Recombinant Chlamydia trachomatis serovar B Deubiquitinase and deneddylase Dub1 (cdu1)

What is the primary structure and localization of Cdu1 in C. trachomatis infections?

Cdu1 is a 418 amino acid protein that contains an N-terminal transmembrane domain and a C-terminal catalytic domain. The protein localizes to the chlamydial inclusion membrane with its active deubiquitinating enzyme domain facing the host cell cytosol . Immunofluorescence studies have confirmed that Cdu1 co-localizes with the inclusion membrane protein IncA, where it is likely anchored by its N-terminal transmembrane domain . The protein's amino acid sequence (MLSPTNSISKTAPVPPQDSSKPVLISEEPQNQLLQKVARTALAVLLVVVTLGLILLFYSF SDLQSFPWCCQTRPSTKEQPTISIPVPLPSPPLAVPRPSTPPPPVISRPSTPPAPTPAIS PPSTPSAPKPSTPPPLPPKAPKPVKTQEDLLPFVPEQVFVEMYEDMARRRIIEALVPAWD SDIIFKCLCYFHTLYQGLIPLETFPPATIFNFKQKIISILEDKKAVLRGEPIKGSLPICC SEENYRRHLQGTTLLPVFMWYHPTPKTLSDTMQTMKQLAIKGSVGASHWLLVIVDIQARR LVYFDSLYNYVMSPEDMKKDLQSFAQQLDQVYPAYDSQKFSVKIAAKEVIQKGSGSSCGA WCCQFLHWYLRDPFTDALNDLPVDSVERHENLASFVQACEAAVQDLPELFWPEAKALF) reveals structural elements that contribute to its enzymatic activities .

How does the structure of Cdu1 compare to mammalian deubiquitinases?

The structure of Cdu1's deubiquitinating domain reveals high similarity to mammalian deubiquitinases but contains a unique α-helix positioned close to the substrate-binding pocket . This structural distinction may explain the substrate specificity of Cdu1 compared to host deubiquitinases. When designing experiments to study Cdu1 function, researchers should consider this structural uniqueness, especially when developing inhibitors or when interpreting differential effects on bacterial versus host deubiquitination pathways.

What are the recommended storage and reconstitution conditions for recombinant Cdu1 protein?

For optimal stability, recombinant His-tagged Cdu1 should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles . For reconstitution, the lyophilized protein should be briefly centrifuged before opening and reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Addition of 5-50% glycerol (final concentration) is recommended for long-term storage at -20°C/-80°C, with 50% glycerol being the standard concentration used in most research protocols . Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided to maintain enzymatic activity .

How can researchers generate and validate Cdu1 mutant strains of C. trachomatis?

Researchers can generate Cdu1 mutant strains using transposon mutagenesis approaches. The Ctr Tn-cdu1 mutant strain has been successfully created and validated in previous studies . Validation of the mutant should include:

• Genome sequencing to confirm the transposon insertion site

• Immunofluorescence analysis to assess Cdu1 protein expression and localization

• Functional assays to confirm altered deubiquitinase activity

• Comparison with control transposon mutants (e.g., insertion in intergenic regions) to distinguish specific effects from general transposon-mediated fitness effects

When using these mutant strains, it's critical to include appropriate genetic controls, such as the IGR::Tn bla (Tn-IGR) strain with a transposon inserted in an intergenic region between converging genes, to account for potential fitness effects contributed by the transposon itself .

What methodological approaches are most effective for studying Cdu1's dual enzymatic activities?

To study the dual deubiquitinase and acetyltransferase activities of Cdu1:

• For deubiquitinase activity:

  • In vitro deubiquitination assays using purified recombinant Cdu1 and ubiquitinated substrates

  • Western blot analysis of ubiquitination levels of target proteins (e.g., Mcl-1) in cells infected with wild-type versus Cdu1-deficient Chlamydia

  • Immunofluorescence microscopy to assess inclusion ubiquitination patterns

• For acetyltransferase activity:

  • Comparative proteomics to identify acetylated proteins in wild-type versus Cdu1-deficient infections

  • Site-directed mutagenesis of key residues in the acetyltransferase domain followed by functional assays

  • Analysis of protein stability for potential Cdu1 targets with and without active acetyltransferase function

Research has demonstrated that these two activities have distinct roles: the acetylase activity (not the DUB activity) protects Cdu1 from ubiquitin-mediated degradation . Therefore, experimental designs should incorporate controls that can distinguish between these two functions.

How can researchers effectively assess the impact of Cdu1 on host cell pathways?

To comprehensively assess Cdu1's impact on host cell pathways:

• Apoptosis pathways:

  • Challenge wild-type and Cdu1-deficient infected cells with apoptosis inducers (e.g., TNFα/CHX) and quantify cell death

  • Assess levels and stability of anti-apoptotic proteins like Mcl-1 using western blotting and pulse-chase experiments

• Immune response pathways:

  • Test sensitivity to IFNγ by treating infected primary cells and measuring bacterial replication

  • Use human fimbriae (Fimb) cells that reflect the natural infection site for C. trachomatis

• Vesicular trafficking:

  • Analyze Golgi vesicle recruitment to the inclusion using fluorescence microscopy

  • Compare vesicular trafficking between wild-type and Cdu1-deficient infections

• Autophagy:

  • Assess recruitment of autophagy receptors to the inclusion membrane

  • Test the effect of autophagy inhibitors on the growth of Cdu1-deficient Chlamydia

A combined approach using these methodologies provides more comprehensive insights into Cdu1's multifaceted roles than any single assay.

How does Cdu1 contribute to Chlamydia survival within host cells?

Cdu1 employs multiple mechanisms to promote Chlamydia survival:

• Stabilization of anti-apoptotic factors: Cdu1 deubiquitinates and stabilizes the anti-apoptotic regulator Mcl-1, which accumulates at the inclusion membrane, contributing to apoptosis resistance in infected cells .

• Protection from IFNγ-mediated clearance: Cdu1-deficient Chlamydia show increased sensitivity to IFNγ treatment, with significantly reduced replication in primary cells after IFNγ exposure .

• Preservation of inclusion membrane integrity: Cdu1 prevents ubiquitination of the inclusion membrane, which could otherwise target the inclusion for autophagy-mediated destruction .

• Facilitation of Golgi vesicle recruitment: Cdu1 supports the recruitment of Golgi vesicles to the inclusion, which is critical for bacterial nutrient acquisition and growth .

• Protection of secreted effector proteins: Through its acetylase activity, Cdu1 protects itself and other chlamydial effector proteins (InaC, IpaM, and CTL0480) from ubiquitin-mediated degradation, thereby maintaining their functions in the host cell .

These functions collectively contribute to creating an optimal replicative niche for Chlamydia within host cells.

What are the consequences of Cdu1 inactivation for Chlamydia infection in vivo?

Inactivation of Cdu1 has significant consequences for Chlamydia infection:

• Impaired infection in mouse models: The Cdu1 transposon mutant shows reduced survival in mouse genital infections, highlighting its importance in vivo .

• Growth defects in primary cells: Cdu1-deficient Chlamydia exhibit growth defects in primary cells, particularly when challenged with immune mediators like IFNγ .

• Altered inclusion properties: Without active Cdu1, the chlamydial inclusion shows increased ubiquitination and recruitment of autophagy receptors, though interestingly, blocking autophagy does not rescue the growth defect .

• Disrupted bacterial exit: Cdu1 and the proteins it protects through its acetylase activity are required for optimal egress of Chlamydia from host cells, affecting the transmission cycle .

These findings suggest that Cdu1 is a critical virulence factor for C. trachomatis, with particular importance during infection of immune-competent hosts where IFNγ responses are active.

How do the deubiquitinase and acetyltransferase activities of Cdu1 differentially contribute to Chlamydia infection?

The dual enzymatic activities of Cdu1 play distinct but complementary roles in Chlamydia infection:

• Deubiquitinase activity:

  • Stabilizes host anti-apoptotic factors like Mcl-1 by preventing their ubiquitin-mediated degradation

  • Reduces ubiquitination of the inclusion membrane, potentially shielding it from recognition by autophagy machinery

  • Modulates host immune responses, particularly IFNγ-mediated clearance mechanisms

• Acetyltransferase activity:

  • Protects Cdu1 itself from ubiquitin-mediated degradation

  • Protects other chlamydial effector proteins (InaC, IpaM, and CTL0480) from degradation

  • Plays a critical role in regulating bacterial exit from host cells

Research has demonstrated that while both activities contribute to infection, they regulate different aspects. The acetylase activity appears specifically important for protein stability and bacterial egress, while the deubiquitinase activity primarily affects host response modulation . When designing experiments to study Cdu1 function, it's crucial to use mutants that selectively disrupt one activity while preserving the other to distinguish their specific contributions.

What is the molecular mechanism by which Cdu1's acetylase activity protects proteins from ubiquitin-mediated degradation?

The molecular mechanism involves:

• Competitive modification: Acetylation of lysine residues by Cdu1 may prevent these residues from being ubiquitinated, as both modifications target lysine residues.

• Structural protection: Acetylation may induce conformational changes that mask ubiquitination sites or disrupt the interaction with E3 ubiquitin ligases.

• Regulation of protein-protein interactions: Acetylation could promote associations with stabilizing factors or disrupt interactions with components of the degradation machinery.

This represents a non-canonical mechanism of pathogen-mediated protection of virulence factors after their delivery into host cells . Experimental approaches to investigate this mechanism could include:

• Site-directed mutagenesis of specific lysine residues to identify critical acetylation sites

• Structural studies to determine conformational changes upon acetylation

• Protein interaction studies to identify host factors that differentially bind to acetylated versus non-acetylated forms of the protected proteins

• Mass spectrometry to map acetylation sites and potential competition with ubiquitination

How can researchers differentiate between the effects of Cdu1's deubiquitinase versus acetyltransferase activities in experimental settings?

To differentiate between Cdu1's dual enzymatic activities:

• Generate activity-specific mutants:

  • Create point mutations that selectively inactivate either the deubiquitinase or acetyltransferase domain while preserving the other function

  • Express these mutants in Cdu1-deficient Chlamydia and assess phenotype rescue

• Use selective inhibitors:

  • Apply specific deubiquitinase inhibitors that do not affect acetyltransferase activity

  • Develop or identify selective acetyltransferase inhibitors

• Design substrate-specific assays:

  • Monitor deubiquitination of known substrates (e.g., Mcl-1)

  • Track acetylation status of target proteins (InaC, IpaM, CTL0480)

• Temporal analysis:

  • Study the kinetics of both activities during the Chlamydia developmental cycle

  • Determine if the activities are differentially regulated at specific infection stages

• Complementation experiments:

  • Use heterologous deubiquitinases or acetyltransferases to determine if they can complement specific defects in Cdu1-deficient strains

These approaches can help attribute specific infection phenotypes to each enzymatic activity of Cdu1 .

What characteristics make Cdu1 a promising target for antichlamydial therapy?

Cdu1 possesses several attributes that make it an attractive therapeutic target:

• Essential for in vivo infection: Cdu1-deficient Chlamydia show impaired infection in mouse models and increased sensitivity to immune clearance, indicating that targeting Cdu1 could enhance natural host defense mechanisms .

• Structural uniqueness: While Cdu1 shares structural similarities with mammalian deubiquitinases, it contains unique features such as the distinctive α-helix near its substrate-binding pocket, which could allow for selective targeting .

• Dual enzymatic activities: The presence of both deubiquitinase and acetyltransferase functions provides multiple intervention points that could be targeted independently or simultaneously .

• Surface accessibility: Cdu1 localizes to the inclusion membrane with its catalytic domain facing the host cytosol, making it potentially accessible to inhibitor molecules without requiring penetration of the bacterial cell wall .

• Role in multiple aspects of pathogenesis: Cdu1 affects apoptosis resistance, immune evasion, vesicular trafficking, and bacterial exit, suggesting that its inhibition could disrupt Chlamydia infection through multiple mechanisms .

What considerations should guide the design of Cdu1 inhibitors?

When designing Cdu1 inhibitors, researchers should consider:

• Structural specificity: Target the unique structural features of Cdu1 (such as the distinctive α-helix near the substrate-binding pocket) to achieve selectivity over human deubiquitinases .

• Activity selectivity: Determine whether to target the deubiquitinase activity, acetyltransferase activity, or both, based on their relative contributions to pathogenesis.

• Pharmacokinetic properties: Design molecules that can penetrate host cells and access the cytosolic face of the inclusion membrane where Cdu1's catalytic domain is exposed.

• Resistance potential: Assess the genetic plasticity of the cdu1 gene to anticipate potential resistance mechanisms and design inhibitors with high barriers to resistance.

• Combination potential: Explore synergistic effects with other antibiotics or host-directed therapies, as Cdu1 inhibition particularly sensitizes Chlamydia to IFNγ-mediated clearance .

• Covalent versus non-covalent approaches: Consider the relative merits of reversible competitive inhibitors versus irreversible covalent inhibitors targeting the catalytic cysteine residue in the deubiquitinase domain .

How can researchers assess the efficacy and specificity of potential Cdu1 inhibitors?

A comprehensive assessment of Cdu1 inhibitors should include:

• In vitro enzymatic assays:

  • Deubiquitinase activity assays using purified recombinant Cdu1 and ubiquitin substrates

  • Acetyltransferase activity assays with appropriate substrates

  • Counter-screening against human deubiquitinases and acetyltransferases to assess specificity

• Cellular infection models:

  • Treatment of Chlamydia-infected cell cultures with inhibitors at various infection stages

  • Measurement of inclusion formation, bacterial replication, and infectious progeny production

  • Assessment of inhibitor effects on Cdu1-dependent processes (e.g., Mcl-1 stabilization, inclusion ubiquitination)

• Primary cell models:

  • Testing in human fimbriae (Fimb) cells that reflect the natural infection site

  • Evaluation of efficacy in the presence of immune mediators such as IFNγ

• Animal infection models:

  • Assessment in mouse genital infection models to determine in vivo efficacy

  • Evaluation of different dosing regimens and administration routes

• Toxicity and specificity assessment:

  • Evaluation of effects on uninfected host cells

  • Assessment of potential off-target effects on host deubiquitinases or acetyltransferases

  • Determination of the therapeutic window between antichlamydial effects and host toxicity