Recombinant Chlamydophila caviae UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2,6-diaminopimelate ligase (murE)

What is UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2,6-diaminopimelate ligase (murE) and what is its function in Chlamydophila caviae?

MurE is a cytoplasmic enzyme that catalyzes a critical step in bacterial peptidoglycan biosynthesis, specifically the addition of meso-diaminopimelic acid (meso-A2pm) to the nucleotide precursor UDP-N-acetylmuramoyl-L-alanyl-D-glutamate . In Chlamydophila caviae, the enzyme functions as part of the peptidoglycan synthesis pathway, which is crucial for bacterial cell wall formation. The enzyme adds the third amino acid residue to the peptide stem during the cytoplasmic phase of peptidoglycan biosynthesis . Unlike some other Chlamydia species where peptidoglycan has been difficult to detect (the "chlamydial peptidoglycan paradox"), C. caviae has been confirmed to incorporate diaminopimelate into its cell wall, verifying the functionality of this enzyme in vivo .

How does the taxonomy of Chlamydophila caviae relate to other Chlamydial species, and why is this relevant for murE research?

Chlamydophila caviae (now often referred to as Chlamydia caviae in updated taxonomy) belongs to the family Chlamydiaceae and is evolutionarily related to other chlamydial pathogens . The genus includes important human pathogens such as C. trachomatis and C. pneumoniae, as well as animal pathogens like C. psittaci, C. pecorum, and C. abortus . C. caviae specifically infects guinea pigs, causing conjunctivitis and genital tract infections that model aspects of human chlamydial disease . This taxonomic relationship is significant for murE research because studying the enzyme in C. caviae provides insights into peptidoglycan synthesis across the Chlamydia genus. C. caviae serves as an experimentally accessible model organism that, unlike some human-specific chlamydial species, can be more easily cultured and manipulated in laboratory settings, making it valuable for comparative studies of cell wall biosynthesis enzymes like murE .

What is known about the genomic context of the murE gene in C. caviae?

The murE gene in C. caviae is part of a complete set of genes necessary for peptidoglycan biosynthesis. Genome surveys of C. caviae have confirmed that the bacterium possesses all the genetic machinery needed to synthesize peptidoglycan de novo . The C. caviae genome is relatively small, containing approximately 1.17 Mbp with 998 protein-coding genes . Additionally, C. caviae strain GPIC contains an extrachromosomal plasmid called pCpGP1, though the murE gene itself is chromosomally encoded . Sequence analysis of murE from C. caviae reveals conservation of key active site residues, including the critical DNPR motif that is involved in substrate binding and catalysis, which is identical across various bacterial species . The genomic presence of a complete peptidoglycan synthesis pathway in C. caviae is particularly interesting given the historical difficulty in detecting peptidoglycan in some chlamydial species, making C. caviae an important model for understanding this aspect of chlamydial biology.

What expression systems have been successfully used for producing recombinant C. caviae murE protein?

Recombinant C. caviae murE has been successfully expressed using baculovirus expression systems . This approach involves cloning the murE open reading frame (ORF) and expressing it in insect cells, which allows for proper protein folding and post-translational modifications. For optimal expression, the following methodological considerations are important:

• PCR amplification of the murE gene from C. caviae genomic DNA using specific primers designed to include appropriate restriction sites

• Cloning into suitable expression vectors compatible with the baculovirus system

• Transfection of insect cells and viral amplification

• Large-scale protein expression followed by purification

The baculovirus expression system has proven effective for producing functional murE enzyme with high purity (>85% as determined by SDS-PAGE) . Alternative approaches documented for related MurE enzymes include expression in E. coli systems, which has been successfully employed for MurE from Verrucomicrobium spinosum, a bacterium related to Chlamydia . When expressing in E. coli, functional complementation assays with E. coli strains harboring mutations in the murE gene can be used to verify enzymatic activity of the recombinant protein in vivo .

What purification strategies are most effective for isolating recombinant C. caviae murE while maintaining its enzymatic activity?

Effective purification of recombinant C. caviae murE requires a multi-step approach that maintains protein stability and enzymatic activity. Based on documented approaches for related MurE enzymes, the following purification strategy is recommended:

• Initial centrifugation of expression culture to pellet cells

• Cell lysis using either sonication or chemical methods in a buffer containing protease inhibitors

• Clarification of lysate by high-speed centrifugation

• Affinity chromatography as the primary purification step, typically using His-tag affinity if the recombinant protein includes a histidine tag

• Secondary purification using ion-exchange chromatography to remove remaining contaminants

• Size-exclusion chromatography as a final polishing step

Throughout the purification process, it's critical to maintain buffer conditions that preserve enzyme activity. For C. caviae murE, this typically includes:

• pH range of 7.5-9.6 (with optimum activity reported at higher pH for related enzymes)

• Presence of divalent cations, particularly magnesium (optimal concentration around 30 mM based on related enzymes)

• Addition of stabilizing agents such as glycerol (5-50%) for storage

• Storage at -20°C/-80°C with aliquoting to avoid repeated freeze-thaw cycles

For long-term storage, the purified enzyme can be maintained as either a liquid form (shelf life approximately 6 months at -20°C/-80°C) or lyophilized form (shelf life approximately 12 months at -20°C/-80°C) .

How can researchers validate the functional activity of purified recombinant C. caviae murE?

Validating the functional activity of purified recombinant C. caviae murE requires both in vivo and in vitro approaches to confirm its enzymatic capabilities. The following methodological workflow is recommended:

• In vivo functional complementation:

  • Transform the recombinant murE gene into an E. coli strain harboring a temperature-sensitive mutation in its native murE gene

  • Assess growth at non-permissive temperatures, where successful complementation indicates functional enzyme activity

• In vitro enzymatic assay:

  • Measure the formation of UDP-MurNAc-tripeptide from UDP-MurNAc-L-Ala-D-Glu and meso-diaminopimelate in the presence of ATP

  • Typical reaction conditions include:

    • Tris-HCl buffer (pH 8.6) or higher pH buffers (up to 9.6)

    • 30 mM MgCl₂

    • 5 mM ATP

    • Substrate concentrations: 0.5 mM UDP-MurNAc-L-Ala-D-Glu and varying concentrations of meso-diaminopimelate

  • Detection methods include:

    • HPLC analysis of reaction products

    • Coupled enzymatic assays measuring ADP release

    • Radiolabeled substrate incorporation

• Substrate specificity analysis:

  • Test activity with alternative substrates structurally related to meso-diaminopimelate

  • Compare kinetic parameters (Km, Vmax) to establish preference for meso-diaminopimelate

• Physical parameter optimization:

  • Determine pH optimum (expected around 9.6 based on related enzymes)

  • Establish magnesium concentration optimum (expected around 30 mM)

  • Evaluate temperature stability and activity profile

The enzyme should demonstrate a strong preference for meso-diaminopimelate with a Km in the micromolar range (approximately 17 μM based on related enzymes) and significantly lower affinity for structural analogs .

What structural features are critical for the catalytic activity of C. caviae murE, and how do they compare with other bacterial MurE enzymes?

The catalytic activity of C. caviae murE depends on several critical structural features that are largely conserved across bacterial MurE enzymes. Based on sequence alignment and homology modeling studies of related MurE enzymes, the following structural elements are essential:

• Three-domain architecture:

  • Domain A: Forms a nucleotide-binding fold that interacts with the UDP portion of the substrate

  • Domain B: Contains the central catalytic core

  • Domain C: Responsible for binding the incoming meso-diaminopimelate substrate

• Key conserved motifs:

  • The DNPR motif (residues 409-412 in V. spinosum MurE, analogous positions in C. caviae) is critical for substrate recognition and is identical across various bacterial species

  • N410 and R412 in this motif form hydrogen bonds with the ε-carboxyl group of meso-diaminopimelate, providing specificity for this substrate

• Active site residues:

  • Approximately 10-16 residues form the active site, with the majority being conserved across different bacterial species

  • These residues create binding pockets for ATP, UDP-MurNAc-L-Ala-D-Glu, and meso-diaminopimelate

• Special structural feature:

  • A carbamylated lysine residue in the active site, which has been observed in MurE from E. coli and is likely present in C. caviae MurE as well

Comparative analysis with other bacterial MurE enzymes shows that C. caviae murE likely shares approximately 37-38% sequence identity with E. coli and M. tuberculosis MurE enzymes, with higher conservation in active site regions . The structural determinants responsible for selecting meso-diaminopimelate as the third amino acid in peptidoglycan are conserved, explaining the enzyme's specificity. This conservation across evolutionary distance highlights the fundamental importance of these structural features for MurE function throughout bacterial evolution.

How do researchers utilize homology modeling and sequence alignment to predict structural features of C. caviae murE?

Researchers employ a systematic approach to predict structural features of C. caviae murE through homology modeling and sequence alignment, as demonstrated in studies of related MurE enzymes:

• Multiple sequence alignment methodology:

  • Perform multiple sequence alignments using tools like ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/)

  • Include experimentally characterized MurE sequences from diverse bacterial species (e.g., E. coli, M. tuberculosis, V. spinosum) along with the C. caviae sequence

  • Identify conserved motifs, particularly the DNPR motif and other catalytically important residues

  • Analyze conservation patterns to identify residues likely involved in substrate binding and catalysis

• Template selection for homology modeling:

  • Conduct PSI-BLAST searches against the Protein Data Bank to identify suitable structural templates

  • Select templates based on sequence identity (typically >30% is preferred) and functional similarity

  • For C. caviae murE, suitable templates would include E. coli MurE (PDB ID: 1E8C) with approximately 37% identity and M. tuberculosis MurE with approximately 38% identity

• Homology model generation:

  • Use specialized software such as SWISS-MODEL (http://swissmodel.expasy.org/)

  • Generate the model based on the selected template

  • Evaluate model quality using metrics such as QMEAN scores (ideally between 0-1, with higher values indicating better models)

• Structural analysis and validation:

  • Analyze domain organization (the three domains A, B, and C)

  • Identify active site pocket and substrate binding regions

  • Compare predicted binding residues with those in experimentally determined structures

  • Validate through comparison with biochemical data on substrate specificity

• Substrate-binding prediction:

  • Overlay the homology model with ligand-bound template structures

  • Analyze potential hydrogen bonds and other interactions between the enzyme and its substrates

  • Identify residues likely responsible for substrate specificity, particularly those that discriminate between meso-diaminopimelate and lysine

The combined approach of sequence alignment and homology modeling allows researchers to make informed predictions about C. caviae murE structure and function, guiding experimental design for site-directed mutagenesis and biochemical characterization studies.

What kinetic parameters characterize C. caviae murE activity, and how do these compare with MurE enzymes from other bacterial species?

The kinetic parameters of C. caviae murE activity provide important insights into its enzymatic efficiency and substrate specificity. While specific parameters for C. caviae murE are not directly provided in the search results, we can infer likely characteristics based on closely related MurE enzymes that have been experimentally characterized:

Table 1: Comparative Kinetic Parameters of MurE Enzymes from Different Bacterial Species

Key observations regarding C. caviae murE kinetics:

The significantly higher specific activity observed in V. spinosum MurE compared to E. coli and P. aeruginosa enzymes (26-fold and 14-fold higher, respectively) suggests that there can be substantial variation in catalytic rates even among related enzymes . This variation might reflect differences in cellular growth rates or regulatory mechanisms controlling peptidoglycan synthesis in different bacterial species.

How does the presence and function of murE in C. caviae relate to the "chlamydial peptidoglycan paradox"?

The presence and function of murE in C. caviae provides significant insights into the longstanding "chlamydial peptidoglycan paradox," which refers to the historical difficulty in detecting peptidoglycan in Chlamydia despite genomic evidence for peptidoglycan synthesis machinery. This paradox has been a central question in chlamydial biology for decades. C. caviae murE contributes to our understanding in several key ways:

• Experimental confirmation of functional peptidoglycan synthesis:

  • Unlike some other chlamydial species, peptidoglycan has been successfully isolated and characterized from C. caviae cultures

  • Analysis revealed the presence of diaminopimelate, confirming that C. caviae incorporates this MurE substrate into its cell wall structure

  • This provides direct evidence that the peptidoglycan synthesis pathway, including MurE activity, is functional in at least some chlamydial species

• Genomic context and pathway completeness:

  • The C. caviae genome contains all genes necessary for de novo peptidoglycan synthesis, including murE

  • The presence of a complete pathway in C. caviae is similar to C. trachomatis, which also possesses the genetic machinery for peptidoglycan synthesis despite difficulties in detecting the end product

• Functional validation across species:

  • The MurE enzyme from C. trachomatis has been experimentally validated as a functional enzyme, even though peptidoglycan detection has been challenging in this species

  • This parallels the situation with C. caviae murE, which has been shown to be an authentic enzyme involved in peptidoglycan synthesis

• Evolutionary implications:

  • The conservation of functional murE and other peptidoglycan synthesis genes across chlamydial species suggests evolutionary pressure to maintain this pathway

  • This indicates that peptidoglycan, or components of its synthetic pathway, must serve important functions in chlamydial biology, even if the final structure differs from typical bacterial peptidoglycan or is produced only under specific conditions

The experimental demonstration that C. caviae produces detectable peptidoglycan containing diaminopimelate strongly suggests that MurE and the entire peptidoglycan synthesis pathway are functionally important in the chlamydial life cycle. This provides a valuable counterpoint to observations in other chlamydial species and helps reconcile genomic evidence with biochemical reality, addressing a key aspect of the chlamydial peptidoglycan paradox.

What are the key similarities and differences between C. caviae murE and the corresponding enzymes in human pathogenic Chlamydia species?

C. caviae murE shares significant similarities with its counterparts in human pathogenic Chlamydia species, but there are also notable differences that may reflect adaptation to different hosts and ecological niches:

Table 2: Comparison of murE Between C. caviae and Human Pathogenic Chlamydia Species

Key similarities:

• Functional conservation:

  • All chlamydial MurE enzymes appear to be functional meso-diaminopimelate adding enzymes

  • The critical DNPR motif and other key catalytic residues are conserved across chlamydial species

• Genomic context:

  • All chlamydial species possess the complete set of genes necessary for peptidoglycan synthesis

  • The murE gene is chromosomally encoded in all species

• Structural features:

  • The three-domain architecture (domains A, B, and C) is preserved across chlamydial MurE enzymes

  • Active site organization is highly similar, reflecting conservation of enzyme mechanism

Key differences:

• Catalytic efficiency:

  • The specific activity of C. caviae murE likely differs from that of human pathogens, potentially correlating with differences in growth rates

  • C. trachomatis MurE showed lower specific activity compared to other bacterial MurE enzymes, reflecting its slow growth as an obligate intracellular organism

• Peptidoglycan detection:

  • Peptidoglycan is more readily detectable in C. caviae than in human pathogenic species

  • This suggests potential differences in regulation, timing of synthesis, or structural modifications that affect detection

• Host adaptation:

  • Differences in MurE properties may reflect adaptation to different host environments (guinea pig vs. human tissues)

  • These adaptations could influence enzyme kinetics or regulatory mechanisms controlling peptidoglycan synthesis

The comparison between C. caviae murE and its counterparts in human pathogenic species highlights both the evolutionary conservation of this essential enzyme and the potential for species-specific adaptations that may contribute to differences in growth, pathogenesis, and interaction with host defenses.

How does recombinant C. caviae murE compare to murE from Verrucomicrobium spinosum, a related bacterium that has been extensively studied?

Comparing recombinant C. caviae murE to its counterpart in Verrucomicrobium spinosum reveals important insights into evolutionary conservation and functional adaptations between these related bacterial lineages:

Table 3: Comparative Analysis of murE from C. caviae and V. spinosum

Key similarities:

• Evolutionary relationship:

  • V. spinosum is evolutionarily related to the genus Chlamydia, making it a valuable comparative model

  • Both organisms employ similar pathways for peptidoglycan synthesis and share key enzymatic components

• Functional characteristics:

  • Both enzymes show specificity for meso-diaminopimelate as the preferred substrate

  • Similar physical parameters including alkaline pH optima and magnesium requirements

  • Conservation of the DNPR motif and other key residues involved in substrate binding

• Structural organization:

  • Three-domain architecture is preserved between both enzymes

  • Similar organization of active site with contributions from all three domains

Key differences:

• Experimental characterization:

  • V. spinosum murE has been extensively characterized biochemically, with detailed kinetic parameters established

  • C. caviae murE characterization is more limited, with many parameters predicted rather than experimentally determined

• Cellular context:

  • V. spinosum is a free-living bacterium with a different lifestyle from the obligate intracellular C. caviae

  • This lifestyle difference may influence enzyme regulation and integration with cellular metabolism

• Pathogenicity:

  • V. spinosum is pathogenic toward invertebrates (Drosophila melanogaster and Caenorhabditis elegans) but not mammals

  • C. caviae has evolved as a mammalian pathogen specifically adapted to guinea pigs

The extensive characterization of V. spinosum murE provides a valuable framework for predicting and understanding the properties of C. caviae murE. The high degree of similarity in key functional and structural features supports the use of V. spinosum as a model system for studying aspects of chlamydial cell wall biosynthesis that may be more challenging to investigate directly in Chlamydia species.

What does the presence of functional murE in C. caviae reveal about chlamydial evolution and adaptation?

The presence of functional murE in C. caviae provides significant insights into chlamydial evolution and adaptation, highlighting both conserved features and specialized adaptations:

• Evolutionary conservation of peptidoglycan synthesis:

  • The retention of functional murE and other peptidoglycan synthesis genes across chlamydial species indicates strong evolutionary pressure to maintain this pathway

  • This conservation suggests that the ability to synthesize peptidoglycan, even if modified or regulated differently from free-living bacteria, remains essential for chlamydial survival and reproduction

• Adaptation to intracellular lifestyle:

  • Despite evolutionary pressures toward genome reduction in obligate intracellular bacteria, chlamydial species have maintained the complete genetic machinery for peptidoglycan synthesis

  • This suggests that cell wall components play crucial roles in the unique developmental cycle of Chlamydia, potentially during transition between elementary bodies and reticulate bodies

• Species-specific regulatory adaptations:

  • The variation in detectability of peptidoglycan between C. caviae (detectable) and human pathogens like C. trachomatis (difficult to detect) suggests species-specific adaptations in regulation or structure

  • These differences may reflect adaptations to different host environments or immune evasion strategies

• Relationship to broader bacterial evolution:

  • The presence of murE in both Chlamydia and related bacteria like Verrucomicrobium provides evidence for the evolutionary relationship between these lineages

  • Comparative analysis of MurE across these bacteria helps reconstruct the evolutionary history of this ancient enzyme family

• Host-pathogen co-evolution:

  • The specificity of C. caviae for guinea pigs (unsuccessful attempts to infect mice, hamsters, rabbits, and gerbils) suggests co-evolutionary adaptation between the pathogen and its natural host

  • This host specificity may be reflected in subtle adaptations of core metabolic processes, potentially including peptidoglycan synthesis

The maintenance of functional murE in C. caviae, along with experimental confirmation of peptidoglycan synthesis, helps resolve the long-standing "chlamydial peptidoglycan paradox" and suggests that peptidoglycan, or components of its synthetic pathway, serve critical functions in the chlamydial life cycle. This provides important context for understanding how these ancient pathogens have evolved and adapted to their specialized intracellular lifestyle while maintaining essential components of bacterial cell biology.

How does research on C. caviae murE contribute to understanding pathogenicity mechanisms in chlamydial infections?

Research on C. caviae murE provides valuable insights into chlamydial pathogenicity mechanisms through several important connections:

• Cell wall integrity and infection process:

  • MurE's role in peptidoglycan synthesis directly impacts cell wall structure, which is critical during the chlamydial developmental cycle

  • The transition between infectious elementary bodies (EBs) and replicative reticulate bodies (RBs) involves changes in cell wall composition, potentially mediated by enzymes like murE

  • Understanding this process helps explain how chlamydial species establish and maintain infections

• Immune response interactions:

  • Peptidoglycan components are recognized by host pattern recognition receptors like NOD1 and NOD2

  • Studies with plasmid-cured C. caviae (strain CC13) have shown that C. caviae activates TLR2-dependent signaling and retains virulence in guinea pig models of infection

  • This contrasts with plasmid-cured C. muridarum and C. trachomatis, which show reduced TLR2 activation, suggesting species-specific differences in immune activation mechanisms

• Model system for human pathogens:

  • C. caviae infection in guinea pigs models aspects of human genital tract infections caused by C. trachomatis

  • The ability to study murE function in this system provides translatable insights for human chlamydial infections

  • Inflammatory pathology induced by C. caviae in guinea pig models closely resembles that seen in human infections

• Genetic tractability and experimental advantages:

  • Recent developments in transformation systems for C. caviae using shuttle vectors enable genetic manipulation that can target murE or related pathways

  • These tools allow for direct testing of hypotheses about the role of murE and peptidoglycan synthesis in pathogenicity

  • The ability to express fluorescent proteins in C. caviae facilitates tracking of infection dynamics and bacterial persistence

• Zoonotic potential:

  • C. caviae has been isolated from human patients with cervicitis and urethritis, suggesting zoonotic transmission potential

  • Understanding conserved pathogenicity mechanisms involving murE and cell wall synthesis across chlamydial species helps predict and manage zoonotic risk

The combination of C. caviae's genetic tractability, established animal models, and the conservation of key pathogenicity factors makes research on its murE enzyme particularly valuable for understanding fundamental aspects of chlamydial infection biology. This research provides mechanistic insights into how these obligate intracellular pathogens establish infection, evade host defenses, and cause inflammatory pathology, with potential applications to preventing and treating both animal and human chlamydial infections.

What potential does C. caviae murE have as a target for developing new antimicrobial strategies?

C. caviae murE presents several promising attributes that position it as a potential target for novel antimicrobial strategies, with implications for both veterinary and human medicine:

• Essential enzymatic function:

  • MurE catalyzes a critical step in peptidoglycan biosynthesis that has no mammalian counterpart

  • The enzyme is essential for bacterial cell wall integrity and survival, making it an attractive target for inhibitor development

  • Targeting murE would interfere with a fundamental process in bacterial cell biology

• Conservation across pathogenic species:

  • The high degree of conservation in catalytic mechanisms and active site architecture between C. caviae murE and enzymes from human pathogenic species (C. trachomatis, C. pneumoniae) suggests that inhibitors could have broad-spectrum activity

  • This conservation extends to other clinically relevant bacteria that rely on peptidoglycan synthesis

• Structural and biochemical characterization:

  • Detailed understanding of murE structure, including key catalytic residues and substrate binding pockets, facilitates structure-based drug design

  • The availability of recombinant protein expression systems enables high-throughput screening for inhibitors

  • Homology models based on related MurE structures provide templates for in silico screening approaches

• Experimental advantages of the C. caviae model:

  • C. caviae is more readily cultured than some other chlamydial species

  • Established guinea pig infection models allow for in vivo testing of potential inhibitors

  • Recent development of transformation systems for C. caviae facilitates genetic manipulation to validate target importance

• Novel therapeutic strategy:

  • Current treatments for chlamydial infections rely primarily on broad-spectrum antibiotics like tetracyclines, macrolides, and fluoroquinolones

  • Targeting murE would represent a novel mechanism of action that could address issues of antimicrobial resistance

  • Inhibitors could potentially be used alone or in combination with existing antibiotics to enhance efficacy

• Addressing unique aspects of chlamydial biology:

  • The continued presence of murE and other peptidoglycan synthesis genes despite the "chlamydial peptidoglycan paradox" suggests these enzymes play crucial roles in chlamydial biology

  • Targeting these pathways may disrupt unique aspects of the chlamydial developmental cycle or stress response mechanisms

The combination of essential function, absence in mammalian hosts, and availability of structural and biochemical data makes C. caviae murE a promising candidate for antimicrobial development. Additionally, the opportunity to test candidate inhibitors in established animal models provides a clear pathway for translating basic research into therapeutic applications for both veterinary and potentially human chlamydial infections.

What are the current challenges and methodological approaches for studying the role of murE in the context of C. caviae's obligate intracellular lifestyle?

Studying murE function in the context of C. caviae's obligate intracellular lifestyle presents several unique challenges that require specialized methodological approaches:

• Genetic manipulation challenges and solutions:

  • Challenge: Traditional genetic methods are difficult to apply to obligate intracellular bacteria

  • Approach: Recently developed transformation systems using shuttle vectors that combine the cryptic plasmid of C. caviae with expression cassettes for fluorescent proteins enable genetic manipulation

  • Method: These systems allow for expression of tagged versions of murE or generation of conditional mutants to study function in the intracellular environment

• Temporal regulation during developmental cycle:

  • Challenge: Chlamydial species transition between distinct developmental forms (elementary bodies and reticulate bodies) with potentially different requirements for murE activity

  • Approach: Time-course experiments with synchronized infections coupled with transcriptomic and proteomic analyses

  • Method: RNA-seq and targeted proteomics at different stages of the developmental cycle can reveal temporal patterns of murE expression and activity

• Visualization of peptidoglycan synthesis:

  • Challenge: Traditional methods for visualizing bacterial cell walls are often ineffective with chlamydial species due to potential modifications or limited synthesis

  • Approach: Metabolic labeling with modified peptidoglycan precursors combined with click chemistry

  • Method: Incorporation of azide- or alkyne-modified D-amino acids followed by fluorescent tagging via click chemistry allows visualization of peptidoglycan synthesis sites within infected cells

• Disentangling host-pathogen interactions:

  • Challenge: The intracellular environment introduces complexities in distinguishing direct effects of murE activity from host responses

  • Approach: Combination of in vitro biochemical studies with cell culture infection models

  • Method: Comparing phenotypes of wild-type and murE-modified strains in various cell types, including cells with modified innate immune sensors (e.g., TLR2, NOD1/2 knockouts)

• Mimicking physiological conditions:

  • Challenge: Laboratory culture conditions may not recapitulate the physiological environment where murE functions in vivo

  • Approach: Development of more sophisticated cell culture models and strategic use of animal models

  • Method: Three-dimensional cell culture systems, co-cultures with immune cells, and targeted experiments in guinea pig infection models to validate findings from simpler systems

• Integration with systems biology:

  • Challenge: Understanding how murE activity integrates with broader metabolic and developmental networks

  • Approach: Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Method: Global analyses of metabolic shifts in response to perturbations of murE activity, potentially using chemical genetics approaches with small molecule inhibitors

These methodological approaches represent the cutting edge of research in this challenging system, allowing investigators to overcome the inherent difficulties of studying obligate intracellular bacteria while gaining meaningful insights into the role of murE in C. caviae biology and pathogenesis.

How can mutagenesis studies of C. caviae murE inform our understanding of structure-function relationships in this enzyme family?

Mutagenesis studies of C. caviae murE offer powerful approaches to elucidate structure-function relationships in this important enzyme family, providing insights that extend beyond chlamydial biology:

• Targeting conserved active site residues:

  • Approach: Site-directed mutagenesis of key catalytic residues identified through sequence alignment and homology modeling

  • Methodology:

    • Focus on the DNPR motif (particularly N and R residues that form critical hydrogen bonds with meso-diaminopimelate)

    • Create conservative and non-conservative substitutions

    • Express and purify mutant proteins for in vitro activity assays

  • Expected outcomes: Quantitative assessment of how specific residues contribute to substrate binding, catalysis, and substrate specificity

• Domain interface mutations:

  • Approach: Target residues at the interfaces between the three domains (A, B, and C)

  • Methodology:

    • Identify residues that participate in interdomain communication based on homology models

    • Create mutations that alter flexibility or communication between domains

    • Analyze changes in enzyme dynamics using techniques like hydrogen-deuterium exchange mass spectrometry

  • Expected outcomes: Understanding how conformational changes during the catalytic cycle are coordinated between domains

• Substrate specificity determinants:

  • Approach: Mutations aimed at altering the preference for meso-diaminopimelate versus related compounds

  • Methodology:

    • Target residues in the binding pocket that interact with the ε-carboxyl group of meso-diaminopimelate

    • Create variants with altered charge distribution or pocket geometry

    • Test activity with meso-diaminopimelate versus L-lysine or other substrate analogs

  • Expected outcomes: Identification of specific structural features that determine substrate selectivity, potentially enabling engineering of enzymes with altered specificity

• Species-specific variations:

  • Approach: Swap non-conserved residues between C. caviae murE and orthologs from other species

  • Methodology:

    • Identify positions that differ between C. caviae and other chlamydial species or more distant bacteria

    • Create chimeric enzymes with domains or specific residues from different species

    • Assess how these changes affect kinetic parameters and substrate preferences

  • Expected outcomes: Understanding how species-specific adaptations influence enzyme function in different bacterial contexts

• In vivo functional analysis:

  • Approach: Express mutated versions of murE in C. caviae using newly developed transformation systems

  • Methodology:

    • Create shuttle vectors expressing mutant versions of murE under native or inducible promoters

    • Transform C. caviae and assess effects on growth, developmental cycle, and peptidoglycan synthesis

    • Combine with metabolic labeling to visualize effects on cell wall formation

  • Expected outcomes: Correlation of biochemical properties with functional importance in the living organism

The results from such mutagenesis studies would significantly advance our understanding of how structure dictates function in MurE enzymes, potentially revealing opportunities for selective inhibition of bacterial versus human pathogenic chlamydial enzymes, and providing fundamental insights into the evolution of substrate specificity in this ancient enzyme family.

What advanced techniques can be applied to investigate the potential interactions between C. caviae murE and other components of the peptidoglycan synthesis machinery?

Investigating interactions between C. caviae murE and other components of the peptidoglycan synthesis machinery requires sophisticated approaches that can detect, characterize, and manipulate protein-protein interactions in this challenging biological system:

• Protein-protein interaction mapping:

  • Bacterial two-hybrid (B2H) systems:

    • Adapt bacterial two-hybrid approaches using split adenylate cyclase or split ubiquitin systems

    • Screen murE against other peptidoglycan synthesis enzymes (MurA-F, MraY, MurG, penicillin-binding proteins)

    • Validate interactions using pull-down assays with purified recombinant proteins

  • Proximity-dependent biotin identification (BioID):

    • Express murE fused to a promiscuous biotin ligase in C. caviae using transformation systems

    • Identify proximal proteins through streptavidin purification and mass spectrometry

    • This approach captures both stable and transient interactions in the native cellular context

• Structural characterization of protein complexes:

  • Cryo-electron microscopy:

    • Reconstitute complexes of murE with interacting partners in vitro

    • Determine structures of these complexes to understand assembly and coordination

    • Focus particularly on potential interactions with MurF, the subsequent enzyme in the pathway

  • Cross-linking mass spectrometry:

    • Use chemical cross-linkers to capture interacting proteins in intact cells

    • Identify cross-linked peptides by mass spectrometry to map interaction interfaces

    • Apply to both recombinant systems and native C. caviae infections

• Functional coordination analysis:

  • Metabolic flux analysis:

    • Use isotopically labeled precursors to track the flow of metabolites through the peptidoglycan synthesis pathway

    • Compare flux in wild-type C. caviae versus strains with modified murE expression

    • Identify rate-limiting steps and metabolic bottlenecks in the pathway

  • In vitro reconstitution:

    • Reconstitute sequential steps of peptidoglycan synthesis with purified enzymes

    • Measure kinetic coupling between murE and other enzymes

    • Test hypotheses about substrate channeling and complex formation

• Spatial organization in cells:

  • Super-resolution microscopy:

    • Express fluorescently tagged versions of murE and other peptidoglycan synthesis enzymes

    • Use techniques like PALM, STORM, or SIM to visualize their spatial distribution in cells

    • Determine if these enzymes colocalize to specific subcellular locations during the developmental cycle

  • Correlative light and electron microscopy (CLEM):

    • Combine fluorescence imaging of tagged enzymes with electron microscopy

    • Correlate enzyme localization with ultrastructural features

    • Particularly useful for visualizing the relationship between peptidoglycan synthesis and membranes

• Systems-level integration:

  • Interactome analysis:

    • Combine multiple protein-protein interaction datasets into a comprehensive network

    • Identify central nodes and potential regulatory hubs

    • Compare the chlamydial peptidoglycan synthesis interactome with those of model bacteria

  • Mathematical modeling:

    • Develop quantitative models of the peptidoglycan synthesis pathway

    • Incorporate experimental data on enzyme kinetics and interactions

    • Predict system behavior under various perturbations and test experimentally

These advanced techniques, particularly when used in combination, can provide unprecedented insights into how murE functions as part of the integrated peptidoglycan synthesis machinery in C. caviae, revealing both conserved features and unique adaptations that have evolved in this specialized intracellular pathogen.