Recombinant Chlamydophila caviae Enolase (eno)

What is the biochemical function of Chlamydophila caviae Enolase?

Chlamydophila caviae Enolase functions primarily as a glycolytic enzyme that catalyzes the conversion of 2-phosphoglycerate (2PGA) to phosphoenolpyruvate (PEP). This reaction represents the ninth step in the glycolytic pathway, which is crucial for bacterial energy metabolism. Experimental verification of C. trachomatis enolase has demonstrated that the purified recombinant enzyme effectively converts 2PGA to PEP in a manner comparable to E. coli enolase . When key conserved residues (such as S44) are mutated, enzymatic activity is abolished, confirming the specificity of this reaction . While Chlamydophila caviae is a distinct species, its enolase shares high sequence homology with other chlamydial enolases and is expected to catalyze the same reaction with similar efficiency.

How is Chlamydophila caviae Enolase regulated post-translationally?

Phosphorylation appears to be a critical regulatory mechanism for Chlamydophila caviae Enolase. Research has specifically detected phosphorylated enolase in elementary bodies (EBs) but not in reticulate bodies (RBs) of Chlamydia caviae . This developmental regulation suggests that enolase phosphorylation may play a role in the chlamydial developmental cycle. Additionally, lysine acetylation has been identified as another post-translational modification that may regulate chlamydial enolase activity . These modifications likely influence the enzyme's catalytic efficiency and potentially its non-glycolytic functions within the bacterial cell.

What methods are most effective for recombinant expression of C. caviae Enolase?

For successful recombinant expression of Chlamydophila caviae Enolase, an E. coli expression system using pET vectors with an N-terminal His-tag has proven effective for chlamydial proteins. Following transformation into expression strains like BL21(DE3), induction with IPTG (typically 0.5-1 mM) at mid-logarithmic phase (OD600 of 0.6-0.8) and growth at 25-30°C for 4-6 hours generally yields optimal protein expression while minimizing inclusion body formation. Purification via nickel affinity chromatography followed by size exclusion chromatography typically produces highly pure, active recombinant enzyme. When expressing chlamydial proteins, codon optimization for E. coli may improve yields, as chlamydial genomes have different codon usage patterns.

What is the relationship between Chlamydophila enolase and the RsbU phosphatase in gene regulation?

The relationship between enolase and RsbU phosphatase represents a novel regulatory mechanism connecting metabolism with gene expression in Chlamydia species. Research has demonstrated that the genes for enolase (eno) and RsbU are adjacently positioned in many Chlamydia genomes and are co-transcribed from the same operon . This genetic arrangement suggests functional coordination between these proteins. Critically, phosphoenolpyruvate (PEP), which is the product of the enolase reaction, has been shown to inhibit RsbU phosphatase activity against both RsbV1 and RsbV2 substrates .

This inhibition has significant downstream effects on gene regulation through the partner-switching mechanism involving RsbW (anti-sigma factor), which normally inhibits transcription by sequestering the sigma subunit of RNA polymerase. When glucose is available, enolase produces PEP, which inhibits RsbU, leading to phosphorylated RsbV that cannot bind RsbW. Consequently, RsbW remains free to sequester sigma factors, inhibiting specific gene transcription. Conversely, when glucose is limited, reduced PEP levels allow RsbU to dephosphorylate RsbV, which then binds RsbW, releasing sigma factors to initiate transcription of their target genes .

How does enolase expression vary throughout the chlamydial developmental cycle?

Temporal regulation of enolase expression follows a specific pattern during the chlamydial developmental cycle. 5' RACE analysis of C. trachomatis-infected cells has revealed that at mid-cycle (18 hours post-infection), transcription primarily occurs from the enolase (eno) promoter. In contrast, at later stages (30 hours post-infection), transcription shifts to preferentially occur from the RsbU promoter . This temporal shift in promoter usage suggests developmental regulation of these genes during the chlamydial life cycle.

Furthermore, phosphorylation status of enolase differs between developmental forms, with phosphorylated enolase detected specifically in elementary bodies (EBs) but not in reticulate bodies (RBs) in Chlamydia caviae . This differential modification suggests that enolase activity may be regulated to accommodate the metabolic requirements of different developmental stages, with potential implications for both energy metabolism and gene regulation.

What experimental approaches can detect the interaction between enolase activity and the RsbU-mediated gene regulation pathway?

To investigate the relationship between enolase activity and RsbU-mediated gene regulation, several experimental approaches can be employed:

• In vitro Phosphatase Assays: Using purified recombinant RsbU phosphatase domain with phosphorylated RsbV1/RsbV2 as substrates in the presence or absence of PEP, 2PGA, and other metabolites to measure phosphatase activity .

• Co-immunoprecipitation Studies: To detect protein-protein interactions between enolase and components of the Rsb system in Chlamydia.

• Reporter Gene Assays: When genetic manipulation is possible, creating reporter constructs with promoters regulated by the Rsb system to monitor gene expression changes in response to glucose availability or enolase inhibition.

• Metabolic Profiling: Measuring intracellular levels of glycolytic intermediates (particularly PEP) under different growth conditions and correlating with gene expression patterns.

• Transcriptome Analysis: RNA-seq or qRT-PCR to measure expression of Rsb-regulated genes following manipulation of glucose availability or enolase activity.

Table 1: Experimental conditions for RsbU phosphatase activity assay

What structural features distinguish Chlamydophila caviae Enolase from other bacterial enolases?

Chlamydophila caviae Enolase shares the characteristic α/β barrel structural fold common to the enolase superfamily but possesses several distinctive features. While detailed crystallographic data specific to C. caviae enolase is limited, comparative analysis with other chlamydial enolases reveals conserved catalytic residues essential for binding the 2PGA substrate and divalent metal ions (typically Mg²⁺).

Particularly notable is the conservation of the S44 residue, which has been shown to be critical for enzymatic activity through mutation studies in C. trachomatis enolase . Substitution of this residue (S44A) completely abolishes enzymatic activity, confirming its essential role in catalysis. The structure likely contains two metal-binding sites at the active center that coordinate the substrate during catalysis.

Chlamydial enolases may possess unique surface-exposed loops that differentiate them from other bacterial enolases, potentially contributing to species-specific functions or interactions. These structural differences could be exploited for developing selective inhibitors or diagnostic tools targeting Chlamydophila caviae.

What methods are most reliable for assessing Chlamydophila caviae Enolase enzymatic activity?

The standard method for assessing Chlamydophila caviae Enolase activity involves a coupled spectrophotometric assay measuring the conversion of 2PGA to PEP. This reaction can be monitored directly by measuring the increase in absorbance at 240 nm due to PEP formation. Alternatively, a coupled assay system using pyruvate kinase and lactate dehydrogenase can be employed, where the oxidation of NADH (monitored at 340 nm) corresponds to enolase activity.

For analyzing enzyme kinetics, reactions should be performed under the following optimized conditions:

• Buffer: 50 mM Tris-HCl, pH 7.4, 100 mM KCl

• Essential cofactor: 10 mM MgCl₂ (or alternative divalent cations)

• Substrate: 2PGA (0.01-10 mM range for Km determination)

• Temperature: 37°C (physiologically relevant)

• Purified enzyme: 0.1-1 μg per reaction

The specific activity should be expressed as μmol PEP formed per minute per mg protein under standard conditions. Potential inhibitors can be added to this assay system to evaluate their effects on enzyme activity, with appropriate controls to account for potential interference with the detection system.

How does glycolytic flux influence the regulatory function of Chlamydophila enolase?

Glycolytic flux exerts significant influence on the regulatory function of Chlamydophila enolase through its impact on PEP production. In the proposed model based on experimental evidence, when glucose is abundantly available to the intracellular bacteria, the glycolytic pathway actively produces 2PGA, which is converted to PEP by enolase . The resulting high levels of PEP inhibit RsbU phosphatase activity, maintaining RsbV in its phosphorylated state. This prevents RsbV from binding to RsbW, leaving RsbW free to sequester sigma factors and inhibit transcription of specific genes .

Conversely, when glucose availability is limited (potentially during stress conditions or certain developmental stages), reduced glycolytic flux leads to lower PEP production. This alleviates the inhibition of RsbU, allowing it to dephosphorylate RsbV. The unphosphorylated RsbV then binds to RsbW, freeing sigma factors to direct transcription of their target genes .

This regulatory mechanism represents a sophisticated way for Chlamydia to sense and respond to host metabolic status, as these obligate intracellular bacteria acquire glucose-6-phosphate directly from the host cell . This creates a direct link between host metabolic state, bacterial glycolytic activity, and gene regulation through the Rsb pathway.

How can recombinant Chlamydophila caviae Enolase be used to study host-pathogen interactions?

Recombinant Chlamydophila caviae Enolase offers several valuable approaches for investigating host-pathogen interactions. As a metabolic enzyme with potential moonlighting functions, enolase may interact with host components during infection. Researchers can use purified recombinant enolase in binding assays to identify host cell receptors or extracellular matrix components that interact with bacterial enolase when it's surface-exposed.

Immunological studies can employ recombinant enolase to investigate host immune responses to this protein during infection. This includes measuring antibody responses in infected hosts, evaluating T-cell epitopes, and determining whether enolase serves as a pathogen-associated molecular pattern (PAMP) that triggers innate immune responses.

Additionally, the connection between enolase activity and gene regulation through the RsbU-RsbV-RsbW pathway provides a tool to investigate how host metabolic status influences chlamydial gene expression . By manipulating host glucose levels and measuring changes in chlamydial gene expression, researchers can elucidate how the pathogen adapts to different metabolic environments within the host cell.

What are the technical challenges in studying enolase function in obligate intracellular pathogens like Chlamydia?

Studying enolase function in obligate intracellular pathogens like Chlamydia presents several significant technical challenges:

• Genetic Manipulation Limitations: Despite recent advances, genetic manipulation of Chlamydia remains challenging, limiting the ability to create knockout mutants or introduce reporter constructs to study enolase function in vivo.

• Isolation of Pure Bacterial Components: Separating bacterial proteins and metabolites from host cell components can be difficult, complicating direct measurement of enolase activity or metabolite levels (like PEP) within bacteria during infection.

• Developmental Cycle Complexity: Chlamydia transitions between multiple developmental forms with different metabolic states, making it challenging to attribute specific functions to enolase at different stages of infection.

• Host Cell Influence: Host metabolism directly affects bacterial metabolism, creating a complex system where isolating the specific contribution of bacterial enolase is difficult.

• Limited Biomass: The intracellular growth habit of Chlamydia limits the amount of bacterial material available for biochemical studies.

To overcome these challenges, researchers typically combine recombinant protein studies with infection models using chemical inhibitors, metabolic manipulation, and advanced microscopy techniques to indirectly assess enolase function.

How might differential phosphorylation of enolase between EBs and RBs contribute to chlamydial pathogenesis?

The differential phosphorylation of enolase between elementary bodies (EBs) and reticulate bodies (RBs) in Chlamydia caviae likely represents a key regulatory mechanism contributing to pathogenesis through multiple pathways:

• Metabolic Adaptation: Phosphorylation may modulate enolase activity to align with the different metabolic requirements of EBs (which are metabolically less active) and RBs (which are metabolically active and replicating). This modification could help optimize energy production during different developmental stages.

• Gene Regulation: Through its connection with the RsbU-RsbV-RsbW pathway, changes in enolase phosphorylation and activity could alter PEP levels, affecting RsbU phosphatase activity and downstream gene expression . This would allow developmental stage-specific gene expression patterns essential for pathogenesis.

• Protein Moonlighting: Beyond its glycolytic function, enolase may serve alternative roles when phosphorylated, potentially including surface exposure on EBs where it could mediate host cell attachment or immune evasion.

• Structural Stability: Phosphorylation may contribute to the structural stability of enolase in EBs, which must survive extracellularly under potentially harsh conditions before infecting new host cells.

This phosphorylation pattern suggests sophisticated regulation of enolase during the developmental cycle, coordinating metabolic activity with gene expression and potentially non-metabolic functions that collectively contribute to the pathogen's ability to establish and maintain infection.

How does Chlamydophila caviae Enolase compare to enolases from other bacterial pathogens in terms of sequence conservation and function?

A distinctive feature of chlamydial enolases is their genomic context and regulatory connection to the Rsb pathway. The adjacency and co-transcription of eno with rsbU appears to be a relatively unique feature of Chlamydia species , suggesting that the metabolic link between glycolysis and gene regulation through the RsbU-RsbV-RsbW pathway may be a specialized adaptation in these obligate intracellular pathogens.

While many bacterial pathogens utilize enolase as a surface-exposed protein for host interaction, the specific details of how C. caviae enolase might function in this capacity, particularly in its differentially phosphorylated states between EBs and RBs , could represent unique adaptations to the chlamydial developmental cycle.

What experimental approaches can determine whether Chlamydophila caviae Enolase exhibits moonlighting functions?

To investigate potential moonlighting functions of Chlamydophila caviae Enolase beyond its glycolytic role, several methodological approaches can be employed:

• Surface Localization Studies: Immunofluorescence microscopy with anti-enolase antibodies on non-permeabilized bacteria can determine if enolase localizes to the bacterial surface, where it could interact with host components.

• Binding Assays: ELISA, surface plasmon resonance, or pull-down assays using purified recombinant enolase to test binding to host components such as plasminogen, extracellular matrix proteins, or cell surface receptors.

• Inhibition Studies: Competitive inhibition experiments using anti-enolase antibodies or recombinant enolase during infection to determine if bacterial adherence or invasion is affected.

• Mass Spectrometry-Based Interaction Studies: Crosslinking followed by immunoprecipitation and mass spectrometry to identify protein-protein interactions involving enolase within the bacterium or with host proteins.

• Mutational Analysis: Creating point mutations that selectively affect either the glycolytic function or putative moonlighting functions to differentiate between these roles (when genetic manipulation is feasible).

• Structural Analysis: Comparing the structure of C. caviae enolase with other bacterial enolases known to have moonlighting functions to identify potential shared binding sites or surface features.

Table 2: Potential moonlighting functions of bacterial enolases and methods to detect them

What is the significance of the co-transcription of enolase and RsbU genes in Chlamydia species?

The co-transcription of enolase (eno) and RsbU genes in Chlamydia species represents a significant evolutionary adaptation linking metabolic activity with gene regulation. Research has demonstrated that these genes are adjacently positioned in many Chlamydia genomes and are co-transcribed from the same operon . This genomic arrangement is particularly noteworthy because it physically and functionally connects a glycolytic enzyme with a regulatory phosphatase.

The significance of this co-transcription lies in several areas:

• Coordinated Expression: The co-transcription ensures that both enzymes are expressed together, maintaining a balanced ratio that is likely optimal for their coordinated function in linking metabolism with gene regulation.

• Temporal Regulation: 5' RACE analysis has shown that at mid-cycle (18 hours post-infection), transcription primarily occurs from the enolase promoter, while at late times (30 hours post-infection), transcription shifts to the RsbU promoter . This suggests developmental stage-specific regulation of these genes.

• Metabolic Sensing Mechanism: The functional connection between these co-transcribed genes creates a sophisticated mechanism for sensing and responding to metabolic conditions. Enolase produces PEP which inhibits RsbU activity , creating a direct link between glycolytic activity and gene regulation.

• Evolutionary Conservation: The conservation of this genetic arrangement across Chlamydia species suggests a selective advantage, likely relating to the ability of these obligate intracellular bacteria to coordinate their gene expression with host metabolic status.

This co-transcription arrangement provides compelling evidence for a novel regulatory mechanism where glycolytic activity directly influences gene expression patterns in Chlamydia, potentially enabling these pathogens to adapt their developmental cycle and virulence in response to host metabolic conditions .

What cutting-edge techniques could advance our understanding of enolase function in Chlamydia pathogenesis?

Several cutting-edge techniques hold promise for deepening our understanding of enolase function in Chlamydia pathogenesis:

• CRISPR Interference (CRISPRi): While traditional gene knockout approaches are challenging in Chlamydia, CRISPRi could provide a method for targeted gene repression to assess the effects of reduced enolase expression on bacterial growth and development.

• Fluorescent Biosensors: Development of genetically-encoded fluorescent biosensors for PEP and other glycolytic intermediates could enable real-time visualization of metabolite dynamics within Chlamydia during infection, revealing how enolase activity fluctuates throughout the developmental cycle.

• Cryo-Electron Tomography: This technique could provide high-resolution structural insights into the localization and potential protein-protein interactions of enolase within intact Chlamydia cells during different developmental stages.

• Proximity Labeling Proteomics: Techniques like BioID or APEX2 fused to enolase could identify proximal protein partners in living bacteria, potentially revealing novel interaction partners beyond the Rsb system.

• Single-Cell RNA-Seq: Applying single-cell transcriptomics to infected host cells could reveal how variability in enolase expression or activity correlates with differential gene expression patterns in individual bacteria within the same inclusion.

• Metabolic Flux Analysis: Using stable isotope-labeled glucose combined with mass spectrometry could quantify how glycolytic flux through enolase changes during the developmental cycle and under different host conditions.

These advanced techniques, especially when used in combination, could provide unprecedented insights into the multifaceted roles of enolase in chlamydial metabolism, gene regulation, and host-pathogen interactions.

How might pharmaceutical targeting of chlamydial enolase affect bacterial viability and gene regulation?

Pharmaceutical targeting of chlamydial enolase could have multifaceted effects on both bacterial viability and gene regulation, making it a potentially valuable therapeutic target:

• Metabolic Disruption: Direct inhibition of enolase enzymatic activity would disrupt glycolysis, potentially depriving the bacteria of essential energy and metabolic intermediates. Since Chlamydia obtains glucose-6-phosphate from the host cell , disrupting its efficient utilization could significantly impair bacterial growth and development.

• Dysregulation of Gene Expression: Given the demonstrated link between enolase activity, PEP production, and inhibition of RsbU phosphatase , enolase inhibitors would likely disrupt the normal regulation of the RsbW pathway. This could lead to inappropriate expression or repression of genes typically regulated by this pathway, potentially disrupting the chlamydial developmental cycle.

• Impact on Developmental Transitions: If the differential phosphorylation of enolase between EBs and RBs is functionally significant for developmental transitions, targeting enolase could interfere with the normal progression between these forms, potentially preventing either replication or infectious particle formation.

• Reduction in Moonlighting Functions: If chlamydial enolase serves additional non-glycolytic functions similar to enolases in other bacteria, inhibitors could also disrupt these activities, potentially including roles in adhesion, immune evasion, or other virulence-related functions.

Potential inhibitors could include small molecules targeting the active site, allosteric modulators, or compounds that disrupt enolase's interaction with other proteins in the Rsb pathway. The ideal therapeutic would selectively target chlamydial enolase without affecting human enolase to minimize side effects.

What experimental approaches would best elucidate the role of host glucose availability in regulating chlamydial gene expression via the enolase-RsbU pathway?

To comprehensively investigate how host glucose availability regulates chlamydial gene expression through the enolase-RsbU pathway, several complementary experimental approaches would be particularly effective:

• Controlled Glucose Modulation: Infecting host cells maintained in media with precisely controlled glucose concentrations, then measuring:

  • Intracellular PEP levels in Chlamydia using targeted metabolomics

  • Phosphorylation state of RsbV using phospho-specific antibodies

  • Expression of genes known to be regulated by the Rsb pathway using qRT-PCR or RNA-seq

• Metabolic Perturbation Studies: Using glucose analogs, glycolytic inhibitors, or host cell lines with altered glucose metabolism to manipulate the system from different angles, then assessing effects on the RsbU pathway and downstream gene expression.

• Real-time Monitoring Systems: Developing fluorescent reporter constructs for Rsb-regulated genes, combined with fluorescent glucose analogs to simultaneously visualize glucose uptake and gene expression changes in living infected cells.

• In vitro Reconstitution: Establishing a cell-free system with purified components of the pathway (enolase, RsbU, RsbV, RsbW, and relevant sigma factors) to directly observe how changing PEP concentrations affect the phosphorylation cascade and sigma factor binding.

• Comparative Transcriptomics Under Metabolic Stress: RNA-seq analysis comparing gene expression patterns in Chlamydia under glucose-replete versus glucose-limited conditions, with particular focus on identifying the complete regulon controlled by this pathway.

• Temporal Analysis Throughout Infection: Assessing how the system responds to glucose availability at different stages of the developmental cycle, potentially revealing stage-specific sensitivities or regulatory patterns.

These approaches would provide a comprehensive understanding of how Chlamydia utilizes the enolase-RsbU pathway as a sophisticated mechanism to sense and respond to host metabolic status, potentially revealing new therapeutic targets that could disrupt this adaptive response.