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

What is Cdu1 and what are its main functions in Chlamydia trachomatis?

Cdu1 is a deubiquitinating enzyme expressed by Chlamydia trachomatis, an obligate intracellular human pathogen. This enzyme possesses both deubiquitinating and deneddylating activities, making it relatively unique among bacterial deubiquitinases. Cdu1 plays crucial roles in the maintenance of chlamydial infection by interfering with host ubiquitination pathways. Its primary functions include stabilizing host anti-apoptotic proteins (particularly Mcl-1) by reducing their ubiquitination and subsequent proteasomal degradation, thus promoting bacterial survival within the host cell. Additionally, Cdu1 contributes to chlamydial protection against interferon-gamma (IFNγ)-mediated host defense mechanisms, which is essential for successful infection in vitro and in vivo .

Where is Cdu1 localized within Chlamydia-infected cells?

Immunofluorescence studies have revealed that Cdu1 predominantly localizes to the chlamydial inclusion membrane, with its active deubiquitinating enzyme domain facing the host cell cytosol. This strategic positioning allows Cdu1 to interact directly with host cell proteins. Using both polyclonal antisera and FLAG-tagged recombinant versions of Cdu1 expressed in Chlamydia, researchers have demonstrated that while some Cdu1 co-localizes with bacterial particles inside the inclusion, the majority is detected at the surface of the inclusion, where it co-localizes with the inclusion membrane protein IncA. This membrane localization is likely facilitated by Cdu1's N-terminal transmembrane domain . Interestingly, contrary to some earlier reports suggesting cytosolic localization, more recent studies have not detected significant amounts of Cdu1 in the host cell cytoplasm, indicating that its primary site of action is at the inclusion membrane interface .

What is the structural composition of Cdu1?

Cdu1 belongs to the C48 family of the CE clan of cysteine proteases. Structural analysis reveals significant similarity to mammalian deubiquitinases, particularly in the catalytically active C-terminal region. The peptidases in this family share common architecture toward the catalytic C-terminus, while the N-terminus shows weak conservation. Notable structural homologs include the Ulp1 protease from Saccharomyces cerevisiae and Sentrin-specific proteases (SENPs) from higher eukaryotes .

A distinctive feature of Cdu1 is an additional α-helix (helix D) not present in other related enzymes. This helix is positioned in proximity to the Ub/Nedd8 C-terminus binding site and is hypothesized to function as a "lid" that may play a critical role in substrate binding and/or recognition. The N-terminally extended α-helix contributes significantly to Cdu1's elevated activity compared to its paralog Cdu2 . The structural similarity between Cdu1, Ulp1, and SENP8 regarding the position of the catalytic triad suggests similar binding mechanisms for ubiquitin and Nedd8 substrates, though the unique structural elements in Cdu1 likely contribute to its specific substrate preferences.

How does Cdu1 differ from its homolog Cdu2?

Despite structural similarities, Cdu1 and Cdu2 exhibit remarkable differences in their enzymatic activities toward poly-ubiquitin chain substrates, as summarized in the following table:

The strikingly elevated activity of Cdu1 compared to Cdu2 can be attributed to the N-terminally extended α-helix present in Cdu1 but absent in Cdu2. This structural difference likely accounts for Cdu1's broader substrate specificity and higher enzymatic efficiency . These functional distinctions suggest that the two deubiquitinases may have evolved to target different host pathways during chlamydial infection.

What are the primary host cell targets of Cdu1?

The anti-apoptotic Bcl-2 family member Mcl-1 has been identified as a major target of Cdu1. Upon infection with C. trachomatis, Mcl-1 protein levels increase significantly starting around 16 hours post-infection (hpi) and remain elevated throughout the chlamydial developmental cycle. This increase coincides with the expression of Cdu1 .

Cdu1 specifically deubiquitinates Mcl-1 at the surface of the inclusion, protecting it from proteasomal degradation. This stabilization is independent of known host factors that regulate Mcl-1 stability, such as the deubiquitinating enzyme USP9X. In cells infected with Cdu1-deficient Chlamydia, Mcl-1 levels are reduced compared to wild-type infection, although they remain higher than in uninfected cells, suggesting that Chlamydia employs multiple mechanisms to regulate Mcl-1 .

While Mcl-1 is a confirmed target, researchers speculate that Cdu1 likely has additional host protein substrates that are deubiquitinated during infection. The enzyme's deneddylating activity also suggests that neddylated proteins may constitute another class of Cdu1 targets, though these remain to be fully characterized .

What experimental approaches have been used to study Cdu1 localization and function?

Researchers have employed multiple complementary techniques to investigate Cdu1, as outlined below:

• Protein Localization Studies:

  • Immunofluorescence microscopy using rabbit polyclonal antisera against recombinant Cdu1

  • Creation of FLAG-tagged Cdu1 expressed in recombinant Chlamydia under control of the original cdu1 promoter

  • Co-localization studies with inclusion membrane markers (e.g., IncA)

• Genetic Manipulation:

  • Development of plasmid constructs (e.g., pAH1) that integrate into the chlamydial genome via homologous recombination

  • Generation of transposon insertion mutants in the Cdu1-encoding gene (Ctr Tn-cdu1)

  • Whole genome sequencing to confirm transposon insertion and screen for additional mutations

• Functional Assays:

  • Ubiquitination analysis of target proteins (e.g., Mcl-1) in infected cells

  • Protein stability assays using cycloheximide chase experiments

  • Apoptosis resistance tests using TNFα/CHX challenge

  • IFNγ sensitivity assays in cell culture models

  • In vivo infection models using trans-cervical infection in mice

• Biochemical Characterization:

  • Structural analysis of the catalytic domain

  • In vitro deubiquitinating activity assays with different ubiquitin chain types (K48, K63)

  • Comparison of enzymatic activities between Cdu1 and Cdu2

These diverse approaches have collectively provided insights into Cdu1's localization, structure, and functions during chlamydial infection.

How does the unique α-helix in Cdu1 contribute to its substrate specificity?

The distinctive α-helix D in Cdu1, which is absent in related enzymes like Ulp1 and SENP8, appears to play a critical role in determining Cdu1's substrate specificity and enzymatic activity. This helix is positioned near the Ub/Nedd8 C-terminus binding site and is hypothesized to function as a specialized "lid" structure .

Structural analyses suggest this helix may assume different orientations depending on substrate binding, potentially creating a dynamic binding interface that contributes to Cdu1's dual specificity for both ubiquitin and Nedd8. When comparing Cdu1 with its paralog Cdu2, the N-terminally extended α-helix present only in Cdu1 has been identified as the key structural element responsible for Cdu1's significantly higher activity toward poly-ubiquitin chains .

The specific mechanism may involve:

• Creating additional binding surfaces for interaction with distal ubiquitin moieties in poly-ubiquitin chains

• Stabilizing enzyme-substrate complexes during catalysis

• Facilitating proper positioning of K48 and K63 linkages within the active site

These structural adaptations likely evolved to optimize Cdu1's activity against specific host targets relevant to chlamydial infection, particularly those involved in apoptosis regulation and immune response pathways.

What is the impact of Cdu1 inactivation on C. trachomatis infection in vitro and in vivo?

Inactivation of Cdu1 through transposon insertion (Ctr Tn-cdu1) has revealed critical roles for this enzyme in chlamydial pathogenesis across multiple experimental systems:

In vitro effects:

• Increased ubiquitination of the chlamydial inclusion

• Reduced stabilization of Mcl-1 at the inclusion membrane

• Decreased Mcl-1 accumulation around the inclusion

• Significantly increased sensitivity to IFNγ treatment, particularly in primary human fimbriae cells that represent the natural infection site

In vivo effects in mouse infection models:

• Substantially reduced bacterial load in the genital tract by day 15 post-infection

• Impaired ability to establish persistent infection

• Decreased pathological impact compared to wild-type infection

Interestingly, the Cdu1-deficient strain did not show increased sensitivity to TNFα/CHX-induced apoptosis compared to wild-type Chlamydia, possibly because other mechanisms (e.g., MEK/ERK signaling) remained active and could partially compensate for reduced Mcl-1 stabilization. This observation highlights the redundancy in Chlamydia's anti-apoptotic strategies .

The table below summarizes the phenotypic differences between wild-type and Cdu1-deficient Chlamydia:

These findings collectively establish Cdu1 as a virulence factor crucial for chlamydial adaptation to the host environment, particularly in countering IFNγ-mediated immune responses.

How does Cdu1-mediated stabilization of Mcl-1 protect Chlamydia from host defense mechanisms?

Cdu1's deubiquitinating activity creates a sophisticated mechanism for Chlamydia to manipulate host cell survival pathways. The process works through multiple coordinated steps:

• Initial Mcl-1 upregulation: Early in infection (before 16 hpi), Chlamydia activates the Raf/MEK/ERK and PI3K/AKT signaling pathways, increasing Mcl-1 expression .

• Sustained Mcl-1 stabilization: As infection progresses, Cdu1 expression begins around 16 hpi, coinciding with the need to maintain elevated Mcl-1 levels. Cdu1 specifically deubiquitinates Mcl-1, preventing its proteasomal degradation .

• Spatial regulation: Cdu1 localizes to the inclusion membrane, creating a microenvironment where Mcl-1 is protected from ubiquitination. This explains the observed accumulation of Mcl-1 around the chlamydial inclusion .

• Anti-apoptotic protection: Stabilized Mcl-1 maintains its anti-apoptotic function, preventing premature host cell death that would terminate the chlamydial developmental cycle. This is particularly important as Chlamydia requires 48-72 hours to complete its intracellular growth phase .

• IFNγ resistance: Beyond apoptosis regulation, Cdu1-stabilized Mcl-1 appears to provide protection against IFNγ-mediated host defense mechanisms. Cdu1-deficient Chlamydia show increased sensitivity to IFNγ treatment, suggesting Mcl-1 may interfere with IFNγ-induced antimicrobial pathways .

This multilayered strategy demonstrates how Chlamydia has evolved to fine-tune host cell processes, creating an optimal environment for its intracellular development while evading host defense mechanisms.

What are the current methods for studying Cdu1 deubiquitinating and deneddylating activities?

Researchers employ various biochemical and cellular approaches to investigate Cdu1's enzymatic activities:

In vitro enzymatic assays:

• Purification of recombinant Cdu1 catalytic domain expressed in E. coli

• Incubation with different ubiquitin substrates:

  • K48-linked poly-ubiquitin chains

  • K63-linked poly-ubiquitin chains

  • Mono-ubiquitin substrates

  • Nedd8-conjugated substrates

• Analysis of reaction products by SDS-PAGE and western blotting

• Quantification of reaction kinetics to determine substrate preferences

Cellular ubiquitination assays:

• Transfection of epitope-tagged ubiquitin (e.g., HA-ubiquitin) into host cells

• Infection with wild-type or Cdu1-deficient Chlamydia

• Immunoprecipitation of target proteins (e.g., Mcl-1)

• Western blot analysis to detect ubiquitinated forms

• Comparison of ubiquitination patterns between different experimental conditions

Inclusion ubiquitination analysis:

• Immunofluorescence staining of infected cells using anti-ubiquitin antibodies

• Co-localization studies with inclusion membrane markers

• Quantitative image analysis to measure ubiquitin accumulation at the inclusion membrane

• Comparison between wild-type and Cdu1-deficient infections

Structure-function relationship studies:

• Creation of Cdu1 variants with mutations in key domains:

  • Catalytic site mutations to abolish enzymatic activity

  • Deletion or modification of the unique α-helix

• Comparison of enzymatic activities between wild-type and mutant proteins

• Analysis of how structural features contribute to substrate specificity

These methodologies have collectively revealed Cdu1's dual enzymatic activities and substrate preferences, providing insights into how this bacterial effector modulates host cell processes during infection.

How do researchers create and validate Cdu1 mutant strains of Chlamydia?

Generation of transposon insertion mutants:

• Utilization of chemical mutagenesis to create a library of C. trachomatis mutants

• Screening for insertions in the Cdu1-encoding gene

• Isolation and purification of the Ctr Tn-cdu1 mutant strain

Plasmid-based genetic complementation:

• Design of non-replicating plasmids (e.g., pAH1) that integrate into the chlamydial genome via homologous recombination

• Construction of plasmids containing FLAG-tagged Cdu1 under control of the native cdu1 promoter

• Transformation of Chlamydia with these constructs to express recombinant Cdu1

Validation procedures:

• Genomic verification:

  • Whole genome sequencing to confirm transposon location and screen for additional mutations

  • PCR amplification of the disrupted gene region

  • Sequence analysis to define the precise nature of the genetic alteration

• Protein expression analysis:

  • Western blotting using anti-Cdu1 antibodies to confirm reduced/altered protein expression

  • Immunofluorescence microscopy to assess changes in Cdu1 localization

  • Detection of truncated Cdu1 proteins in mutant strains

• Functional characterization:

  • Assessment of inclusion ubiquitination in cells infected with mutant strains

  • Measurement of Mcl-1 stabilization and localization

  • Evaluation of sensitivity to IFNγ treatment

  • In vivo infection studies to determine impact on pathogenesis

Control strains are essential in these studies, such as the transposon mutant Tn-IGR, which has a transposon inserted in an intergenic region between two converging genes (CT383/384 or CTL0639/40) and serves as a genetic control for potential fitness effects contributed by the transposon itself .

These rigorous approaches ensure that observed phenotypes can be specifically attributed to Cdu1 deficiency rather than to other genetic alterations or non-specific effects of the genetic manipulation technologies employed.

What is the relationship between Cdu1 activity and IFNγ-mediated host defense mechanisms?

The relationship between Cdu1 and IFNγ-mediated immunity represents a critical aspect of chlamydial pathogenesis with significant implications for persistent infection:

Experimental evidence:

• Cdu1-deficient Chlamydia (Ctr Tn-cdu1) exhibit significantly increased sensitivity to IFNγ treatment in vitro, particularly in primary human fimbriae cells that represent the natural infection site .

• In mouse infection models, Cdu1-deficient strains show substantially reduced bacterial loads by day 15 post-infection, indicating an impaired ability to establish persistent infection .

Proposed mechanisms:

• Indirect protection via Mcl-1: Cdu1-stabilized Mcl-1 may interfere with IFNγ-induced cell death pathways or alter cellular metabolic responses that would otherwise restrict chlamydial growth.

• Direct deubiquitination of immune signaling components: Cdu1 could potentially target and modulate the ubiquitination status of proteins involved in IFNγ signaling pathways, such as:

  • STAT1 (Signal Transducer and Activator of Transcription 1)

  • IRF1 (Interferon Regulatory Factor 1)

  • Components of the JAK-STAT signaling pathway

• Protection against IFNγ-induced antimicrobial mechanisms: IFNγ induces multiple effector mechanisms that restrict chlamydial growth, including:

  • Indoleamine 2,3-dioxygenase (IDO), which depletes tryptophan

  • Guanylate-binding proteins (GBPs)

  • Immunity-related GTPases (IRGs)

  Cdu1 may interfere with the induction or function of these restriction factors, potentially through deubiquitination of key regulators.

The experimental data clearly establish that Cdu1 provides significant protection against IFNγ-mediated host defense, although the precise molecular mechanisms remain to be fully elucidated. This aspect of Cdu1 function appears particularly important for chlamydial persistence in vivo, highlighting its potential as a therapeutic target for treating chronic chlamydial infections.

How can recombinant expression systems be optimized for studying Cdu1?

Optimization of recombinant expression systems is crucial for detailed biochemical and structural studies of Cdu1. Several strategies have been implemented with varying degrees of success:

Bacterial expression systems:

• E. coli-based expression:

  • Expression of the catalytic domain (lacking the transmembrane region) improves solubility

  • Fusion tags (His, GST, MBP) enhance protein stability and facilitate purification

  • Low-temperature induction (16-18°C) minimizes inclusion body formation

  • Co-expression with chaperones may improve folding

Chlamydial expression systems:

• Promoter selection:

  • Using the native cdu1 promoter maintains natural expression timing and levels

  • Inducible promoters allow controlled expression for toxicity studies

• Plasmid design considerations:

  • Non-replicating plasmids (e.g., pAH1) that integrate via homologous recombination

  • Verification of integration and expression using epitope tags (e.g., FLAG tag)

• Expression verification methods:

  • Western blotting to confirm protein expression

  • Immunofluorescence to assess proper localization

  • Functional assays to verify enzymatic activity

Mammalian cell expression:
For certain applications, expressing Cdu1 in mammalian cells may be beneficial:

• Transfection of Cdu1-encoding plasmids with appropriate eukaryotic promoters

• Stable cell lines expressing Cdu1 under inducible promoters

• Careful monitoring for potential cytotoxic effects

Purification considerations:

• Two-step affinity purification for higher purity

• Size-exclusion chromatography to ensure monomeric state

• Activity tests with model substrates to confirm proper folding

When recombinant expression is used to study Cdu1 in the context of infection, it's important to verify that the recombinant protein doesn't alter chlamydial infectivity, as assessed by the efficiency and duration of the developmental cycle . These optimized expression systems enable detailed biochemical characterization and structure-function analyses that advance our understanding of Cdu1's role in chlamydial pathogenesis.

What challenges exist in determining the complete set of Cdu1 substrates?

Identifying the full complement of Cdu1 substrates presents several technical and biological challenges:

Technical limitations:

• Substrate transience: Deubiquitination reactions are often rapid and transient, making it difficult to capture enzyme-substrate interactions.

• Localization constraints: Cdu1's predominant localization at the inclusion membrane creates a spatially restricted environment for interactions, complicating proteome-wide screening approaches .

• Genetic manipulation challenges: The obligate intracellular lifestyle of Chlamydia limits the genetic tools available for substrate identification.

Biological complexity:

• Redundant mechanisms: Chlamydia employs multiple strategies to manipulate host processes, as evidenced by the partial retention of Mcl-1 upregulation in Cdu1-deficient strains . This redundancy makes it difficult to isolate Cdu1-specific effects.

• Temporal regulation: Cdu1 expression begins around 16 hpi and continues through the developmental cycle . Different substrates may be targeted at different time points, requiring time-course analyses.

• Substrate specificity overlap: The dual deubiquitinating and deneddylating activities of Cdu1 expand the potential substrate pool and necessitate distinguishing between these two activities.

Current approaches and limitations:

Despite these challenges, combining multiple approaches with advances in proteomics and targeted genetic manipulation of Chlamydia is gradually expanding our understanding of Cdu1's substrate repertoire beyond the well-characterized Mcl-1.