Recombinant Protochlamydia amoebophila UPF0176 protein pc0378 (pc0378)

What is Protochlamydia amoebophila UPF0176 protein pc0378 and what organism does it originate from?

Protochlamydia amoebophila UPF0176 protein pc0378 is a protein belonging to the uncharacterized protein family UPF0176 encoded by the pc0378 gene in Protochlamydia amoebophila. This organism is an obligate intracellular bacterium that exists as an endosymbiont in free-living Acanthamoeba species . Protochlamydia amoebophila belongs to the Parachlamydiaceae family, which is related to but distinct from the pathogenic Chlamydiaceae family that includes human pathogens . Unlike its pathogenic relatives, Protochlamydia amoebophila has evolved as a symbiont of amoebae rather than as a pathogen of vertebrates .

The UPF0176 designation indicates that this protein belongs to an uncharacterized protein family, meaning its precise function remains to be fully elucidated. Based on bioinformatic analysis, it contains a rhodanese domain, which is typically associated with sulfurtransferase activity in other proteins .

How does Protochlamydia amoebophila interact with its host cell, and what role might UPF0176 proteins play in this relationship?

Protochlamydia amoebophila exists in a complex symbiotic relationship with its Acanthamoeba host. As an obligate intracellular bacterium, it has evolved sophisticated mechanisms to survive within the amoeba:

• Metabolic integration: P. amoebophila has lost several metabolic pathways throughout evolution and become dependent on host-derived metabolites, as evidenced by specialized nucleotide transporter (NTT) proteins that mediate exchange of essential molecules with the host cell .

• Developmental cycle: Similar to pathogenic chlamydiae, P. amoebophila exhibits a biphasic developmental cycle involving conversion between infectious elementary bodies (EBs) and replicative reticulate bodies (RBs) .

• Specialized protein secretion: Proteomic analyses have identified numerous proteins involved in host interaction .

While the specific role of UPF0176 protein pc0378 in host interactions remains uncharacterized, its potential functions may include:

• Participation in metabolic regulation

• Involvement in stress response mechanisms

• Potential role in protein-protein interactions within the inclusion membrane

• Possible involvement in sulfur metabolism if the rhodanese domain is functionally active

Interestingly, proteome analysis of P. amoebophila elementary bodies revealed that they contain a surprisingly complete set of proteins for transcription, translation, and energy generation, suggesting a more metabolically independent state compared to pathogenic chlamydiae .

What expression systems are most effective for recombinant production of Protochlamydia amoebophila UPF0176 protein pc0378?

Recombinant Protochlamydia amoebophila UPF0176 protein pc0378 can be expressed in multiple heterologous systems, each with distinct advantages and limitations:

Research has shown that for most research applications, expression in E. coli or yeast systems provides the optimal balance of yield and proper folding . For Protochlamydia amoebophila UPF0176 protein pc0378 specifically, the yeast expression system has been documented to produce protein with >85% purity as assessed by SDS-PAGE .

Methodological considerations for successful expression include:

• Codon optimization for the respective expression host

• Selection of appropriate fusion tags (His, GST, MBP) to enhance solubility and facilitate purification

• Optimization of induction parameters (temperature, inducer concentration, timing)

• Supplementation with relevant cofactors if the rhodanese domain is to be functionally preserved

What purification strategies yield the highest purity of recombinant Protochlamydia amoebophila UPF0176 protein pc0378?

Purification of recombinant Protochlamydia amoebophila UPF0176 protein pc0378 typically employs a multi-step chromatographic approach to achieve high purity (>85% by SDS-PAGE) . The following purification workflow has proven effective:

• Initial Capture:

  • For His-tagged protein: Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA or Co-based resins

  • Buffer composition: Typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10-20 mM imidazole for binding, with elution using 250-300 mM imidazole

• Intermediate Purification:

  • Ion Exchange Chromatography (IEX): Based on the theoretical pI of the protein

  • Size Exclusion Chromatography (SEC): To separate monomeric protein from aggregates and remove residual impurities

• Polishing Step:

  • Hydrophobic Interaction Chromatography (HIC): Particularly useful if the protein contains hydrophobic regions

For optimal results when working with the rhodanese domain-containing protein:

• Include reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) in all buffers to maintain potential catalytic cysteine residues in a reduced state

• Consider including 5-10% glycerol to improve protein stability

• Maintain temperature at 4°C throughout purification to minimize degradation

The recombinant protein is typically available in either lyophilized powder form or as a liquid, with recommended storage at -20°C to -80°C. Lyophilized protein generally maintains stability for up to 12 months, while liquid formulations are stable for approximately 6 months under proper storage conditions.

What experimental approaches should be used to determine the structure of Protochlamydia amoebophila UPF0176 protein pc0378?

The structural characterization of Protochlamydia amoebophila UPF0176 protein pc0378 presents several challenges due to its uncharacterized nature. A comprehensive approach would include:

Currently, no three-dimensional structure has been experimentally determined for this protein. The absence of resolved structural data limits mechanistic insights into its function and interactions.

A methodological approach for structural studies would include:

• Expression optimization to produce milligram quantities of pure, homogeneous protein

• Stability screening to identify buffer conditions that enhance protein stability

• Limited proteolysis to identify stable domains if the full-length protein proves recalcitrant to structural studies

• Tag removal assessment to determine if fusion tags interfere with structural studies

What methodologies can elucidate the potential function of the UPF0176 protein pc0378 in Protochlamydia amoebophila?

Determining the function of UPF0176 protein pc0378 requires a multifaceted approach combining in silico prediction, biochemical characterization, and cellular studies:

• Comparative Genomics and Bioinformatics:

  • Analyze UPF0176 homologs in other chlamydial species to identify conserved functional domains

  • Employ structural prediction algorithms to identify potential active sites

  • Use gene neighborhood analysis to identify functionally related genes

• Biochemical and Enzymatic Characterization:

  • Test for rhodanese (sulfurtransferase) activity using standard thiosulfate:cyanide sulfurtransferase assays

  • Perform substrate screening to identify potential physiological substrates

  • Conduct isothermal titration calorimetry (ITC) to identify binding partners

• Functional Genomics:

  • Generate gene knockouts or knockdowns in model organisms expressing homologous proteins

  • Perform complementation studies to verify function

  • Conduct transcriptomic and proteomic studies under different growth conditions to identify co-regulated genes

• Cellular Localization and Interaction Studies:

  • Develop antibodies against the recombinant protein for immunolocalization studies

  • Use fluorescently tagged versions for live-cell imaging

  • Perform pull-down assays combined with mass spectrometry to identify interaction partners

• Membrane Integration Analysis:

  • Test substrate transport, membrane integration, or enzymatic activity using lipid bilayer or host-interaction models

  • Utilize liposome reconstitution assays if the protein is involved in membrane processes

Unlike well-characterized proteins in P. amoebophila such as nucleotide transporters (NTTs) or inclusion membrane proteins (e.g., IncA, IncQ), the localization and precise function of pc0378 remain unverified, making it an important target for fundamental research.

How can recombinant Protochlamydia amoebophila UPF0176 protein pc0378 be utilized in comparative studies between environmental and pathogenic chlamydiae?

Recombinant Protochlamydia amoebophila UPF0176 protein pc0378 serves as a valuable tool for comparative studies between environmental chlamydiae (like Protochlamydia) and pathogenic chlamydiae (like Chlamydia trachomatis). Such comparative approaches yield insights into chlamydial evolution and host adaptation mechanisms:

• Evolutionary Analysis:

  • Examine the presence/absence and sequence divergence of UPF0176 homologs across the chlamydial phylum

  • Reconstruct the evolutionary history of this protein family to understand its acquisition, retention, or loss

  • Compare selection pressures on UPF0176 proteins in environmental versus pathogenic lineages

• Functional Comparisons:

  • Assess differences in biochemical properties between UPF0176 proteins from different chlamydial species

  • Compare protein interaction networks to identify lineage-specific adaptations

  • Evaluate differences in cellular localization patterns across species

• Host Interaction Studies:

  • Compare the effects of UPF0176 proteins from different chlamydial species on host cell processes

  • Evaluate potential differences in immunogenicity and immune response modulation

  • Investigate host specificity determinants

Experimental data have revealed both "fundamental differences and convergent evolution between pathogenic and symbiotic chlamydiae" , making comparative studies particularly valuable. For example, proteomic analysis of P. amoebophila elementary bodies showed they are "remarkably well equipped with proteins involved in transcription, translation, and energy generation" compared to Chlamydiaceae, suggesting different adaptation strategies.

When designing comparative studies, researchers should consider:

• Using standardized expression and purification protocols across all protein variants

• Employing consistent assay conditions to ensure valid comparisons

• Including appropriate controls, such as proteins with known functions from each organism

What role can recombinant Protochlamydia amoebophila proteins play in understanding bacterial adaptation to intracellular lifestyles?

Recombinant proteins from Protochlamydia amoebophila, including UPF0176 protein pc0378, provide unique opportunities to study bacterial adaptation to intracellular lifestyles. The evolutionary position of P. amoebophila—related to but distinct from pathogenic chlamydiae—makes it an excellent model for understanding transitions between different host-microbe relationships:

• Metabolic Adaptations:

  • Investigate the role of UPF0176 proteins in potential metabolic integration with the host

  • Compare with metabolically impaired pathogenic chlamydiae that are "intimately connected with their host cell's metabolism in a surprisingly complex manner"

  • Explore potential involvement in nutrient acquisition or energy parasitism

• Host Range Determinants:

  • Study how UPF0176 proteins might contribute to host specificity or range

  • Compare protein function in the context of amoeba hosts versus vertebrate hosts

  • Investigate potential roles in host cell manipulation or evasion of host defenses

• Developmental Cycle Regulation:

  • Examine expression patterns throughout the developmental cycle

  • Compare with expression patterns of homologs in pathogenic chlamydiae

  • Investigate potential roles in elementary body to reticulate body transitions

• Experimental Approaches:

  • Heterologous expression in model bacterial systems

  • Protein localization during infection using fluorescently tagged proteins

  • Transcriptomic and proteomic profiling under different growth conditions

Proteomic analysis of P. amoebophila has already provided valuable insights, revealing that it "exploits its additional genetic repertoire (compared with the Chlamydiaceae)" . These findings suggest that studying proteins unique to or divergent in P. amoebophila can reveal alternative strategies for adaptation to intracellular lifestyles.

What challenges exist in developing genetic manipulation systems for studying UPF0176 protein pc0378 function in Protochlamydia amoebophila?

Developing genetic manipulation systems for Protochlamydia amoebophila presents significant challenges that must be addressed to study UPF0176 protein pc0378 function directly in its native context:

• Technical Barriers to Genetic Manipulation:

  • Obligate intracellular lifestyle complicates standard genetic techniques

  • Lack of established transformation protocols specifically for P. amoebophila

  • Complex developmental cycle with alternating forms (elementary bodies and reticulate bodies)

  • Requirement for amoeba host cells for propagation

• Potential Adaptation Strategies from Related Organisms:

  • Transformation methods developed for Chlamydia trachomatis could potentially be adapted, as demonstrated by successful transformation of a plasmid-free C. trachomatis isolate

  • Key modifications required might include:

    • Centrifugation steps to enhance bacterial entry into host cells

    • CaCl₂ treatment protocols similar to those used for C. trachomatis

    • Selection markers suitable for P. amoebophila

• Alternative Approaches:

  • Heterologous expression systems to study protein function

  • Development of cell-free assays for specific activities

  • Computational prediction combined with interactome studies

  • CRISPR-interference approaches if transformation can be achieved

The development of genetic systems for chlamydiae has been challenging historically, with major breakthroughs only occurring relatively recently. For example, transformation of C. trachomatis required specialized protocols including "a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants" . Similar principles might apply to P. amoebophila, though significant optimization would likely be necessary.

How might differential expression of UPF0176 protein pc0378 across different developmental stages of Protochlamydia amoebophila be experimentally determined?

Determining the differential expression of UPF0176 protein pc0378 across the developmental stages of Protochlamydia amoebophila requires specialized techniques that account for the organism's obligate intracellular lifestyle:

• Transcriptomic Approaches:

  • RNA-Seq analysis of synchronized infections at different time points

  • Quantitative RT-PCR targeting pc0378 mRNA

  • In situ hybridization to visualize transcript localization

  • Dual RNA-Seq to simultaneously monitor host and bacterial transcriptional changes

• Proteomic Methods:

  • Quantitative proteomics (e.g., SILAC, TMT labeling) of synchronized infections

  • Western blot analysis using antibodies against recombinant pc0378

  • Comparison with known stage-specific markers

  • Subcellular fractionation to determine protein localization at different stages

• Experimental Design Considerations:

  • Synchronization of infection is critical—typically achieved through low-temperature incubation followed by temperature shift

  • Careful separation of elementary bodies from reticulate bodies for stage-specific analysis

  • Appropriate normalization strategies to account for changes in bacterial numbers

  • Controls for host cell contamination

• Visualization Techniques:

  • Immunofluorescence microscopy using antibodies against recombinant pc0378

  • Combined with markers for different developmental stages

  • Super-resolution microscopy to precisely localize the protein

  • Correlative light and electron microscopy for ultrastructural context

Previous proteome studies of P. amoebophila elementary bodies identified 472 proteins representing 23.2% of all encoded proteins , providing a methodological framework for such analyses. This approach allowed researchers to demonstrate that elementary bodies are "remarkably well equipped with proteins involved in transcription, translation, and energy generation" , distinguishing them from their counterparts in pathogenic chlamydiae.

What course-based undergraduate research experiences (CUREs) could be designed around Protochlamydia amoebophila UPF0176 protein pc0378 to engage students in authentic research?

Designing course-based undergraduate research experiences (CUREs) around Protochlamydia amoebophila UPF0176 protein pc0378 offers an excellent opportunity to engage students in authentic research while addressing open scientific questions:

• Expression and Purification Module:

  • Students clone, express, and purify recombinant pc0378 from different expression systems

  • Compare yields, purity, and activity across systems

  • Learn core techniques: PCR, transformation, protein purification, SDS-PAGE, Western blotting

  • Assessment: Students submit purification reports with yield and purity data

  Expression System	Expected Yield (mg/L culture)	Typical Purity
  E. coli (BL21)	5-10 mg/L	75-85%
  Yeast (Pichia pastoris)	2-5 mg/L	>85%
  Insect cells	0.5-2 mg/L	>90%

• Structural Characterization Module:

  • Students perform bioinformatic analyses to predict secondary structure

  • Conduct CD spectroscopy to determine actual secondary structure content

  • Perform thermal shift assays to identify stabilizing buffer conditions

  • Assessment: Students prepare structure prediction posters comparing computational and experimental results

• Functional Analysis Module:

  • Students test for potential rhodanese activity using various substrates

  • Perform pull-down assays with amoeba lysates to identify binding partners

  • Conduct comparative analyses with homologs from related species

  • Assessment: Students prepare scientific manuscripts describing their findings

• Implementation Framework:

  • Structure the course as a 10-week research experience with 2-3 lab sessions per week

  • Incorporate elements of successful CUREs as described in the literature :

    • Discovery and relevance: Working on uncharacterized protein with unknown function

    • Collaboration: Students work in teams and share data across class

    • Iteration: Multiple experimental replicates and approaches

    • Scientific practices: Data analysis, troubleshooting, scientific communication

This CURE model follows successful implementations where "students at the end of the course identified experimental repetition, data analysis, and collaboration as important elements of thinking like a scientist" . The design also incorporates key elements from high-enrollment CUREs that have shown "positive impact on the development of students' conceptions and practice of scientific thinking" .

Student outcomes from similar CUREs have demonstrated "that this course-embedded research experience has a positive impact on the development of students' conceptions and practice of scientific thinking" , with assessment data showing "consistent student gains in both research skills and content knowledge" .

How could studies of Protochlamydia amoebophila UPF0176 protein pc0378 contribute to understanding bacterial evolution and horizontal gene transfer?

Studying Protochlamydia amoebophila UPF0176 protein pc0378 provides a unique window into bacterial evolution and potential horizontal gene transfer events, given the organism's evolutionary position and endosymbiotic lifestyle:

• Evolutionary Origins Analysis:

  • Comprehensive phylogenetic analysis of UPF0176 family across bacterial phyla

  • Comparison of gene and species trees to identify potential horizontal gene transfer events

  • Analysis of codon usage patterns to detect recent gene acquisitions

  • Assessment of genomic context conservation across species

• Endosymbiont Evolution Insights:

  • Comparison with free-living relatives and other endosymbionts

  • Analysis of selection pressures on the pc0378 gene

  • Investigation of potential gene family expansion or contraction

  • Correlation with host adaptation mechanisms

• Methodological Approaches:

  • Comparative genomics across the chlamydial phylum

  • Ancestral sequence reconstruction to infer evolutionary trajectories

  • Molecular clock analyses to date divergence events

  • Functional complementation in distantly related bacteria

The evolutionary significance of P. amoebophila is highlighted by its position as an early-branching member of the Chlamydiales order, which "evolved to survive within protist hosts, such as Acanthamobae, 700 million years ago" . This ancient association with eukaryotic hosts makes P. amoebophila proteins particularly valuable for studying long-term evolutionary processes in host-associated bacteria.

Furthermore, genomic analysis has revealed that "a genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system" , suggesting that horizontal gene transfer might have played an important role in the evolution of this organism.

What implications might the study of Protochlamydia amoebophila UPF0176 protein pc0378 have for understanding potential virulence factors in pathogenic chlamydiae?

While Protochlamydia amoebophila is not itself pathogenic to humans, studying its UPF0176 protein pc0378 can provide significant insights into potential virulence mechanisms in pathogenic chlamydiae through comparative approaches:

• Evolutionary Retention or Loss Analysis:

  • Determine whether homologs exist in pathogenic chlamydiae

  • Analyze selective pressure differences between environmental and pathogenic lineages

  • Identify potential functional replacement in pathogens if direct homologs are absent

  • Correlate evolutionary patterns with host range shifts

• Functional Comparisons with Pathogenic Homologs:

  • Express and characterize homologs from pathogenic species

  • Compare biochemical properties and substrate specificities

  • Assess differences in cellular localization patterns

  • Evaluate differential effects on host cell processes

• Host Response Differences:

  • Compare host immune responses to UPF0176 proteins from environmental versus pathogenic chlamydiae

  • Assess potential immunomodulatory effects

  • Evaluate interaction with host defense mechanisms

  • Investigate contribution to persistence mechanisms

• Research Applications:

  • Development of diagnostic tools differentiating environmental and pathogenic chlamydiae

  • Potential therapeutic target identification

  • Vaccine development considerations

Proteomic analyses of P. amoebophila have already revealed the presence of "putative virulence-associated proteins" , suggesting that environmental chlamydiae carry proteins that could be related to virulence factors in pathogenic relatives. Further, experimental evidence shows that while "Protochlamydia induces apoptosis in human-immortal HEp-2 cells," it "had no effect on the survival of primary PBMCs" , indicating complex and selective interactions with human cells that could inform our understanding of pathogenic mechanisms in related species.

Studies comparing pathogenic and environmental chlamydiae provide "valuable insights into chlamydial evolution and mechanisms for microbe-host interaction" , making comparative analysis of proteins like UPF0176 pc0378 particularly informative for understanding the molecular basis of chlamydial pathogenicity.