Recombinant Protochlamydia amoebophila Holo-[acyl-carrier-protein] synthase (acpS)

Basic Research Questions

• What is Protochlamydia amoebophila Holo-[acyl-carrier-protein] synthase (acpS)?

  Protochlamydia amoebophila AcpS is an enzyme that catalyzes the transfer of the 4′-phosphopantetheine moiety from coenzyme A (CoA) onto a conserved serine residue of apo-acyl carrier protein (apo-ACP), resulting in the conversion to functional holo-ACP. This post-translational modification is essential for fatty acid biosynthesis as the resulting holo-ACP carries growing fatty acid chains during elongation via its 4'-phosphopantetheine prosthetic group. P. amoebophila is a symbiont of Acanthamoeba spp. and shows characteristic features of chlamydiae, including dependency on host-derived metabolites and the ability to thrive as an energy parasite within its eukaryotic host .

• What is the function of AcpS in the metabolism of P. amoebophila?

  AcpS plays a crucial role in P. amoebophila metabolism by activating acyl carrier protein through phosphopantetheinylation. This activation is essential for:

  • Fatty acid biosynthesis and membrane phospholipid production

  • Supporting the developmental cycle between replicative bodies (RBs) and elementary bodies (EBs)

  • Enabling metabolic activity in EBs that contributes to maintaining infectivity

  • Facilitating central carbon metabolism, including glucose catabolism via the pentose phosphate pathway and TCA cycle

  Recent research has demonstrated that P. amoebophila EBs maintain respiratory activity and can metabolize D-glucose, with substrate uptake, synthesis of labeled metabolites, and release of labeled CO₂ from ¹³C-labeled D-glucose . The holo-ACP formed through AcpS activity mediates the transfer of acyl intermediates by the covalent attachment of acyl groups to the thiol of the 4′-phosphopantetheine moiety .

• What is the biochemical mechanism of AcpS catalysis?

  The biochemical mechanism of AcpS catalysis involves:

  1. Binding of CoA to the AcpS active site

  2. Recognition and binding of apo-ACP as substrate

  3. Nucleophilic attack by the serine hydroxyl group of apo-ACP on the phosphopantetheine-CoA bond

  4. Formation of a phosphodiester bond between the serine residue and phosphopantetheine

  5. Release of holo-ACP and 3',5'-ADP

  This reaction requires divalent metal ions, typically Mg²⁺, as a cofactor. The holo-ACP formed contains a flexible phosphopantetheine arm with a terminal thiol group that serves as the attachment site for fatty acyl intermediates during biosynthesis . The reaction is generally irreversible under physiological conditions, ensuring that once apo-ACP is converted to holo-ACP, it remains in the active form for fatty acid synthesis.

• How does the metabolic activity of P. amoebophila elementary bodies relate to AcpS function?

  Contrary to the traditional view that chlamydial elementary bodies (EBs) are metabolically inert, research has revealed that P. amoebophila EBs maintain significant metabolic activity, including:

  • Respiratory activity detectable through CTC reduction assays

  • D-glucose uptake and metabolism via the pentose phosphate pathway

  • Host-independent activity of the tricarboxylic acid (TCA) cycle

  • Synthesis of labeled metabolites from ¹³C-labeled glucose

  AcpS function is crucial for these metabolic activities as it ensures the availability of active holo-ACP needed for fatty acid metabolism and membrane maintenance. Notably, when D-glucose is replaced with non-metabolizable L-glucose, there is a rapid decline in the proportion of active bacteria and infectious particles, demonstrating that metabolic activity in the extracellular stage is critical for maintaining infectivity . This establishes a direct link between metabolic capabilities enabled by functional AcpS and the biological fitness of P. amoebophila.

Experimental Design and Methodology

• What expression systems are most effective for producing recombinant P. amoebophila AcpS?

  Based on successful approaches with analogous proteins, the following expression systems should be considered:

  1. E. coli BL21(DE)3-based expression:

     • Advantages: High protein yields, well-established protocols, easy genetic manipulation

     • Optimization strategies: Lower induction temperature (16-25°C), reduced IPTG concentration (0.1-0.5 mM), co-expression with chaperones

  2. Alternative expression hosts:

     • Bacillus subtilis: For cases where E. coli-expressed protein is inactive

     • Cell-free protein synthesis: For rapid screening of expression conditions

  3. Expression vector selection:

     • pET series vectors: Provide tight control of expression with T7 promoter

     • pGEX vectors: Expression as GST fusion for improved solubility

     • pMAL vectors: Expression as MBP fusion for enhanced solubility and easy purification

  4. Affinity tag strategies:

     • N-terminal His₆ tag: Standard approach for metal affinity purification

     • GST fusion: Enhanced solubility but larger tag size

     • SUMO fusion: Improved folding with cleavable tag

  For example, successful expression of TC0668 protein from Chlamydia muridarum was achieved by amplifying the gene with specific primers, cloning into pGEX-4T-1, transforming into E. coli BL21/T7E, and purifying using GST-Agarose Resin . This approach could be adapted for P. amoebophila AcpS with appropriate modifications based on protein properties.

• How can the activity of recombinant P. amoebophila AcpS be assayed in vitro?

  Multiple complementary approaches can be employed to assay AcpS activity:

  1. Electrophoretic methods:

     • Conformationally sensitive urea-PAGE: Holo-ACP migrates faster than apo-ACP due to conformational differences

     • Quantification through densitometry using ImageJ or similar software

     • Example protocol: Run samples on 20% polyacrylamide gels containing 2.5 M urea at 100V for 2-3 hours

  2. Mass spectrometry-based assays:

     • ESMS analysis to detect mass shift (+340 Da) upon phosphopantetheinylation

     • Standard reaction mixture: 50 mM Tris-HCl buffer, 10 mM MgCl₂, 1 mM DTT, 300 μM CoA, 5 μM AcpS, and 50-100 μM apo-ACP

     • Incubation at room temperature for 1 hour before analysis

  3. Functional coupling assays:

     • Testing ability of modified holo-ACP to accept acyl groups from acyl-ACP synthetases

     • Monitoring formation of acyl-ACP using conformationally sensitive gels or mass spectrometry

  4. Spectrophotometric assays:

     • Coupled enzyme assays monitoring CoA release or consumption

     • Fluorescence-based detection using environmentally sensitive probes

  These approaches have been successfully used to characterize AcpS enzymes from various bacteria and should be applicable to P. amoebophila AcpS with appropriate controls and optimization.

• What methods can be used to investigate the structure-function relationship of P. amoebophila AcpS?

  Comprehensive investigation of structure-function relationships requires multiple complementary approaches:

  1. Computational analysis:

     • Homology modeling based on crystal structures of related AcpS enzymes

     • Molecular docking studies to predict substrate binding modes

     • Sequence alignment to identify conserved and variable regions

  2. Mutagenesis studies:

     • Site-directed mutagenesis of predicted catalytic residues and substrate binding sites

     • Creation of chimeric proteins with other AcpS enzymes to identify functional domains

     • Alanine-scanning mutagenesis of conserved residues

  3. Biophysical characterization:

     • Circular dichroism (CD) spectroscopy to assess secondary structure

     • Thermal shift assays to evaluate protein stability

     • Nuclear magnetic resonance (NMR) for structural analysis in solution

  4. Functional correlation:

     • Activity assays of mutant enzymes to correlate structural features with function

     • Binding studies using isothermal titration calorimetry or surface plasmon resonance

  This approach has proven effective for other phosphopantetheinyl transferases and would provide insights into the unique features of P. amoebophila AcpS that reflect its adaptation to the intracellular environment of amoeba hosts .

• How can researchers investigate the role of AcpS in P. amoebophila's developmental cycle?

  Investigating AcpS role in P. amoebophila's developmental cycle requires specialized approaches due to its obligate intracellular lifestyle:

  1. Temporal expression analysis:

     • qRT-PCR to monitor acpS transcript levels throughout the developmental cycle

     • Western blotting with specific antibodies to track AcpS protein levels

     • Normalize data to established markers of developmental stages (e.g., 16S rRNA for bacterial load)

  2. Localization studies:

     • Immunofluorescence microscopy with anti-AcpS antibodies

     • Co-localization with stage-specific markers (similar to HSP60 and PGP3 used for C. muridarum)

     • Super-resolution microscopy for precise subcellular localization

  3. Functional inhibition approaches:

     • Small molecule inhibitors targeting AcpS activity

     • Antisense RNA strategies if genetic manipulation is possible

     • Host cell RNA interference targeting pathways that interact with AcpS function

  4. Metabolomic analysis:

     • Track changes in fatty acid profiles at different developmental stages

     • Isotope labeling to follow AcpS-dependent metabolic fluxes

     • Correlation of metabolite levels with AcpS activity

  For example, researchers studying C. muridarum TC0668 used IFA to determine protein localization during the chlamydial life cycle, finding it could be detected at 8h post-infection with peak expression at 16h, coinciding with RB multiplication and chlamydial differentiation from RBs into EBs . Similar approaches could reveal the temporal and spatial dynamics of AcpS during P. amoebophila development.

Data Analysis and Interpretation

• How can researchers distinguish between active and inactive forms of recombinant P. amoebophila AcpS?

  Distinguishing active from inactive forms requires multiple complementary approaches:

  1. Enzymatic activity assays:

     • Direct measurement of phosphopantetheinyl transfer to apo-ACP

     • Comparison with positive controls (known active AcpS enzymes)

     • Quantitative assessment of reaction kinetics

  2. Structural integrity analysis:

     • Circular dichroism (CD) spectroscopy to assess secondary structure content

     • Thermal shift assays to evaluate protein stability

     • Size-exclusion chromatography to detect aggregation or oligomerization

  3. Substrate binding studies:

     • Isothermal titration calorimetry to measure CoA binding

     • Surface plasmon resonance to evaluate apo-ACP interaction

     • Fluorescence-based assays using environment-sensitive probes

  4. Cofactor analysis:

     • Metal content determination (typically Mg²⁺ is required)

     • Assessment of redox state for any critical cysteine residues

  For example, researchers studying AcpS from other bacteria have used ESMS analysis to verify the ability of purified enzymes to convert apo-ACP substrates to holo-ACP, incubating reaction mixtures containing buffer, MgCl₂, DTT, CoA, AcpS, and apo-ACP substrates at room temperature before analysis . Similar approaches would be applicable to P. amoebophila AcpS.

• How can metabolomic data be analyzed to understand the role of AcpS in P. amoebophila metabolism?

  Comprehensive metabolomic analysis to understand AcpS role should include:

  1. Experimental design considerations:

     • Isotope labeling strategies (e.g., ¹³C-glucose) to track metabolic fluxes

     • Temporal sampling to capture developmental stage-specific metabolism

     • Comparative analysis between wild-type and AcpS-inhibited conditions

  2. Analytical platforms:

     • LC-MS for targeted metabolite profiling

     • ICR/FT-MS for high-resolution untargeted metabolomics

     • UPLC-MS for complex lipid analysis

     • Isotope-ratio mass spectrometry (IRMS) for flux analysis

  3. Data processing approaches:

     • Multivariate statistical analysis (PCA, PLS-DA) to identify patterns

     • Pathway enrichment analysis to contextualize findings

     • Integration with transcriptomic and proteomic data

  4. Interpretation strategies:

     • Focus on metabolites involved in ACP-dependent pathways

     • Trace carbon flux through central metabolic pathways

     • Compare with metabolic profiles of related organisms

  Research on P. amoebophila EBs has successfully employed a combined metabolomics approach, including fluorescence microscopy-based assays, IRMS, ICR/FT-MS, and UPLC-MS, with a particular focus on central carbon metabolism . This approach revealed that P. amoebophila EBs metabolize D-glucose via the pentose phosphate pathway and TCA cycle, demonstrating the power of metabolomics for understanding the metabolic capabilities of these bacteria.

• How can researchers analyze the impact of environmental conditions on P. amoebophila AcpS activity?

  Analyzing environmental impacts on AcpS activity requires systematic approaches:

  1. In vitro enzyme characterization under varying conditions:

     • Temperature range relevant to host environment (10-40°C)

     • pH range encompassing physiological variation (pH 5.0-9.0)

     • Ionic strength and specific ion effects (particularly Mg²⁺, K⁺, Na⁺)

     • Redox conditions mimicking intracellular environment

  2. Host-pathogen interaction models:

     • Infection of Acanthamoeba under controlled environmental conditions

     • Monitoring AcpS activity and fatty acid profiles in response to environmental changes

     • Assessment of developmental cycle progression under different conditions

  3. Data analysis approaches:

     • Response surface methodology to model multifactorial effects

     • Time-course analysis to capture dynamic responses

     • Correlation of AcpS activity with measures of bacterial fitness

  4. Specific environmental factors to consider:

     • Nutrient availability (particularly glucose)

     • Oxygen tension (microaerophilic vs. aerobic conditions)

     • Host cell density and metabolic state

  Previous research demonstrated that D-glucose availability significantly affects P. amoebophila EB activity. When D-glucose was replaced with non-metabolizable L-glucose, the proportion of metabolically active EBs decreased from 27.0% to 6.0% after 40h incubation . This approach of substrate manipulation could be extended to investigate other environmental factors affecting AcpS-dependent metabolism.

Biological Significance and Applications

• How does AcpS activity contribute to P. amoebophila's ability to maintain infectivity?

  AcpS activity contributes to P. amoebophila infectivity through several mechanisms:

  1. Metabolic support during extracellular phase:

     • Enables fatty acid metabolism necessary for membrane integrity

     • Supports energy generation via ACP-dependent pathways

     • Maintains functional capacity during transition between hosts

  2. Developmental cycle regulation:

     • Facilitates fatty acid synthesis required for RB replication

     • Supports membrane remodeling during EB formation

     • Contributes to the formation of infectious EB membrane structure

  3. Experimental evidence:

     • D-glucose deprivation leads to rapid decline in infectious particles

     • Similar effects observed in C. trachomatis, suggesting a conserved mechanism

     • Metabolic activity in the extracellular stage is critical for maintaining infectivity

  Research has demonstrated that when P. amoebophila EBs were incubated in medium where D-glucose was replaced with non-metabolizable L-glucose, there was a significant decrease in the proportion of active bacteria and a corresponding decline in infectivity . This direct correlation between metabolic activity and infectious potential highlights the critical role of metabolic enzymes like AcpS in maintaining bacterial viability during the host-free stage.

• What are the potential applications of recombinant P. amoebophila AcpS in biotechnology?

  Recombinant P. amoebophila AcpS has several potential biotechnological applications:

  1. Tool for carrier protein activation:

     • Production of functional holo-ACPs for in vitro fatty acid synthesis

     • Activation of carrier proteins for polyketide and non-ribosomal peptide synthesis

     • Engineering novel biosynthetic pathways requiring phosphopantetheinylated carrier proteins

  2. Target for antimicrobial development:

     • Design of inhibitors targeting AcpS to disrupt chlamydial metabolism

     • Platform for screening compound libraries for novel anti-chlamydial agents

     • Structure-based drug design for specific inhibition of chlamydial AcpS

  3. Metabolic engineering applications:

     • Enhancement of fatty acid production in engineered microorganisms

     • Incorporation into synthetic biology circuits for controlled lipid synthesis

     • Production of specialized fatty acids and derivatives

  4. Research tool for studying host-pathogen interactions:

     • Probe for investigating metabolic dependencies of obligate intracellular bacteria

     • Model system for understanding evolutionary adaptations in host-associated bacteria

  The applicability of AcpS in biotechnology is supported by research on related phosphopantetheinyl transferases. For example, studies in Dictyostelium discoideum identified two functionally distinct phosphopantetheinyl transferases, with DiSfp being specific to Type I multifunctional PKS/fatty acid synthase proteins . Similar functional specificity in P. amoebophila AcpS could be exploited for selective activation of carrier proteins in biotechnological applications.

• How does P. amoebophila AcpS contribute to our understanding of evolutionary adaptations in obligate intracellular bacteria?

  P. amoebophila AcpS provides important insights into evolutionary adaptations of obligate intracellular bacteria:

  1. Metabolic streamlining versus retention:

     • Despite genome reduction common in obligate intracellular bacteria, P. amoebophila has retained relatively complete sets of proteins for energy metabolism

     • AcpS represents an essential function that has been conserved despite evolutionary pressure for genome minimization

     • Suggests the critical importance of fatty acid metabolism even in host-dependent bacteria

  2. Host adaptation signatures:

     • Comparison with AcpS from other bacteria can reveal adaptation to specific host environments

     • Substrate specificity may reflect co-evolution with host metabolic systems

     • Kinetic parameters may be optimized for the amoeba intracellular environment

  3. Evolutionary relationships:

     • P. amoebophila represents the Parachlamydiaceae family, which diverged from pathogenic Chlamydiaceae

     • Comparative analysis of AcpS across chlamydial lineages can provide insights into the evolution of host dependence

     • Position as a symbiont of protozoa rather than a pathogen of mammals offers perspective on the evolution of chlamydial metabolism

  4. Metabolic independence:

     • The ability of P. amoebophila EBs to maintain metabolic activity challenges traditional views of obligate intracellular bacteria

     • AcpS-dependent metabolism represents a degree of metabolic independence that may be an ancestral trait partially lost in more specialized pathogens

     • Suggests a more complex view of the evolutionary trajectory toward obligate intracellular lifestyle

  Analysis of the complete genome sequence of P. amoebophila UWE25 demonstrated that it shows many characteristic features of chlamydiae while having distinct differences from its closest relatives . These differences, which may include AcpS functionality, provide insights into the evolutionary history and adaptations of this important group of bacteria.