Recombinant Legionella pneumophila Non-canonical purine NTP pyrophosphatase (lpp2548)

What is the physiological role of lpp2548 in Legionella pneumophila metabolism?

Lpp2548 functions as a non-canonical purine NTP pyrophosphatase that hydrolyzes non-standard purine nucleoside triphosphates. This enzyme likely serves as a "housekeeping" enzyme that maintains nucleotide pool quality by preventing the incorporation of potentially mutagenic non-canonical nucleotides into DNA or RNA. This function may be particularly important during stress conditions such as antibiotic exposure or host immune responses, where damaged nucleotides might accumulate. The enzyme performs hydrolysis of phosphorus-containing anhydrides in non-canonical purine nucleotides, which would otherwise disrupt normal cellular processes through misincorporation into genetic material.

How does lpp2548 distinguish between canonical and non-canonical purine nucleotides?

The specificity of lpp2548 for non-canonical purine nucleotides likely depends on structural recognition of altered hydrogen bonding patterns. Non-canonical base pairs have "hydrogen bonding patterns which differ from the patterns observed in Watson-Crick base pairs" . The enzyme's binding pocket likely contains specific residues that recognize these distinctive patterns in the nucleotide bases. Each of the nucleobases has "distinct edge-specific distribution patterns of their respective hydrogen bond donor and acceptor atoms" , and lpp2548 has evolved to recognize these patterns specifically in modified or damaged purine nucleotides while discriminating against standard adenine and guanine nucleotides.

What regulatory mechanisms control lpp2548 expression during different growth phases?

While specific regulatory mechanisms for lpp2548 aren't directly addressed in the available data, research on L. pneumophila persistence provides relevant context. During the amoeba infection cycle, L. pneumophila exhibits distinct growing and non-growing subpopulations, with the latter potentially representing persister cells . Regulation of lpp2548 likely differs between these populations, with potentially increased expression during stress conditions when non-canonical nucleotides might accumulate. The expression may be tied to growth phase, as "two subpopulations of L. pneumophila Paris bacteria were identified during amoeba infection" , suggesting differential gene expression patterns associated with distinct physiological states.

What expression systems yield optimal activity for recombinant lpp2548?

For optimal expression of recombinant lpp2548, consider the following protocol:

Post-expression analysis should include activity verification using non-canonical purine nucleotides as substrates, with phosphate release measured using malachite green assay or HPLC-based product analysis.

What purification strategy yields the highest specific activity for lpp2548?

A multi-step purification approach ensures high specific activity:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Equilibration buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Elution buffer: Same with 250 mM imidazole

  • Include 1-5 mM β-mercaptoethanol throughout

• Intermediate purification: Ion exchange chromatography

  • Q-Sepharose column for anion exchange

  • Buffer A: 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT

  • Buffer B: Same with 1 M NaCl

  • Linear gradient elution (0-100% Buffer B)

• Polishing: Size exclusion chromatography

  • Superdex 75 or 200 column

  • Buffer: 25 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

Throughout purification, monitor specific activity using an appropriate enzyme assay to track enrichment and ensure proper folding is maintained.

How can enzyme kinetics of lpp2548 be accurately determined?

For accurate enzymatic characterization of lpp2548:

• Prepare reaction mixtures containing:

  • 50 mM Tris-HCl or HEPES buffer (pH 7.5-8.0)

  • 5-10 mM MgCl2 (or other divalent cations for metal dependency studies)

  • 1 mM DTT

  • Varying concentrations of non-canonical purine NTP substrates (0.05-2.0 mM)

  • Purified lpp2548 (10-100 nM)

• Monitor reaction progress using:

  • Malachite green assay for inorganic phosphate release

  • HPLC analysis of substrate consumption and product formation

  • Coupled enzyme assays linking pyrophosphate release to NAD(P)H oxidation

• Calculate kinetic parameters:

  • Plot initial velocities versus substrate concentration

  • Fit data to Michaelis-Menten equation to determine Km and kcat

  • Compare parameters across different non-canonical substrates

Additional controls should include testing canonical purine NTPs to confirm specificity and using known inhibitors to validate the assay system.

What techniques can distinguish lpp2548 activity from other nucleotide-processing enzymes in cellular extracts?

To specifically measure lpp2548 activity in complex cellular extracts:

• Develop substrate selectivity profiles:

  • Test various non-canonical nucleotide substrates to identify those with highest specificity for lpp2548

  • Create activity fingerprints based on relative activities with different substrates

• Implement immunological approaches:

  • Use anti-lpp2548 antibodies for immunodepletion to compare activities before and after depletion

  • Perform activity staining following native PAGE separation

• Genetic approaches:

  • Compare extracts from wild-type and lpp2548 knockout strains

  • Complementation with recombinant lpp2548 to confirm activity differences

• Inhibitor-based discrimination:

  • Identify selective inhibitors that target lpp2548 but not other nucleotide-processing enzymes

  • Use differential inhibition profiles to distinguish enzyme activities

These approaches are particularly important given that "highly variable genomes between ST groups make difficult the identification of persistence pathways involved in L. pneumophila" .

How does lpp2548 activity correlate with persister cell formation in L. pneumophila?

While direct evidence linking lpp2548 to persistence isn't provided in the search results, a methodological approach to investigate this correlation would include:

• Comparing lpp2548 expression levels in growing versus non-growing subpopulations:

  • Use the Timer bac system that "allows us to identify potential persister cells"

  • Isolate RNA from sorted subpopulations for RT-qPCR analysis of lpp2548 expression

• Creating and characterizing lpp2548 knockout mutants:

  • Evaluate persister frequency using "biphasic killing kinetics using ofloxacin stress"

  • Compare persistence phenotypes between wild-type and knockout strains

  • Complementation studies to confirm phenotype specificity

• Analyzing lpp2548 activity during stress conditions:

  • Measure enzyme activity in cell extracts before and after antibiotic exposure

  • Correlate activity levels with persister frequency

The search results indicate that "persister formation appears to be strain or ST (sequence type) dependent" , suggesting potential variation in lpp2548 expression or activity across different L. pneumophila strains.

What is the effect of lpp2548 deletion on intracellular replication within amoebae and macrophages?

To determine how lpp2548 affects L. pneumophila's intracellular lifecycle:

• Construct lpp2548 deletion mutants and fluorescently labeled strains:

  • Use techniques similar to those described for the Timer bac system, which was "set up in the L. pneumophila Paris reference strain and all clinical isolates"

• Perform infection assays in:

  • Amoeba models like Acanthamoeba polyphaga, as mentioned in the search results

  • Human macrophage cell lines or primary alveolar macrophages

• Quantify replication parameters including:

  • Uptake efficiency

  • Intracellular growth rate

  • Maximum bacterial load

  • Proportion of non-growing bacteria within the population

• Analyze stress response:

  • Evaluate sensitivity to host-generated reactive oxygen and nitrogen species (ROS/NOS)

  • The search results highlight the importance of understanding "the inducible mechanism of persistence in relation to stress generated towards intracellular bacteria (i.e. ROS/NOS molecules)"

Is lpp2548 expression altered in clinical isolates from recurring legionellosis cases?

Based on the methodology described in the search results for analyzing clinical isolates from recurring legionellosis:

• Compare lpp2548 sequence and expression between paired isolates:

  • Analyze "7 pairs of L. pneumophila clinical isolates, with isolate pairs corresponding to two episodes (early and late) of legionellosis in the same patient"

  • Perform RT-qPCR to measure lpp2548 expression levels

• Correlate with persistence phenotypes:

  • The search results indicate that "the proportion of non-growing cells was stable within each pair of isolates (early and late)" for most sequence types

  • Analyze if lpp2548 expression correlates with this stability or with exceptions like ST48

• Functional analysis of lpp2548 variants:

  • Express and purify lpp2548 from different clinical isolates

  • Compare enzymatic properties and substrate specificities

  • Evaluate if functional differences might contribute to persistence variation

The search results note that "recurring Legionnaires' disease is often the result of relapse rather than reinfection" , highlighting the importance of understanding potential molecular mechanisms like lpp2548 function in disease recurrence.

How do stress conditions affect lpp2548 activity and expression?

To assess how environmental and host-induced stresses impact lpp2548:

• Expose L. pneumophila cultures to relevant stressors:

  • Antibiotic stress (e.g., ofloxacin at sub-MIC concentrations)

  • Oxidative stress (H2O2, paraquat)

  • Nutrient limitation

  • Temperature stress

• Monitor lpp2548 expression changes:

  • RT-qPCR to measure transcript levels

  • Western blotting to assess protein levels

  • Reporter gene constructs (e.g., lpp2548 promoter fused to GFP)

• Measure enzyme activity in extracts from stressed cells:

  • Use specific activity assays with non-canonical purine substrates

  • Compare with housekeeping enzymes as controls

• Correlate with persistence phenotypes:

  • The search results indicate that "enhanced persister formation during the host cell infection" occurs

  • Determine if lpp2548 expression changes correlate with increased persister frequency

This approach would help determine if lpp2548 is part of a stress response mechanism that contributes to the "transient reversible phenotypic status of persistence" described in the search results.

What structural features determine substrate specificity of lpp2548?

While specific structural data for lpp2548 isn't provided in the search results, a methodological approach to determine substrate specificity determinants would include:

• Homology modeling based on related NTP pyrophosphatases:

  • Identify conserved catalytic residues

  • Predict substrate binding pocket architecture

• Site-directed mutagenesis of key residues:

  • Target amino acids in the predicted binding pocket

  • Focus on residues that might interact with non-canonical base modifications

  • Create a panel of mutants with altered substrate specificity

• Structural analysis techniques:

  • X-ray crystallography of lpp2548 with bound substrate analogs

  • Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Computational docking studies:

  • Compare binding energies of canonical versus non-canonical purine nucleotides

  • Identify key interaction points that determine specificity

The search results mention that nucleobases can be "schematically represented as triangles with one of their vertices linked to the sugar, and the three sides accounting for three edges through which they can form hydrogen bonds" , which could inform modeling of substrate recognition by lpp2548.

How conserved is lpp2548 across different Legionella species and sequence types?

To analyze lpp2548 conservation:

• Perform comparative genomic analysis:

  • Conduct BLAST searches across Legionella genomes

  • Create multiple sequence alignments of lpp2548 homologs

  • Calculate percent identity and similarity

• Analyze conservation patterns:

  • Identify highly conserved domains (likely catalytic regions)

  • Map variable regions that might confer strain-specific properties

  • Compare with other housekeeping genes to determine relative conservation rate

• Correlate with phylogenetic relationships:

  • The search results mention "7 different sequence types (ST)" of L. pneumophila

  • Determine if lpp2548 conservation follows ST classification patterns

• Functional comparison of variants:

  • Express and characterize lpp2548 from different species/STs

  • Compare substrate specificity, catalytic efficiency, and inhibitor sensitivity

What genomic context surrounds lpp2548 and does it suggest functional relationships?

A methodological approach to analyze lpp2548's genomic context:

• Examine the operon structure:

  • Determine if lpp2548 is co-transcribed with other genes

  • Identify transcription start sites and terminators

• Analyze neighboring genes:

  • Identify functions of adjacent genes

  • Look for other nucleotide metabolism or stress response genes

• Perform comparative genomic analysis:

  • Compare gene arrangements around lpp2548 across Legionella strains

  • Identify conserved gene clusters that might indicate functional relationships

• Analyze co-expression patterns:

  • Conduct RNA-seq under various conditions

  • Identify genes with expression patterns similar to lpp2548

How do post-translational modifications affect lpp2548 activity?

To investigate potential post-translational modifications (PTMs) of lpp2548:

• Identify potential PTM sites:

  • In silico prediction of phosphorylation, acetylation, or other modification sites

  • Comparison with known modifications in homologous proteins

• Mass spectrometry-based approaches:

  • Purify native lpp2548 from L. pneumophila under different conditions

  • Perform LC-MS/MS analysis to identify and quantify modifications

  • Compare modification patterns between growing and non-growing subpopulations

• Functional impact assessment:

  • Generate site-directed mutants mimicking or preventing modifications

  • Compare enzymatic properties with wild-type protein

  • Assess impact on substrate specificity and catalytic efficiency

• In vivo significance:

  • Create knock-in strains expressing modification-mimicking variants

  • Evaluate persistence phenotypes and stress responses

How can lpp2548 be used as a biomarker for detecting persistent L. pneumophila infections?

While the search results don't directly address lpp2548 as a biomarker, a methodological approach based on L. pneumophila detection principles would include:

• Developing lpp2548-specific detection methods:

  • PCR-based detection targeting lpp2548 sequence

  • Antibody-based assays to detect the protein in clinical samples

  • Activity-based probes using non-canonical substrates with fluorescent products

• Clinical validation:

  • Test sensitivity and specificity in samples from patients with confirmed legionellosis

  • Compare with current detection methods like "Buffered charcoal yeast extract (BCYE) agar containing 0.1% alpha-ketoglutarate"

• Correlation with persistence phenotypes:

  • Determine if lpp2548 expression levels or variants correlate with recurrence risk

  • The search results note that "recurring Legionnaires' disease is often the result of relapse rather than reinfection"

• Point-of-care application development:

  • Design rapid diagnostic tests based on lpp2548 detection

  • Optimize for clinical laboratory implementation

This approach could potentially address the challenge noted in the search results that "investigations into persistence in a clinical context and the mechanisms involved may allow us to combat this issue" of treatment failure in legionellosis.

Could lpp2548 serve as a target for anti-persister therapeutics against L. pneumophila?

To evaluate lpp2548 as a therapeutic target:

• Validate the importance of lpp2548 in persistence:

  • Create knockout mutants and assess persistence capacity

  • Determine if chemical inhibition of lpp2548 affects persister formation or reactivation

• Develop and screen inhibitors:

  • Design substrate analogs that bind but aren't hydrolyzed

  • Perform high-throughput screening of chemical libraries

  • Assess selectivity for bacterial versus human homologs

• Evaluate efficacy in infection models:

  • Test inhibitors in amoeba and macrophage infection models

  • Use the Timer bac system described in the search results to monitor effects on non-growing subpopulations

  • Assess synergy with conventional antibiotics

• Address potential challenges:

  • The search results note that "highly variable genomes between ST groups make difficult the identification of persistence pathways"

  • Confirm inhibitor efficacy across diverse clinical isolates

This approach could potentially address the treatment failure issue mentioned in the search results: "Treatment failure in legionellosis is a serious issue as infections have a 5-10% mortality rate" .

What experimental models best capture the role of lpp2548 in L. pneumophila environmental persistence?

Based on methodologies described in the search results:

• Amoeba infection model:

  • Use Acanthamoeba polyphaga as described in the search results

  • Implement the Timer bac system to distinguish growing from non-growing bacteria

  • Compare wild-type and lpp2548 mutant strains

• Biofilm formation model:

  • Establish L. pneumophila biofilms on appropriate surfaces

  • Monitor lpp2548 expression in different biofilm regions

  • Assess impact of lpp2548 deletion on biofilm formation and persistence

• Environmental microcosm systems:

  • Create artificial water systems mimicking cooling towers or water distribution systems

  • Apply procedures described for "the recovery of Legionella from the environment"

  • Track population dynamics and persistence over extended periods

• Stress resistance assays:

  • Subject bacteria to environmental stressors (temperature fluctuation, desiccation, chlorination)

  • Compare survival of wild-type and lpp2548 mutants

  • Measure lpp2548 expression changes in response to stressors

These approaches would help determine if lpp2548 contributes to the environmental persistence of L. pneumophila, which is described in the search results as "ubiquitous in freshwater environments" .

How can high-throughput approaches advance our understanding of lpp2548 function?

Modern high-throughput methodologies to study lpp2548 include:

• Genome-wide interaction screens:

  • Transposon mutagenesis coupled with lpp2548 deletion

  • CRISPR interference screening to identify genetic interactions

  • Synthetic genetic array analysis to map functional relationships

• Chemical genomics approaches:

  • Screen for small molecules that specifically affect lpp2548 mutants

  • Identify pathways that become essential in the absence of lpp2548

• Proteomics-based interactome mapping:

  • Affinity purification-mass spectrometry to identify protein interaction partners

  • Crosslinking mass spectrometry to capture transient interactions

  • Compare interactomes under normal growth versus persistence-inducing conditions

• Transcriptomics in clinical isolates:

  • Analyze gene expression differences between the "7 pairs of L. pneumophila clinical isolates" mentioned in the search results

  • Correlate lpp2548 expression with global transcriptional patterns

  • Identify co-regulated genes that might function in the same pathway

These approaches could advance understanding of lpp2548's role in the "transient reversible phenotypic status of persistence" described in the search results, potentially revealing new strategies to combat recurring legionellosis.