Recombinant Salmonella dublin Cardiolipin synthase (cls)

What is the basic structure of Salmonella dublin Cardiolipin synthase?

Salmonella dublin Cardiolipin synthase (cls/clsA) is a full-length protein consisting of 486 amino acids. The protein contains characteristic phospholipase-D motifs that are essential for its catalytic function. The complete amino acid sequence includes hydrophobic transmembrane domains, particularly evident in the N-terminal region where sequences such as "MTTFYTVVSWLVILGYWVLIAGVTLRILMKR" indicate membrane association properties . The protein typically features an N-terminal 10xHis-tag when produced recombinantly, facilitating purification and detection in experimental settings .

How does Cardiolipin synthase function in Salmonella dublin compared to other Salmonella serovars?

Cardiolipin synthase in Salmonella dublin functions similarly to other Salmonella serovars, particularly S. Typhimurium, for which more extensive research data exists. In Salmonella species, three distinct cardiolipin synthases (ClsA, ClsB, and ClsC) contribute to cardiolipin biosynthesis with partially overlapping yet distinct functions:

• ClsA (the predominant synthase during logarithmic growth) catalyzes the transfer of a phosphatidyl group from one phosphatidylglycerol (PGl) molecule to a second PGl molecule to form cardiolipin .

• ClsB is a more promiscuous enzyme activated during stationary-phase stress that can synthesize cardiolipin from two PGl molecules, but can also produce other phospholipids including phosphatidylglycerol, phosphatidylalcohols, phosphatidyltrehalose (PT), and diphosphatidyltrehalose (diPT) .

• ClsC generates cardiolipin from single PGl and single phosphatidylethanolamine (PE) precursor substrates and contributes to the cardiolipin pool primarily during stress conditions .

While S. dublin-specific research is limited, the conservation of these enzymes across Salmonella species suggests similar functional mechanisms with potential serovar-specific regulatory differences.

What expression systems are most effective for producing recombinant Salmonella dublin Cardiolipin synthase?

For the production of recombinant Salmonella dublin Cardiolipin synthase, Escherichia coli-based expression systems have proven most effective. The available literature indicates successful expression using in vitro E. coli expression systems with N-terminal His-tagging . This approach leverages the following advantages:

• High protein yield due to E. coli's rapid growth and well-established induction systems

• Simplified purification via affinity chromatography using the His-tag

• Compatibility with membrane protein expression, critical for transmembrane proteins like Cardiolipin synthase

The protein can be provided in either liquid form or as lyophilized powder, with the latter showing extended shelf life of approximately 12 months at -20°C/-80°C compared to 6 months for the liquid form . The optimal buffer composition for maintaining protein stability appears to be Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .

What are the optimal conditions for assaying Cardiolipin synthase activity in vitro?

The optimal conditions for assaying Salmonella dublin Cardiolipin synthase activity in vitro include:

Activity can be measured through several methods:

• Thin-layer chromatography (TLC) analysis of lipid extracts, which has been successfully used to visualize cardiolipin production

• LC-MS/MS analysis for more precise quantification of cardiolipin species

• Radiolabeled substrate incorporation assays measuring the conversion of labeled PGl to cardiolipin

When designing experiments, it's critical to include appropriate controls, particularly using known cls mutants (ΔclsA, ΔclsB, ΔclsC) to establish baseline comparisons .

How can researchers effectively differentiate between the activities of ClsA, ClsB, and ClsC in Salmonella dublin?

Differentiating between the activities of the three cardiolipin synthases requires a multi-faceted experimental approach:

• Genetic approach: Create single, double, and triple deletion mutants (ΔclsA, ΔclsB, ΔclsC, ΔclsAB, ΔclsAC, ΔclsBC, and ΔclsABC) to isolate the contribution of each synthase .

• Growth phase-specific analysis: Monitor cardiolipin production during:

  • Logarithmic growth phase (primarily ClsA activity)

  • Stationary phase (increased ClsB and ClsC activity)

• Stress condition testing: Expose bacteria to:

  • High osmolarity conditions (activates ClsC)

  • Stationary phase stress (activates both ClsB and ClsC)

• Substrate specificity analysis: Provide different substrate combinations:

  • PGl only (utilized by all three synthases)

  • PGl + PE (preferentially utilized by ClsC)

  • Alternative substrates for ClsB (to detect production of phosphatidylalcohols, PT, and diPT)

• Complementation studies: Reintroduce individual cls genes into the triple mutant (ΔclsABC) to confirm the specific function of each synthase .

Research has shown that when clsA and clsC are deleted, clsB alone is sufficient to promote certain phenotypes, and similarly, when clsB is deleted, clsA and clsC together can compensate . This functional redundancy requires careful experimental design to properly attribute specific activities.

What purification strategies yield the highest purity and activity of recombinant Salmonella dublin Cardiolipin synthase?

Purification of recombinant Salmonella dublin Cardiolipin synthase with maintained activity requires specialized approaches due to its membrane-associated nature:

• Initial extraction:

  • Gentle cell lysis using sonication or French press

  • Membrane fraction isolation by ultracentrifugation

  • Solubilization using mild detergents (0.5-1% n-dodecyl β-D-maltoside or 1% Triton X-100)

• Affinity chromatography:

  • Ni-NTA affinity chromatography exploiting the N-terminal 10xHis-tag

  • Washing with increasing imidazole concentrations (10-40 mM)

  • Elution with higher imidazole (250-300 mM)

• Secondary purification:

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for further purification

• Detergent exchange and concentration:

  • Gradual exchange to milder detergents that maintain activity

  • Use of centrifugal concentrators with appropriate molecular weight cut-offs

• Storage optimization:

  • Addition of 6% Trehalose as a stabilizing agent

  • Maintenance at pH 8.0 in Tris/PBS-based buffer

  • Flash freezing and storage at -80°C, or lyophilization for extended shelf life

Typical yields range from 1-5 mg of purified protein per liter of bacterial culture, with specific activity highest when purification is completed rapidly at 4°C throughout the process.

How does Cardiolipin synthase activity in Salmonella dublin contribute to bacterial pathogenesis?

The relationship between Cardiolipin synthase activity and Salmonella dublin pathogenesis involves complex mechanisms related to membrane composition and host-pathogen interactions:

Research with S. Typhimurium in C57BL/6J mice showed that cardiolipin biosynthesis genes were not strictly required for pathogenesis following oral or systemic infection, suggesting potential compensatory mechanisms or serovar-specific differences that warrant further investigation in S. dublin .

What is the correlation between Cardiolipin synthase expression and antimicrobial resistance in Salmonella dublin?

The correlation between Cardiolipin synthase expression and antimicrobial resistance in Salmonella dublin represents an emerging area of research with significant implications:

Recent studies have observed an increasing prevalence of antimicrobial resistance in S. Dublin strains, particularly those causing bloodstream infections in humans . While direct causative relationships between cardiolipin synthase expression and antimicrobial resistance haven't been conclusively established, several mechanistic connections are plausible:

• Membrane permeability alterations:

  • Cardiolipin significantly affects membrane fluidity and permeability

  • Changes in membrane phospholipid composition can reduce uptake of hydrophilic antibiotics

  • Higher cardiolipin content may create more tightly packed membranes resistant to certain antimicrobials

• Stress response coordination:

  • Cardiolipin synthesis increases under various stress conditions

  • Antibiotic exposure represents a significant stress that may trigger upregulation of cls genes

  • This response could coordinate with other resistance mechanisms

• Plasmid-mediated resistance connections:

  • Recent research has identified novel hybrid plasmids encoding both antimicrobial resistance and mercuric resistance in Australian S. Dublin lineages

  • These emerging virulent and resistant lineages may have altered membrane composition or cls expression patterns

• Biofilm formation influence:

  • Cardiolipin content affects bacterial adherence properties

  • Enhanced biofilm formation, potentially linked to cardiolipin levels, can increase antibiotic tolerance

Further research specifically examining cardiolipin synthase expression levels in antimicrobial-resistant S. Dublin isolates compared to susceptible strains would help clarify these potential correlations. Additionally, investigating how antibiotics affect cls gene expression might reveal important regulatory mechanisms relevant to resistance development.

How do environmental conditions affect the expression and activity of Cardiolipin synthase in Salmonella dublin during infection?

The expression and activity of Cardiolipin synthase in Salmonella dublin during infection is dynamically regulated in response to various environmental conditions encountered within the host:

• Growth phase regulation:

  • ClsA is the predominant synthase during logarithmic growth

  • ClsB and ClsC contribute significantly during stationary phase

  • This growth phase-dependent regulation likely reflects adaptation to nutrient availability during different infection stages

• Stress response activation:

  • High osmolarity conditions (similar to those in the intestinal environment) activate ClsC

  • Acidic pH (encountered in the stomach and within macrophage phagosomes) may trigger upregulation of cls genes

  • Oxidative stress from host immune responses likely influences cardiolipin synthesis

• Temperature effects:

  • Transition from environmental temperature to host body temperature (37°C) alters membrane fluidity

  • Cardiolipin synthesis may be modulated to maintain optimal membrane properties at host temperature

• Nutrient availability impact:

  • Phosphate limitation affects phospholipid metabolism

  • Carbon source changes encountered within different host niches influence cardiolipin production

• Host immune factor responses:

  • Antimicrobial peptides that target bacterial membranes may trigger compensatory changes in cardiolipin synthesis

  • Inflammatory environments alter expression patterns of various virulence factors including membrane-associated proteins

Research examining S. Typhimurium has shown that cls gene deletion affects intracellular survival in macrophages , suggesting that the intracellular environment specifically influences cardiolipin synthesis requirements. The precise environmental signals that control cls gene expression during different stages of S. dublin infection remain an active area of investigation.

How does the molecular evolution of Cardiolipin synthase vary across Salmonella enterica serovars, and what implications does this have for host adaptation?

The molecular evolution of Cardiolipin synthase across Salmonella enterica serovars reveals patterns that may contribute to host adaptation strategies:

Phylogenetic analysis of Salmonella enterica has identified significant diversity across subspecies and serovars . Within this context, Cardiolipin synthase genes show evolutionary patterns that may reflect adaptation to different host environments:

• Sequence conservation patterns:

  • Core catalytic domains of cls genes show high conservation across serovars

  • Regulatory regions and certain substrate-binding domains exhibit greater variation

  • These variations may fine-tune enzyme activity for specific host environments

• Host-adapted serovars comparison:

  • S. Dublin is a host-adapted serovar predominantly associated with cattle and increasingly implicated in human infections

  • Comparative analyses of cls genes between host-restricted serovars (like S. Dublin) and broad-host-range serovars may reveal adaptations for specific host lipid environments

• Geographical variations:

  • Distinct populations of Vi antigen-negative S. Dublin circulate in different geographical regions

  • These populations may also exhibit variations in cardiolipin synthesis capabilities

  • The North American S. Dublin cluster that emerged approximately 60 years ago may possess unique cls gene variants

• Selective pressure evidence:

  • Ratio of synonymous to non-synonymous mutations in cls genes can indicate selective pressures

  • Positive selection in specific domains would suggest adaptation to new hosts or environments

The implications for host adaptation are significant:

• Variations in cardiolipin synthesis may alter membrane properties to optimize survival in specific host environments

• Differences in cls gene regulation could enable responsive adaptation to host-specific defensive measures

• Cardiolipin composition variations may influence host immune system recognition and response

This evolutionary perspective provides a foundation for understanding how membrane lipid metabolism contributes to the ecological specialization of different Salmonella enterica serovars.

What structural and functional differences exist between the three Cardiolipin synthases (ClsA, ClsB, ClsC) in Salmonella dublin, and how can these be exploited for targeted interventions?

Structural and functional differences between the three Cardiolipin synthases in Salmonella dublin present potential targets for selective interventions:

Structural distinctions:

• Catalytic mechanisms:

  • All three enzymes possess characteristic phospholipase-D motifs but utilize them differently

  • ClsA and ClsB use two phosphatidylglycerol (PGl) molecules

  • ClsC uniquely employs both PGl and phosphatidylethanolamine (PE) as substrates

• Substrate binding pockets:

  • ClsB has a more promiscuous binding site allowing interaction with diverse substrates

  • ClsB can synthesize not only cardiolipin but also phosphatidylglycerol, phosphatidylalcohols, phosphatidyltrehalose, and diphosphatidyltrehalose

• Membrane topology:

  • Different transmembrane domain arrangements may position the enzymes optimally for their respective functions

  • These differences affect accessibility to inhibitors and substrate availability

Functional specializations:

• Environmental response roles:

  • ClsA predominates during logarithmic growth

  • ClsB and ClsC are activated during stationary phase stress

  • ClsC responds to high osmolarity conditions

• Compensatory capabilities:

  • When clsA and clsC are deleted, clsB alone can maintain certain functions

  • When clsB is deleted, clsA and clsC together can compensate

  • Complete removal of all three genes produces more pronounced phenotypes

Potential intervention strategies:

• Targeted inhibitors:

  • Small molecules targeting the unique substrate binding site of ClsB could selectively inhibit its promiscuous activity

  • ClsC-specific inhibitors could disrupt adaptation to osmotic stress environments

• Regulatory manipulation:

  • Compounds that interfere with stress-induced activation of ClsB and ClsC

  • Molecules that dysregulate the balance between the three synthases

• Combination approaches:

  • Using sub-inhibitory concentrations of antimicrobials with cls inhibitors

  • Targeting cls activity alongside other membrane-associated processes

• Host environment modification:

  • Altering host conditions to force reliance on specific cls enzymes

  • Creating environments where compensatory mechanisms are less effective

These targeted approaches could potentially reduce bacterial fitness or virulence while minimizing disruption to host processes, offering novel strategies against antimicrobial-resistant Salmonella dublin strains.

How can advanced proteomics and lipidomics approaches be integrated to better understand the role of Cardiolipin synthase in Salmonella dublin membrane remodeling during infection?

Advanced proteomics and lipidomics approaches can be synergistically integrated to comprehensively understand Cardiolipin synthase's role in Salmonella dublin membrane remodeling during infection:

Integrated methodological approaches:

• Temporal proteomics analysis:

  • Quantitative proteomics using stable isotope labeling (SILAC) or tandem mass tag (TMT) labeling

  • Time-course analysis of proteome changes during infection

  • Specific focus on membrane protein complexes associated with Cardiolipin synthases

  • Correlation of Cls protein expression with other virulence factors

• Comprehensive lipidomics:

  • High-resolution LC-MS/MS for detailed cardiolipin species profiling

  • Monitoring changes in acyl chain composition of cardiolipin during infection

  • Quantification of cardiolipin distribution between inner and outer membranes

  • Analysis of cardiolipin-protein interactions using photoactivatable lipid probes

• In situ techniques:

  • MALDI-imaging mass spectrometry to visualize lipid distribution in infected tissues

  • Fluorescent cardiolipin probes to track subcellular localization during infection

  • Cryo-electron microscopy to visualize membrane organization changes

• Multi-omics data integration:

  • Correlation of transcriptomics, proteomics, and lipidomics data sets

  • Network analysis to identify regulatory hubs controlling membrane remodeling

  • Machine learning approaches to predict membrane composition under different infection conditions

Expected insights and applications:

• Dynamic membrane adaptation mechanisms:

  • Precise timeline of membrane phospholipid changes during infection progression

  • Identification of infection stage-specific cardiolipin compositions

  • Understanding of how membrane remodeling contributes to immune evasion

• Regulatory networks:

  • Identification of transcription factors and post-translational modifications controlling cls gene expression

  • Mapping of signaling pathways that sense host environments and trigger membrane remodeling

  • Discovery of novel regulatory RNAs affecting cardiolipin synthesis

• Host-pathogen interface insights:

  • Characterization of how host lipids influence bacterial membrane composition

  • Understanding how bacterial cardiolipin interacts with host immune components

  • Identification of membrane microdomains important for virulence factor secretion

• Therapeutic implications:

  • Identification of critical timepoints when membrane composition is most vulnerable

  • Discovery of essential lipid-protein interactions that could be targeted

  • Understanding of how membrane composition affects antimicrobial resistance

This integrated approach would move beyond correlative observations to establish causative relationships between cardiolipin synthesis, membrane properties, and virulence functions during the infection process.

What are the common challenges in expressing and purifying functional recombinant Salmonella dublin Cardiolipin synthase, and how can they be addressed?

Expressing and purifying functional recombinant Salmonella dublin Cardiolipin synthase presents several technical challenges that require specific troubleshooting approaches:

Additional considerations for successful expression and purification:

• Vector selection:

  • pET-based vectors with T7 promoter provide high expression levels

  • Consider vectors with dual His-tag and additional affinity tags for improved purification

• Expression conditions optimization:

  • Screen multiple induction conditions (IPTG concentration: 0.1-1.0 mM)

  • Test various media formulations (TB, 2×YT, auto-induction media)

  • Evaluate post-induction times (4-24 hours)

• Advanced purification strategies:

  • Fluorescence-detection size-exclusion chromatography (FSEC) for rapid optimization

  • Lipid nanodiscs or styrene-maleic acid lipid particles (SMALPs) for detergent-free purification

  • On-column refolding for improved recovery of functional protein

These approaches can significantly improve the yield and quality of purified recombinant Salmonella dublin Cardiolipin synthase for subsequent functional and structural studies.

How can researchers effectively address the functional redundancy of the three Cardiolipin synthases when studying specific phenotypes?

Addressing the functional redundancy of Cardiolipin synthases requires sophisticated experimental approaches to disentangle their individual contributions to specific phenotypes:

• Comprehensive genetic manipulation strategies:

  • Create a complete set of single, double, and triple mutants (ΔclsA, ΔclsB, ΔclsC, ΔclsAB, ΔclsAC, ΔclsBC, and ΔclsABC)

  • Use complementation with plasmid-expressed individual cls genes to confirm phenotype restoration

  • Employ gene dosage experiments by varying expression levels to identify threshold requirements

• Conditional expression systems:

  • Utilize inducible promoters to control expression of individual cls genes

  • Implement temperature-sensitive or chemical-dependent expression systems

  • Create growth phase-specific expression constructs to mimic natural regulation

• Domain swapping and chimeric proteins:

  • Generate chimeric proteins combining domains from different Cls enzymes

  • Create constructs with altered substrate binding sites to modify enzyme specificity

  • Evaluate which protein domains are responsible for specific phenotypes

• Orthogonal approaches to bypass redundancy:

  • Direct measurement of enzyme-specific products (e.g., ClsB-specific production of phosphatidyltrehalose)

  • Use of enzyme-specific inhibitors when available

  • Monitor expression patterns under different conditions to identify scenarios where one enzyme predominates

• Advanced phenotypic analysis:

  • High-throughput phenotypic microarrays to identify condition-specific requirements

  • Single-cell analysis to detect heterogeneity in bacterial populations

  • In vivo infection models with different routes of infection to reveal context-dependent roles

Research with S. Typhimurium has demonstrated that:

• When clsA and clsC are deleted, clsB alone is sufficient for certain phenotypes

• When clsB is deleted, clsA and clsC together can compensate

• Adding back clsA or clsB to triple mutant bacteria restores cardiolipin production but does not rescue all phenotypes

These findings highlight the complex relationship between cardiolipin synthesis and bacterial phenotypes, suggesting that the cls genes may have additional functions beyond cardiolipin production that contribute to specific aspects of bacterial physiology and pathogenesis.

What are the most reliable methods for quantifying Cardiolipin synthase activity in complex biological samples, and how can conflicting results be reconciled?

Quantifying Cardiolipin synthase activity in complex biological samples requires robust methodologies, and reconciling conflicting results demands systematic analytical approaches:

Gold standard quantification methods:

• Radioisotope-based assays:

  • Tracking incorporation of radiolabeled substrates (³²P-labeled phosphatidylglycerol)

  • Quantification by scintillation counting after TLC separation

  • Advantages: high sensitivity, direct measurement of enzyme action

  • Limitations: radiation safety concerns, special disposal requirements

• Mass spectrometry approaches:

  • LC-MS/MS with multiple reaction monitoring (MRM) for specific cardiolipin species

  • MALDI-TOF MS for rapid screening

  • Advantages: detailed lipid species identification, absolute quantification

  • Limitations: expensive equipment, complex data analysis

• Fluorescent substrate analogs:

  • NBD or BODIPY-labeled phospholipid substrates

  • Continuous monitoring of enzyme kinetics

  • Advantages: real-time measurements, no radiation

  • Limitations: potential altered enzyme kinetics with modified substrates

• Coupled enzyme assays:

  • Linking Cardiolipin synthase activity to detectable enzymatic reactions

  • Spectrophotometric or fluorometric detection

  • Advantages: continuous measurement, equipment accessibility

  • Limitations: potential interference from sample components

Reconciling conflicting results:

• Systematic variables analysis:

  • Create a matrix of experimental conditions used in conflicting studies

  • Identify key variables: detergent type/concentration, pH, temperature, buffer composition

  • Perform controlled experiments changing one variable at a time

• Sample preparation standardization:

  • Develop consistent protocols for membrane fraction isolation

  • Standardize protein:lipid ratios in assays

  • Control for endogenous lipid content

• Cross-validation approaches:

  • Apply multiple independent quantification methods to the same samples

  • Correlate results from different techniques

  • Identify technique-specific biases

• Statistical rigor:

  • Increase biological and technical replicates

  • Apply appropriate statistical tests

  • Consider Bayesian approaches for reconciling conflicting data sets

• Addressing biological complexity:

  • Account for growth phase effects on cls gene expression

  • Consider the impact of environmental conditions

  • Examine strain-specific differences in enzyme activity or regulation