Recombinant Salmonella newport Phosphatidylserine decarboxylase proenzyme (psd)

What is phosphatidylserine decarboxylase (PSD) and what is its function in Salmonella newport?

PSD is a critical enzyme in phospholipid metabolism that catalyzes the decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine (PE), a major component of bacterial membranes. In Salmonella newport, as in other bacteria, PE constitutes a significant portion of membrane phospholipids and is essential for proper membrane function, including permeability, protein folding, and cellular division.

The reaction catalyzed by PSD is:
Phosphatidylserine → Phosphatidylethanolamine + CO₂

This reaction is fundamental to bacterial membrane biogenesis, as PE typically comprises 70-80% of the membrane phospholipids in gram-negative bacteria like Salmonella. Proper membrane composition is crucial for bacterial survival, particularly under stress conditions encountered during host infection .

How does the PSD proenzyme undergo activation?

PSD is initially synthesized as an inactive proenzyme that undergoes autoproteolytic cleavage to form the active enzyme. This self-processing event is essential for catalytic activity. The activation process involves:

• The proenzyme undergoes self-cleavage at a conserved motif (typically LGST in bacteria, though some variations exist)

• This cleavage results in the formation of two non-identical subunits: an α-subunit and a β-subunit

• During the cleavage, a pyruvoyl prosthetic group is created at the N-terminus of the α-subunit

• The pyruvoyl group serves as the essential catalytic center, forming a Schiff base with the substrate phosphatidylserine during the decarboxylation reaction

Site-directed mutagenesis studies on similar PSDs have demonstrated that mutations in the cleavage site motif can completely abolish enzyme activity by preventing formation of the pyruvoyl prosthetic group. For example, in Plasmodium falciparum PSD (PfPSD), which has a VGSS motif (positions 314-317), mutations showed dramatically different effects on enzyme processing and activity .

How is PSD genetically regulated in Salmonella newport?

Based on studies of PSD regulation in other bacteria, several mechanisms likely control PSD expression and activity in Salmonella newport:

The gene encoding PSD in Salmonella enterica is likely subject to similar regulatory mechanisms as in related bacteria, potentially involving stress responses that adjust membrane composition during infection and environmental adaptation .

What experimental methods are most effective for expressing and purifying recombinant Salmonella newport PSD?

Based on successful approaches with other bacterial PSDs, the following methods are recommended:

Expression Systems:

Optimized Expression Strategy:

• Use pET or pMAL vectors with fusion tags (MBP-His₆ recommended)

• Express at lower temperatures (16-20°C) to enhance proper folding

• Induce with lower IPTG concentrations (0.1-0.25 mM)

• Utilize TB or 2xYT media supplemented with glucose

Purification Protocol:

• Cell disruption via sonication or French press

• Membrane fraction isolation through differential centrifugation

• Solubilization with mild detergents (DDM, LDAO, or Triton X-100)

• Two-step chromatography:

  • IMAC for His-tagged proteins or amylose resin for MBP fusion

  • Size exclusion chromatography for final purification

After purification, enzyme activity should be verified using either a fluorescence-based assay with distyrylbenzene-bis-aldehyde (DSB-3) for PE detection or radiometric assay using ¹⁴C-labeled PS. The fluorescence assay allows selective monitoring of PE production (λex = 403 nm, λem = 508 nm) with strong discrimination against the PS substrate .

How do mutations in the active site of Salmonella newport PSD affect enzymatic activity?

Mutations in the active site of PSD can profoundly impact its enzymatic activity. Based on site-directed mutagenesis studies of similar enzymes, we can predict the effects of key mutations:

Critical Residues and Their Functions:

Effects of Specific Mutations:
Studies on the VGSS motif in Plasmodium falciparum PSD show that:

• V314A: Reduced proenzyme processing efficiency by 75%

• G315A: Completely abolished processing and activity

• S316A: Severely impaired processing and reduced activity by 95%

• S317A: Reduced processing efficiency by 50%

Structure-guided mutagenesis analyses have confirmed key residues involved in phospholipid recognition, decarboxylation of PS, and maturation of PSD. The processing of the proenzyme into two non-identical subunits (α and β) is particularly sensitive to mutations in the conserved motif region .

What approaches are most effective for screening inhibitors of Salmonella newport PSD?

High-throughput screening for PSD inhibitors can employ several complementary approaches:

Fluorescence-Based Assay Platform:
A recently developed fluorescence assay using distyrylbenzene-bis-aldehyde (DSB-3) provides significant advantages for inhibitor screening:

• High sensitivity for PE detection (λex = 403 nm, λem = 508 nm)

• Strong discrimination against PS substrate

• Compatible with 96- and 384-well plate formats

• Works with purified enzyme, crude extracts, and membrane fractions

Workflow for Inhibitor Screening Campaign:

• Primary Screen:

  • Assay optimization for Z' factor >0.7

  • Screening at single concentration (10-20 μM)

  • Hit threshold: >50% inhibition

• Secondary Validation:

  • Dose-response analysis (IC₅₀ determination)

  • Counter-screening against mammalian PSDs

  • Orthogonal confirmation (radiometric assay)

• Mechanism Studies:

  • Enzyme kinetics to determine inhibition type

  • Binding studies (thermal shift, ITC)

  • Structural studies with bound inhibitors

Compound Libraries to Consider:

• FDA-approved drug libraries (repurposing approach)

• Natural product collections

• Focused libraries targeting enzymes with pyruvoyl prosthetic groups

• Fragment libraries for structure-based optimization

This approach is readily amenable to high-throughput screening and should prove useful for identifying inhibitors of PSD enzymes across diverse phyla, including Salmonella newport .

How does the kinetic profile of Salmonella newport PSD compare to PSDs from other organisms?

While specific kinetic data for Salmonella newport PSD must be experimentally determined, comparison with characterized PSDs provides valuable insights:

Comparative Kinetic Parameters:

*Estimated values based on related bacterial PSDs; requires experimental confirmation

Factors Affecting Kinetic Measurements:

• Detergent choice significantly impacts measured parameters

• Substrate presentation (liposomes vs. mixed micelles)

• Membrane composition in cellular assays

• Assay methodology (fluorescence vs. radiometric)

What technical challenges exist in crystallizing Salmonella newport PSD for structural studies?

Membrane proteins like PSD present significant challenges for crystallization. The following technical hurdles must be addressed:

Major Crystallization Challenges:

• Membrane protein solubilization:

  • Identifying optimal detergents that maintain protein folding

  • Balancing detergent concentration to effectively solubilize without denaturing

• Protein heterogeneity:

  • Mixed populations of proenzyme and processed enzyme

  • Conformational flexibility inherent to membrane proteins

• Crystal contacts:

  • Limited hydrophilic surfaces for crystal formation

  • Detergent micelles interfering with protein-protein contacts

Successful Strategies from Recent PSD Structures:
Recent successful crystallization of bacterial PSDs employed:

• Detergent screening (DDM, LDAO, OG)

• Lipid addition during purification (PE, PC)

• Surface engineering to enhance crystallizability

• Lipidic cubic phase (LCP) crystallization methods

Alternative Structural Approaches:
When crystallization proves challenging, alternative methods include:

• Cryo-electron microscopy (cryo-EM)

• NMR of specific domains

• Molecular dynamics simulations based on homology models

X-ray crystal structures of bacterial PSDs have been achieved at resolutions of 1.90 and 2.63 Å for apo states, and 2.12 and 2.70 Å for PE-bound states, providing valuable templates for modeling Salmonella newport PSD .

How can genomic and proteomic approaches be integrated to study PSD's role in Salmonella newport pathogenesis?

A comprehensive multi-omics approach can provide deep insights into PSD's role in pathogenesis:

Integrated Research Framework:

• Genomic Analysis:

  • Comparative genomics across Salmonella serovars to identify PSD variations

  • CRISPR-Cas9 gene editing for precise mutations in PSD

  • Transcriptomic profiling (RNA-seq) during infection stages

• Proteomic Investigation:

  • Quantitative proteomics to measure PSD levels in infection models

  • Analysis of the processing efficiency under various conditions

  • Protein-protein interaction studies to identify functional partners

• Lipidomic Integration:

  • Mass spectrometry-based analysis of membrane phospholipid composition

  • Correlation between PE/PS ratios and virulence phenotypes

• Functional Validation:

  • In vivo infection models with PSD-modified strains

  • Host cell interaction studies

Analytical Workflow for S. newport Virulence Studies:

This integrated approach can reveal how PSD activity contributes to Salmonella virulence, particularly in multidrug-resistant strains like the Newport MDR-AmpC isolates that have been implicated in numerous outbreaks .

How does Salmonella newport PSD activity relate to antibiotic resistance mechanisms?

While direct evidence linking PSD to antibiotic resistance is limited, several potential mechanisms exist:

Membrane-Related Resistance Mechanisms:

• Permeability barrier:

  • PE composition affects membrane fluidity and permeability

  • Altered PE:PS ratios may reduce antibiotic penetration

  • Modified membrane architecture can enhance physical barrier function

• Efflux pump function:

  • Membrane phospholipid composition affects assembly and function of efflux systems

  • These pumps actively export antibiotics, contributing to multidrug resistance

  • Optimal pump function depends on appropriate membrane environment

Evidence from Salmonella newport MDR Strains:
Multidrug-resistant S. newport isolates (MDR-AmpC) show resistance to at least nine antimicrobials, including extended-spectrum cephalosporins. Studies of these strains reveal:

• 60% of studied S. newport isolates were identified as MDR-AmpC

• These isolates contained transferable resistance genes

• Class 1 integrons containing resistance genes were present in 40% of isolates

Recent outbreaks of S. Newport with decreased susceptibility to azithromycin demonstrate the clinical significance of these resistance mechanisms. Whole genome sequencing has been valuable for tracking these resistant strains and identifying potential transmission routes .

Research Approaches to Investigate This Relationship:

• Comparative lipidomics of susceptible vs. resistant strains

• PSD expression studies in response to antibiotic exposure

• Testing whether PSD inhibitors could potentiate antibiotic action

Understanding how membrane composition contributes to resistance could identify new strategies for combating multidrug-resistant S. newport infections .

What are the most effective methods for detecting and measuring PSD activity?

Several complementary methods are available for measuring PSD activity, each with specific advantages:

Fluorescence-Based Assay:
A novel fluorescence assay using distyrylbenzene-bis-aldehyde (DSB-3) offers significant advantages:

• Selective monitoring of PE production (λex = 403 nm, λem = 508 nm)

• Strong discrimination against the PS substrate

• Compatible with 96/384-well plate formats

• Works with purified enzyme, crude extracts, and membrane fractions

This method is particularly valuable for high-throughput applications and provides greater convenience than traditional approaches .

Radiometric Assay:
The traditional method using radiolabeled PS as substrate:

• [¹⁴C]PS substrate with measurement of released ¹⁴CO₂

• Highly specific but requires special handling of radioactive materials

• Less amenable to high-throughput screening

• Standard in the field for over 50 years

Chromatographic Methods:

• HPLC or TLC separation of PS and PE

• Can use fluorescently labeled lipids for detection

• More cumbersome but provides direct product measurement

Comparison of Detection Methods:

For most research applications, the fluorescence-based assay offers the best combination of sensitivity, throughput, and ease of use, making it particularly valuable for inhibitor screening campaigns .

How can PSD function be studied in the context of live Salmonella newport infections?

Studying PSD function during actual infection requires specialized approaches:

In Vivo Infection Models:

• Animal infection models:

  • Mouse typhoid model (systemic infection)

  • Calf enteritis model (intestinal infection)

  • Assessment of bacterial loads in tissues

• Cell culture infection models:

  • Macrophage infection assays (RAW264.7, J774)

  • Epithelial cell invasion (Caco-2, HT-29)

  • Monitoring intracellular survival and replication

Techniques for Studying PSD During Infection:

• Genetic approaches:

  • Conditional expression systems (tetracycline-responsive)

  • Point mutations affecting activity but not stability

  • Reporter fusions to monitor expression in vivo

• Molecular tools:

  • RNA isolation from infected tissues for expression analysis

  • Membrane isolation from recovered bacteria

  • Immunoprecipitation to capture PSD complexes

• Imaging methods:

  • Fluorescent lipid probes to track membrane composition

  • Immunofluorescence microscopy to localize PSD

  • Live cell imaging during infection

Recent Applications:
Whole genome sequencing has been valuable for tracking outbreak strains of Salmonella Newport with resistance to multiple antibiotics, including those with decreased susceptibility to azithromycin. These approaches could be adapted to specifically study PSD function in clinical isolates .

The recent persistent strain of Salmonella Newport (REPJJP01) that has caused outbreaks linked to travel to Mexico and beef products provides an important model system for studying virulence and resistance mechanisms in clinically relevant contexts .

What are the most promising approaches for developing selective inhibitors of Salmonella newport PSD?

Several strategic approaches show potential for developing selective PSD inhibitors:

Target-Based Design Strategies:

• Structure-based design:

  • Virtual screening against bacterial PSD models

  • Fragment-based discovery targeting the active site

  • Design of transition state mimics

• Mechanism-based inhibitors:

  • Compounds targeting the pyruvoyl prosthetic group

  • Suicide substrates that irreversibly modify the active site

  • Allosteric inhibitors affecting enzyme processing

Selectivity Considerations:
To achieve selective inhibition of bacterial PSD over mammalian counterparts:

• Target differences in the cleavage site motif (LGST vs variants)

• Exploit differences in membrane localization

• Design compounds with preferential uptake by bacteria

Promising Chemical Scaffolds:
Based on what is known about PSD's mechanism:

• Phosphonate analogs of phosphatidylserine

• Compounds with reactive groups targeting the pyruvoyl center

• Peptidomimetics targeting the processing site

The development of a fluorescence-based assay system for measuring PSD activity represents a significant advance that makes high-throughput screening more accessible. This assay is highly sensitive and provides strong discrimination against the PS substrate, making it ideal for inhibitor screening campaigns .

How might genetic variation in PSD affect pathogenicity and drug susceptibility in Salmonella newport?

Genetic variation in PSD could substantially impact virulence and treatment outcomes:

Impact of PSD Variants on Virulence:

• Catalytic efficiency:

  • Mutations affecting Km or kcat could alter PE production

  • Changes in PE:PS ratio may affect membrane properties

• Regulation of expression:

  • Promoter variants affecting transcriptional regulation

  • Changes in translation efficiency

• Post-translational processing:

  • Mutations near the cleavage site affecting activation

  • Altered rate of proenzyme processing

Connection to Drug Susceptibility:
The membrane composition influenced by PSD activity may affect:

• Permeability to antibiotics, particularly hydrophobic compounds

• Function of membrane-associated resistance mechanisms

• Survival under antimicrobial pressure

Research Approaches:

• Sequencing PSD in diverse clinical isolates

• Correlating sequence variants with virulence phenotypes

• Experimental introduction of mutations using CRISPR-Cas9

The genomic diversity of Salmonella Newport is evident in subtyping studies, which have identified numerous sequence types even within outbreak isolates. Among 84 isolates in one study, 38 different sequence types were defined using CRISPR-multi-virulence-locus sequence typing (CRISPR-MVLST) .