Recombinant Chlamydophila caviae Phosphoglucosamine mutase (glmM)

What is the function of phosphoglucosamine mutase (glmM) in Chlamydophila caviae?

Phosphoglucosamine mutase (GlmM) in C. caviae catalyzes the essential conversion of glucosamine-6-phosphate (GlcN6P) to glucosamine-1-phosphate (GlcN1P), a critical step in the biosynthetic pathway for UDP-N-acetylglucosamine, which is required for bacterial cell wall synthesis . This conversion is particularly important in the context of C. caviae's metabolism, as it plays a role in glycogen utilization. The enzyme operates via a ping-pong mechanism that involves glucosamine-1,6-diphosphate as an intermediate . Unlike some other bacterial species, C. caviae shows specific adaptations in its GlmM function related to its obligate intracellular lifestyle .

How can I express and purify recombinant C. caviae phosphoglucosamine mutase?

Methodology:

• Vector Selection: Use a pET-based expression system with a 6xHis-tag for efficient purification

• Expression Host: BL21(DE3) E. coli strain is recommended due to its reduced protease activity

• Induction Conditions: 0.5-1mM IPTG, 18°C overnight incubation minimizes inclusion body formation

• Purification Protocol:

  • Lyse cells in buffer containing 50mM Tris-HCl pH 8.0, 300mM NaCl, 10mM imidazole

  • Use Ni-NTA affinity chromatography with an imidazole gradient (10-250mM)

  • Further purify using size exclusion chromatography if needed

Based on published protocols, expect protein yields of approximately 5-10mg per liter of culture. The CCA00034 gene (C. caviae ortholog of CT295) is approximately 1.5kb in length and can be amplified from genomic DNA using appropriate primers .

What assays can I use to measure phosphoglucosamine mutase activity?

Multiple complementary approaches can be employed to assess GlmM activity:

A. Direct Activity Measurement:

• HPAEC-PAD Analysis: High-performance anion exchange chromatography with pulsed amperometric detection provides direct quantitative measurement of substrate (GlcN6P) to product (GlcN1P) conversion

• Coupled Enzymatic Assay: Link GlmM activity to NADH oxidation through auxiliary enzymes for spectrophotometric detection

B. Complementation Assay:

• Use PGM1-deficient human fibroblasts to test GlmM functionality

• Transfect cells with GlmM expression construct and measure restoration of ICAM-1 expression as indicator of functional complementation

C. Autophosphorylation Detection:

• Incubate purified enzyme with [γ-32P]ATP

• Analyze phosphorylation state by SDS-PAGE followed by autoradiography

When conducting these assays, it's critical to include glucose-1,6-diphosphate as a cofactor for optimal enzyme activity .

What are the methodological considerations for studying GlmM localization and secretion in C. caviae?

Investigating GlmM localization and secretion requires specialized approaches due to C. caviae's obligate intracellular lifestyle:

A. Immunofluorescence Microscopy Protocol:

• Infect host cells on coverslips with C. caviae

• Fix at various time points post-infection (recommended: 24, 48, 72 hours)

• Permeabilize with 0.1% Triton X-100 or 0.05% saponin (differential permeabilization can distinguish inclusion lumen vs. bacterial cytoplasm localization)

• Use anti-GlmM antibodies and appropriate fluorescent secondary antibodies

• Counterstain with DAPI and anti-chlamydial antibodies

• Analyze using confocal microscopy

B. Type III Secretion Testing:
Evidence indicates that GlmM may be secreted via the Type III Secretion System (T3SS). Utilize heterologous secretion systems like Shigella flexneri to verify T3S signals:

• Fuse N-terminal regions (first 20-50 amino acids) of C. caviae GlmM to reporter proteins

• Express constructs in S. flexneri wild-type and T3SS-deficient (mxiD) strains

• Analyze secretion profiles by western blotting of bacterial pellets and culture supernatants

C. Sequence Analysis:
Compare C. caviae GlmM with orthologs from other Chlamydia species that differ in glycogen accumulation patterns. The following table summarizes key differences:

This correlation between T3S signal presence and glycogen accumulation supports the secretion hypothesis .

How does the mechanism of C. caviae GlmM autophosphorylation differ from other bacterial phosphoglucomutases?

C. caviae GlmM, like other bacterial phosphoglucomutases, requires phosphorylation for activity, but has distinct characteristics:

A. Autophosphorylation Analysis:

• C. caviae GlmM can self-phosphorylate in the presence of ATP

• Experimental protocol: Incubate purified GlmM with [γ-32P]ATP, analyze by SDS-PAGE and autoradiography

• Key controls: Include samples with phosphatase treatment and non-hydrolyzable ATP analogs

B. Site-Directed Mutagenesis Approach:

• Identify conserved serine residue (typically Ser102 or equivalent) likely involved in phosphorylation

• Create S→A mutant using site-directed mutagenesis

• Assess activity of wild-type vs. mutant protein using HPAEC-PAD analysis

• Quantify phosphorylation levels using mass spectrometry or 32P incorporation

C. Comparative Analysis:
While the E. coli phosphoglucomutase has been extensively characterized , C. caviae GlmM shows some unique features:

• Acts in the specialized environment of the inclusion lumen

• May have evolved specific regulatory mechanisms related to its role in glycogen metabolism

• Has acquired a type III secretion signal not present in all orthologs

For comprehensive analysis, researchers should compare phosphorylation kinetics between C. caviae GlmM and other bacterial phosphoglucomutases using Michaelis-Menten parameters.

How do I analyze and interpret contradictory data regarding GlmM function in different Chlamydia species?

Research on chlamydial GlmM has produced seemingly contradictory results across species. A systematic approach to resolving these contradictions includes:

A. Evolutionary Analysis:
Despite genomic similarities, functional differences exist between Chlamydia species. Analysis shows:

B. Statistical Approach to Contradictory Data:
When facing contradictory results in GlmM studies:

C. Resolving Species-Specific Differences:
When conflicting data arise from different Chlamydia species:

• Consider differences in host adaptation (C. caviae naturally infects guinea pigs, while C. trachomatis infects humans)

• Examine genomic context of glmM and associated regulatory elements

• Assess differences in experimental conditions (particularly cell types and growth media)

• Evaluate host-specific factors that may interact with GlmM

The guinea pig model using C. caviae should be carefully evaluated before extrapolating results to human C. trachomatis infections, as there are significant differences despite similarities in pathogenesis .

What are the critical considerations for designing gene knockout or complementation studies of glmM in C. caviae?

Genetic manipulation of obligate intracellular bacteria like C. caviae presents unique challenges:

A. Knockout Strategy Considerations:

• Essentiality Assessment: Since glmM is likely essential, consider conditional knockdown approaches

• CRISPR Interference: Adapt dCas9-based systems for chlamydial expression

• Antisense RNA Approach: Design antisense constructs targeting glmM mRNA

B. Complementation Design:

• Vector Selection: Use chlamydial shuttle vectors with appropriate promoters

• Expression Control: Consider inducible systems (e.g., tetracycline-responsive)

• Tagging Strategy: C-terminal tags are preferred as N-terminal may interfere with T3S signals

• Verification Methods:

  • Western blotting to confirm expression

  • Immunofluorescence to verify localization

  • Functional assays to demonstrate enzyme activity

C. Alternative Approaches:
For species like C. caviae where genetic systems are less developed:

• Use heterologous expression in more genetically tractable chlamydial species

• Employ chemical genetics with specific inhibitors

• Utilize dominant-negative mutants

• Consider plasmid-cured strains (e.g., CC13) that may show altered metabolism

D. Validation Strategy:

• Combine multiple independent approaches

• Include appropriate positive and negative controls

• Use qPCR to quantify effects on bacterial replication

• Assess effects on glycogen accumulation using biochemical and microscopic approaches

How can I investigate the potential role of C. caviae GlmM as a virulence factor?

Recent evidence suggests GlmM may function beyond its metabolic role:

A. Comprehensive Experimental Design:

• Infection Models:

  • Cell culture: HeLa or HEp-2 cells

  • Animal model: Guinea pig genital tract infection

  • Measurement parameters: Bacterial load, inclusion size, inflammatory markers

• Comparative Approach:

  • Compare wild-type C. caviae with strains expressing modified GlmM

  • Assess effects of GlmM overexpression or inhibition

  • Evaluate impact on glycogen accumulation and bacterial fitness

B. Host Response Analysis:

• Transcriptomics: RNA-Seq of infected vs. uninfected cells

• Cytokine Profiling: Measure IL-8, IL-1β, TNF-α in supernatants

• Pathway Analysis: Focus on innate immune signaling pathways

• Statistical Analysis: Use GLMMs with appropriate random effects structure

C. Secretion and Subcellular Localization:
Evidence suggests C. caviae GlmM may be secreted via the Type III secretion system, similar to CT295 in C. trachomatis. Key experimental approaches include:

• Immunofluorescence with differential permeabilization

• Subcellular fractionation of infected cells

• Mass spectrometry analysis of inclusion contents

• Heterologous secretion assays in Shigella

These investigations should distinguish between direct effects of GlmM on virulence and indirect effects through its role in glycogen metabolism.