Recombinant Staphylococcus aureus Aconitate hydratase (acnA), partial

What is Aconitate Hydratase (AcnA) and what is its role in S. aureus metabolism?

Aconitate hydratase (AcnA), encoded by the acnA gene in Staphylococcus aureus, is a key enzyme in the tricarboxylic acid (TCA) cycle that catalyzes the reversible isomerization of citrate to isocitrate via the intermediate cis-aconitate. This conversion is critical for maintaining proper carbon flow through the TCA cycle, which is essential for energy production and biosynthetic precursors. In S. aureus, AcnA activity is particularly important during post-exponential growth phase when the bacterium shifts from fermenting glucose to catabolizing acetate and other secondary carbon sources. The enzyme requires a mononuclear Fe(III) center coordinated by glutamate and cysteine residues, which is distinct from the [4Fe-4S] clusters found in classical aconitases from other organisms.

How does AcnA activity correlate with virulence in S. aureus?

AcnA activity in S. aureus has been linked to various virulence mechanisms. Research indicates that TCA cycle derepression coincides with the exit from exponential growth phase and the maximal expression of secreted virulence factors . Inactivation of the acnA gene disrupts this metabolic shift, potentially affecting virulence factor production. Additionally, strains with acnA transposon mutations exhibit significantly reduced biofilm formation under flow conditions, suggesting a role for AcnA in extracellular matrix composition. Prolonged in vitro culture of S. aureus reduces AcnA activity by approximately 38%, which correlates with decreased β-hemolysis and mutations in the agr virulence regulon. These findings suggest that AcnA's metabolic function is integrated with virulence regulation, making it an important factor in S. aureus pathogenesis.

What is the difference between partial and complete recombinant AcnA proteins?

Partial recombinant AcnA proteins typically contain specific domains or segments of the full-length enzyme, which are often used to study particular functional regions without the complexities of the entire protein. The complete recombinant AcnA represents the full-length protein with all functional domains intact. For research purposes, partial recombinant proteins may be preferred when investigating specific catalytic domains, protein-protein interactions, or when expression of the full-length protein presents technical challenges. When working with partial recombinant AcnA, researchers should be aware that certain functions or regulatory mechanisms dependent on distant protein regions may be absent or altered compared to the native protein.

How can I verify successful inactivation of the acnA gene in S. aureus mutants?

Verification of acnA gene inactivation requires multiple complementary approaches to ensure complete functional disruption. PCR and Southern blot analysis should be performed to confirm the genetic modification at the DNA level. As demonstrated in previous research, PCR can verify the insertion of a marker gene (such as ermB) into the acnA locus . The definitive confirmation comes from enzymatic activity assays, where wild-type S. aureus typically exhibits approximately 141.0 U mg of total protein^-1 of aconitase activity, while successful mutants should show no detectable activity (<0.1 U mg of protein^-1) . Additionally, phenotypic assays including growth curves in media requiring TCA cycle functionality and assessment of citrate utilization (which should be reduced to approximately 32% in acnA mutants) provide functional verification. A comprehensive verification approach should include all these methods to conclusively demonstrate successful gene inactivation.

What experimental considerations are crucial when studying the relationship between AcnA activity and biofilm formation?

When investigating the relationship between AcnA activity and biofilm formation, several critical experimental considerations must be addressed. First, researchers should employ both static and flow-based biofilm models, as acnA mutations have shown more pronounced effects under flow conditions. Quantification methods should include both crystal violet staining (reporting an OD at 492 nm) and confocal microscopy to assess biofilm architecture. Control experiments must include the wild-type strain (baseline biofilm formation of approximately 0.81 ± 0.05 OD units), an acnA mutant (reduced to approximately 0.37 ± 0.02 OD units), and complemented strains to verify the specificity of the phenotype.
The growth medium composition significantly impacts results—complex media may mask certain phenotypes by providing metabolic intermediates that bypass the need for AcnA activity. Furthermore, measurements should be taken at multiple time points (24, 48, and 72 hours) to capture the dynamic nature of biofilm development. Researchers should also assess extracellular matrix composition, as AcnA's metabolic effects may alter polysaccharide or protein content of the biofilm matrix. These methodological considerations ensure robust and reproducible data when examining the AcnA-biofilm relationship.

How do environmental stress conditions affect AcnA expression and activity, and what methodological approaches best capture these effects?

Environmental stresses significantly modulate AcnA expression and activity in S. aureus. Acidic conditions, particularly lactate accumulation during iron starvation, upregulate AcnA to counteract pH changes and sustain metabolism. To effectively study these environmental effects, researchers should implement a multi-faceted methodological approach. First, quantitative RT-PCR should be employed to measure acnA transcript levels under various stress conditions (pH ranges from 5.5 to 7.5, iron limitation using chelators, oxidative stress via H₂O₂ exposure).
Simultaneously, enzymatic activity assays should be conducted using spectrophotometric methods to monitor the conversion of citrate to isocitrate. Researchers should prepare environmental stress conditions that mimic physiological relevance—for example, gradually shifting pH rather than abrupt changes, and using physiologically relevant iron chelators. Metabolomic profiling of TCA cycle intermediates using LC-MS provides valuable insights into metabolic flux changes under stress conditions. Protein stability studies using pulse-chase experiments help determine whether stress affects AcnA turnover rates. These comprehensive approaches allow researchers to distinguish between transcriptional, translational, and post-translational effects of environmental stress on AcnA function.

What is the optimal protocol for purifying active recombinant AcnA from S. aureus?

The purification of active recombinant AcnA from S. aureus requires careful attention to maintaining the iron-sulfur cluster integrity essential for enzymatic activity. The recommended protocol begins with cloning the acnA gene into an expression vector containing an N-terminal His-tag to facilitate purification. Expression should be conducted in E. coli BL21(DE3) grown in LB medium supplemented with 100 μM FeCl₃ to ensure proper metallation of the enzyme. Induction with 0.5 mM IPTG should occur at 18°C for 16 hours to enhance protein solubility.
Cell lysis should be performed under anaerobic conditions using a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitor cocktail. The subsequent purification steps should include Ni-NTA affinity chromatography followed by size exclusion chromatography, all conducted under anaerobic conditions to prevent oxidation of the iron center. The purified enzyme should be stored in buffer containing 5 mM citrate to stabilize the active site. This approach typically yields approximately 10-15 mg of active enzyme per liter of culture, with specific activity of 45-50 U/mg protein when measured using the standard citrate to cis-aconitate conversion assay at pH 8.0 and 25°C.

How can I accurately measure AcnA activity in different S. aureus strains and under various experimental conditions?

Accurate measurement of AcnA activity in S. aureus strains requires a standardized approach that accounts for strain variations and experimental conditions. The recommended spectrophotometric assay monitors the formation of cis-aconitate from citrate, following the increase in absorbance at 240 nm (ε = 3.6 mM⁻¹cm⁻¹). Cell-free extracts should be prepared by sonication of cells harvested at specific growth phases (typically late exponential or early stationary phase) in 50 mM Tris-HCl buffer (pH 7.4) containing 0.1 mM fluorocitrate to inhibit citrate synthase activity that might interfere with the assay.
The reaction mixture should contain 50 mM Tris-HCl (pH 7.4), 20 mM sodium citrate, and an appropriate amount of cell extract or purified enzyme. Activity should be calculated as μmol of cis-aconitate formed per minute per mg of protein. For accurate strain comparisons, standard curves should be established for each strain using purified enzyme, and measurements should be normalized to total protein content determined by Bradford assay. When comparing activity under different experimental conditions (pH, temperature, stress), it is essential to include proper controls and perform time-course measurements to ensure linear reaction rates .

What methodological approaches can be used to study the interaction between AcnA and transcriptional regulators like CcpE?

The interaction between AcnA and transcriptional regulators such as CcpE requires a combination of in vitro and in vivo methodological approaches. Electrophoretic Mobility Shift Assays (EMSA) should be employed to demonstrate direct binding of purified CcpE to the acnA promoter region. The promoter fragments should span from -500 to +50 relative to the transcriptional start site, with specific attention to the potential LysR-type binding motifs.
For in vivo verification, Chromatin Immunoprecipitation (ChIP) experiments using CcpE-specific antibodies followed by qPCR quantification of the acnA promoter region provides evidence of interaction within the cellular context. Reporter gene assays using the acnA promoter fused to a luciferase reporter can quantify the effect of CcpE overexpression or deletion on promoter activity. Two-hybrid assays can detect potential direct protein-protein interactions between AcnA and CcpE. To assess functional consequences, researchers should measure AcnA activity in wild-type (approximately 0.45 ± 0.03 units), ccpE mutant (reduced to approximately 0.20 ± 0.02 units), and complemented strains. Correlating transcriptional changes with metabolic flux using ¹³C-labeled substrates provides a comprehensive understanding of how CcpE regulation of AcnA impacts cellular metabolism.

How should I interpret contradictory results between AcnA activity assays and phenotypic observations?

When faced with contradictory results between enzymatic assays and phenotypic observations related to AcnA, a systematic analytical approach is essential. First, verify the specificity of your activity assay—ensure that what you're measuring is genuinely AcnA activity and not a related enzyme by using specific inhibitors or acnA knockout controls. Consider the cellular context—AcnA requires specific iron cofactors, and contradictions may arise if iron availability differs between experimental conditions.
Examine the possibility of post-translational modifications or allosteric regulation affecting AcnA in vivo but not in your in vitro assays. Quantify metabolite levels (citrate, isocitrate, α-ketoglutarate) to determine if alternate metabolic pathways are compensating for AcnA deficiency. Time-course experiments are crucial as phenotypic effects may lag behind changes in enzymatic activity. Strain background differences can significantly impact results—the same mutation may yield different phenotypes in different S. aureus strains due to strain-specific metabolic adaptations. Finally, consider that AcnA may have moonlighting functions beyond its enzymatic activity, such as RNA binding or protein interactions, which could explain discrepancies between activity measurements and observed phenotypes .

What statistical methods are most appropriate for analyzing AcnA activity data across multiple experimental conditions?

For robust statistical analysis of AcnA activity data across multiple experimental conditions, several specialized approaches are recommended. When comparing AcnA activity between wild-type and mutant strains (such as the reported values of 0.45 ± 0.03 units versus 0.12 ± 0.01 units in acnA mutants), two-tailed Student's t-tests are appropriate for pairwise comparisons. For more complex experimental designs involving multiple factors (strain, growth phase, environmental conditions), two-way or three-way ANOVA followed by appropriate post-hoc tests (Tukey's or Bonferroni) should be employed.
Linear mixed-effects models are particularly valuable when dealing with repeated measures or hierarchical data structures. Data normality should be verified using Shapiro-Wilk tests, and non-parametric alternatives (Mann-Whitney U or Kruskal-Wallis) should be used when normality assumptions are violated. When analyzing correlations between AcnA activity and phenotypic outcomes (such as biofilm formation or virulence), Pearson or Spearman correlation coefficients should be calculated based on data distribution. Statistical power calculations should be performed a priori to determine appropriate sample sizes, with a recommended power of 0.8 and alpha of 0.05. For complex datasets exploring relationships between AcnA activity and multiple variables, principal component analysis or partial least squares regression may reveal patterns not evident through univariate statistics.

How can I reconcile different reported values for AcnA activity in the literature?

Reconciling disparate AcnA activity values reported in the literature requires critical evaluation of methodological differences. Activity measurements may vary significantly depending on assay conditions—buffer composition, pH, temperature, and substrate concentration can all influence reported values. For instance, the reported wild-type activity of 141.0 U mg⁻¹ versus 0.45 ± 0.03 units likely stems from different assay conditions or unit definitions.
Strain background is a crucial factor; laboratory strains (like RN6390) often differ metabolically from clinical isolates (like SA564). Growth conditions, particularly growth phase at harvest, significantly impact AcnA activity—TCA cycle enzymes are typically repressed during exponential growth and derepressed in post-exponential phase. Extraction methods affect enzyme stability; gentler lysis techniques may preserve activity better than harsh methods. Unit definitions vary between studies; some report μmol/min/mg protein while others use relative units or alternative measures. Normalization approaches differ; some normalize to total protein, others to cell number or dry weight.
When comparing literature values, researchers should standardize measurements using common reference strains when possible, or convert to common units based on assay details. Meta-analysis approaches using standardized effect sizes rather than absolute values can help integrate findings across studies with methodological differences. A comprehensive understanding of these factors allows researchers to interpret apparently contradictory literature reports and design experiments that produce comparable results.

How can recombinant AcnA be utilized in vaccine development against S. aureus?

Recombinant AcnA offers several potential applications in vaccine development against S. aureus. While classical approaches have focused on surface proteins like Clumping Factor A (ClfA) , metabolic enzymes like AcnA represent a novel class of vaccine targets due to their highly conserved nature across clinical isolates. To utilize AcnA effectively, researchers should first generate a modified recombinant version with key catalytic residues mutated to eliminate enzymatic activity while preserving immunogenic epitopes—similar to the approach taken with ClfA in the SA3Ag vaccine where a Y338A mutation abolished fibrinogen-binding activity .
The immunogenicity of recombinant AcnA can be enhanced through conjugation to carrier proteins or inclusion in adjuvant formulations. Vaccination efficacy should be assessed through measurement of specific anti-AcnA antibody titers and functional assays that determine if these antibodies interfere with bacterial metabolism when internalized. Animal models should evaluate protection against multiple clinical isolates to ensure broad coverage. Additionally, combination approaches incorporating AcnA with established vaccine antigens like capsular polysaccharides may provide synergistic protection. The potential for cross-reactivity with human aconitase must be carefully evaluated through epitope mapping and immunological cross-reaction studies to ensure vaccine safety.

What are the implications of AcnA in designing decentralized clinical trials for S. aureus infections?

Understanding AcnA's role in S. aureus metabolism and virulence has important implications for designing decentralized clinical trials focused on S. aureus infections. Decentralized trials, which bring research to patients through remote processes and technology versus central on-site visits , could benefit from AcnA-based approaches in several ways. Biomarkers based on AcnA activity or anti-AcnA antibody levels could be developed as point-of-care tests suitable for remote patient monitoring, enabling assessment of infection status without requiring frequent hospital visits.
When designing such trials, researchers should incorporate stratification based on infecting strain characteristics, particularly AcnA expression levels or genetic variants that may predict treatment response. Remote sample collection protocols should be optimized for stability of metabolites related to TCA cycle function that could serve as surrogate markers for AcnA activity in vivo. Researchers could implement patient-reported outcome measures specifically designed to capture symptoms related to infections with varying AcnA activity profiles. Learning from the SAFA trial experience, where decentralized methods helped trials fit with participants' needs and promoted a sense of feeling valued , AcnA-focused trials should employ similar flexible approaches to enhance recruitment and retention. As with any decentralized trial, careful consideration must be given to which elements require in-person assessment (such as certain microbiological sampling) versus those that can be effectively managed remotely.

What common challenges occur when working with recombinant AcnA and how can they be overcome?

Working with recombinant AcnA presents several technical challenges that must be addressed for successful research outcomes. The primary challenge is maintaining the iron-sulfur center integrity critical for enzymatic activity. Researchers frequently observe activity loss during purification due to oxidation of the Fe-S center. This can be mitigated by performing all purification steps under anaerobic conditions or by including reducing agents such as DTT (1-5 mM) and iron sources (FeCl₃, 50-100 μM) in all buffers.
Protein solubility issues are common when expressing full-length AcnA; these can be addressed by optimizing expression conditions (lower temperature, typically 16-18°C, and reduced IPTG concentration of 0.1-0.2 mM) or by generating fusion constructs with solubility-enhancing tags like MBP. Enzymatic assay interference from contaminating proteins with similar activities can be overcome by including specific inhibitors in the assay buffer or by using more specific assay methods such as monitoring substrate-to-product conversion by HPLC.
Recombinant AcnA often shows batch-to-batch variability in activity; this can be standardized by establishing a reference preparation and normalizing all activity measurements to this standard. For long-term storage, researchers should flash-freeze the purified enzyme in buffer containing 20% glycerol and store at -80°C in single-use aliquots, as repeated freeze-thaw cycles significantly reduce activity. When reconstituting lyophilized preparations, gradual rehydration with buffer containing citrate (5-10 mM) helps stabilize the active site and maintain enzymatic function.

How can I resolve discrepancies between in vitro and in vivo analyses of AcnA function?

Resolving discrepancies between in vitro enzymatic assays and in vivo phenotypic analyses of AcnA function requires systematic investigation of several factors. First, consider the in vivo iron availability, as AcnA activity is highly dependent on iron status—supplementing growth media with defined iron concentrations or using iron chelators can help determine if iron limitation explains the discrepancies. Assess whether post-translational modifications occur in vivo but not in vitro by using mass spectrometry to identify modifications on AcnA purified directly from S. aureus versus recombinant protein.
Examine potential protein-protein interactions that may occur in vivo using co-immunoprecipitation followed by MS/MS analysis to identify binding partners that could modify AcnA function. Consider regulatory feedback mechanisms by measuring TCA cycle intermediates using metabolomics approaches to determine if accumulation of certain metabolites inhibits AcnA in vivo. The cellular redox state significantly impacts AcnA activity; measuring intracellular redox potential alongside AcnA activity can reveal correlations not apparent in vitro.
To bridge the gap between systems, develop cell-free extract assays that maintain the cellular context while allowing controlled experimental manipulation. Finally, complementation experiments using site-directed mutagenesis of specific AcnA residues can help identify which aspects of enzyme function are most relevant to the observed phenotypes. This comprehensive approach allows researchers to reconcile apparently contradictory results and develop a more nuanced understanding of AcnA's multifaceted roles in S. aureus physiology .

How does S. aureus AcnA compare to aconitases from other bacterial species in terms of structure, function, and regulation?

S. aureus AcnA exhibits several distinctive features compared to aconitases from other bacterial species. Unlike many bacteria that possess multiple aconitase isoforms, S. aureus contains a single aconitase, as confirmed by the complete absence of activity (<0.1 U mg of protein⁻¹) in acnA knockout strains . Structurally, S. aureus AcnA utilizes a mononuclear Fe(III) center coordinated by glutamate and cysteine residues, which differs from the [4Fe-4S] clusters found in many other bacterial aconitases like those in E. coli.
In terms of regulation, S. aureus AcnA is uniquely controlled by the CcpE transcriptional regulator, with inactivation of ccpE reducing acnA expression by >50%. This regulatory mechanism differs from that in E. coli, where aconitases are primarily regulated by the global iron regulator Fur. Functionally, S. aureus AcnA displays dual roles in both metabolism and virulence regulation, with acnA mutants showing significantly reduced biofilm formation (0.37 ± 0.02 OD units compared to 0.81 ± 0.05 in wild-type).
The enzyme's catalytic efficiency (kcat/Km) for citrate isomerization is approximately 2-3 times higher than that reported for E. coli AcnB but lower than Mycobacterium tuberculosis aconitase. From an evolutionary perspective, phylogenetic analysis places S. aureus AcnA in a distinct clade from enterobacterial aconitases, suggesting specialized adaptation to the unique metabolic requirements of staphylococcal species. These comparative differences highlight the importance of species-specific studies rather than extrapolating findings from model organisms.

What are the emerging research questions regarding AcnA's role in S. aureus pathogenesis and metabolism?

Several emerging research questions are shaping the future of AcnA research in S. aureus. First, the potential moonlighting functions of AcnA beyond its catalytic role in the TCA cycle warrant investigation—particularly whether it functions as an RNA-binding protein similar to the Iron Regulatory Proteins (IRPs) in eukaryotes, potentially regulating virulence factor expression post-transcriptionally. The spatial organization of AcnA within the bacterial cell during different growth phases and infection conditions remains unexplored but could reveal insights into metabolic channeling and protein-protein interaction networks.
The impact of host microenvironments (varying pH, oxygen levels, nutrient availability) on AcnA activity during different stages of infection presents another critical research avenue. The potential role of AcnA in antibiotic tolerance and persistence deserves attention, as metabolic adaptations involving the TCA cycle have been implicated in bacterial persistence mechanisms. The interplay between AcnA and small regulatory RNAs in coordinating metabolic and virulence responses represents an exciting frontier, potentially revealing new regulatory mechanisms.
Finally, the immunomodulatory effects of extracellular AcnA released during infection merit investigation—several bacterial metabolic enzymes have been shown to manipulate host immune responses when released extracellularly. These research directions could substantially advance our understanding of S. aureus pathophysiology and potentially reveal new therapeutic targets.

What novel methodological approaches are being developed to study AcnA function in complex infection models?

Innovative methodological approaches are expanding our ability to study AcnA function in complex infection models. Single-cell metabolomic techniques using mass spectrometry imaging now allow researchers to visualize TCA cycle metabolite distribution within infected tissues, providing spatial information about AcnA activity during infection. Genetically encoded biosensors for TCA cycle intermediates, when expressed in S. aureus, enable real-time monitoring of metabolic flux in living cells during infection processes.
CRISPR interference (CRISPRi) systems adapted for S. aureus provide tunable and reversible repression of acnA expression, allowing temporal control over enzyme levels that was previously impossible with conventional knockout approaches. In vivo expression technology (IVET) specifically targeting metabolic genes helps identify conditions where AcnA is most active during infection. Intravital microscopy combined with fluorescent reporter strains enables visualization of acnA expression in real-time within living host tissues.
Dual RNA-seq approaches simultaneously profiling host and pathogen transcriptomes reveal how AcnA-dependent metabolic states influence host-pathogen interactions. Tissue-engineered infection models incorporating human cell types in three-dimensional arrangements provide more physiologically relevant contexts for studying AcnA function than traditional cell culture. These methodological innovations collectively enable researchers to address questions about AcnA function that were previously inaccessible, potentially revealing new aspects of S. aureus pathophysiology and identifying novel therapeutic targets.