other bacterial pathogens reveals important insights:

L. pneumophila shows extensive evidence of recombination events and horizontal gene transfer (HGT) throughout its genome . For example, a 65-kb pathogenicity island described first in L. pneumophila strain Philadelphia is present in several L. pneumophila strains and also in other Legionella species like L. anisa . This genomic plasticity suggests that ProS function may be integrated into species-specific regulatory networks.

What can be learned from studying ProS across different Legionella species?

Studying ProS across different Legionella species can provide valuable insights into:

• Evolutionary conservation: Determining core functions preserved across the genus versus species-specific adaptations.

• Host range determinants: Correlating ProS sequence variations with host range differences among Legionella species.

• Virulence adaptations: Identifying how ProS function may be optimized for different ecological niches or host cell types.

• Regulatory network evolution: Understanding how ProS integrates into species-specific regulatory circuits.

• Drug target evaluation: Assessing ProS as a potential broad-spectrum target for anti-Legionella therapeutics.

This comparative approach is particularly relevant given the genomic diversity within the Legionella genus, where even effector proteins critical to pathogenicity show poor conservation across species . Notably, while most Legionnaires' disease cases (95-98%) are caused by L. pneumophila , the genus includes over 60 known species with varying pathogenic potential .

What are the optimal expression systems for producing recombinant L. pneumophila ProS?

Optimal expression systems for producing recombinant L. pneumophila ProS include:

• E. coli BL21(DE3) derivatives: These strains lack certain proteases and provide tight control of T7 promoter-driven expression. Codon optimization may be necessary due to differences in codon usage between L. pneumophila and E. coli.

• Expression vectors: pET series vectors with His-tags or other affinity tags facilitate purification. Including a cleavable tag can help obtain native protein after purification.

• Expression conditions:

  • Temperature: 16-18°C often yields more soluble protein than 37°C

  • Induction: Low IPTG concentrations (0.1-0.5 mM) may reduce inclusion body formation

  • Media: Rich media (such as TB or 2XYT) can improve yields

  • Additives: Including 5-10% glycerol and 50-100 mM NaCl in the growth medium can improve solubility

• Legionella-based expression systems: For studying in vivo function, expression from its native promoter on a plasmid like pMMB207c following a ptac promoter can be used with 1 mM IPTG for induction .

• Cell-free expression systems: When dealing with potentially toxic proteins, cell-free systems based on E. coli extracts offer an alternative approach.

How can researchers accurately measure ProS activity and tRNA charging levels in L. pneumophila?

Researchers can accurately measure ProS activity and tRNA charging levels in L. pneumophila using these methods:

• In vitro aminoacylation assays:

  • Reaction components: Purified ProS, tRNA^Pro, ATP, Mg²⁺, and radiolabeled proline

  • Measurement: Incorporation of radioactivity into TCA-precipitable material

  • Controls: Heat-inactivated enzyme and no-tRNA controls

• tRNA charging level determination:

  • Acid-urea gel electrophoresis separation of charged and uncharged tRNAs

  • Northern blot analysis with tRNA^Pro-specific probes

  • Quantification of aminoacyl-tRNA versus total tRNA^Pro

• In vivo charging state analysis:

  • Acid extraction of total RNA under conditions that preserve the aminoacyl bond

  • Oxidation with periodate to distinguish charged from uncharged tRNAs

  • Quantitative RT-PCR analysis of tRNA^Pro species

• Mass spectrometry approaches:

  • LC-MS/MS analysis of nuclease-digested tRNAs for direct measurement of modified nucleosides

  • Intact mass analysis of purified tRNA^Pro species

• Reporter systems:

  • Construct reporter genes with proline codon-rich leader sequences

  • Monitor reporter expression as a proxy for tRNA^Pro charging levels

These approaches have been successfully used to demonstrate that conditions like proline limitation and hyperosmotic stress can decrease levels of proline-charged tRNA, which serves as a regulatory signal affecting gene expression .

What emerging technologies could advance our understanding of ProS function in L. pneumophila?

Emerging technologies that could advance our understanding of ProS function in L. pneumophila include:

• CRISPR interference (CRISPRi): For tunable repression of proS expression without complete deletion, allowing study of partial loss-of-function phenotypes.

• Ribosome profiling: For genome-wide analysis of translation efficiency at proline codons under various conditions.

• Time-resolved cryo-EM: To capture conformational changes during the aminoacylation reaction.

• Proximity labeling proteomics: Using enzyme-catalyzed proximity labeling (BioID or APEX) fused to ProS to identify interaction partners in vivo.

• tRNA sequencing (tRNA-seq): For comprehensive analysis of tRNA modifications and charging states.

• Nanopore direct RNA sequencing: For real-time monitoring of tRNA charging states without amplification biases.

• Microfluidics: For single-cell analysis of ProS activity and its correlation with virulence gene expression.

• Super-resolution microscopy: To visualize subcellular localization and dynamics of ProS during different growth phases and host cell infection.

• AlphaFold and related AI approaches: For improved structural predictions and identification of potential regulatory or interaction sites.

What are the key unresolved questions about ProS in L. pneumophila pathogenesis?

Key unresolved questions about ProS in L. pneumophila pathogenesis include:

• Regulatory mechanisms: Does ProS or proline-charged tRNA^Pro directly participate in regulating virulence gene expression similar to the mechanisms observed in Salmonella ?

• Stress response integration: How is ProS activity modulated during environmental stress and host cell infection?

• Host interaction: Do host factors directly influence ProS activity during intracellular replication?

• Moonlighting functions: Does ProS perform additional non-canonical functions beyond aminoacylation, as observed for some other aminoacyl-tRNA synthetases?

• Species differences: How do variations in ProS across Legionella species contribute to differences in host range and virulence?

• Therapeutic potential: Can ProS be targeted for antimicrobial development without affecting host cell protein synthesis?

• Evolutionary adaptations: Has ProS evolved specific features to accommodate the intracellular lifestyle of L. pneumophila?

• Integration with other pathways: How does ProS function intersect with other regulatory systems such as the RelA-mediated stringent response that affects stationary phase phenotypes in L. pneumophila ?

• Horizontal gene transfer implications: Given L. pneumophila's capacity for extensive recombination and horizontal gene transfer , has ProS acquired unique features through these evolutionary mechanisms?