The acetylase activity of Cdu1 protects Chlamydia effectors from degradation to regulate bacterial exit from infected cells

Reviewer #1 (Public Review):

The initial goal of this work was to study how the activity of the C. trachomatis effector Cdu1 impacts on the number and nature of ubiquitinated proteins in infected host cells, and how this is related to a previously described function of Cdu1 in promoting Golgi distribution around the Chlamydia vacuole, known as inclusion.

The authors generated a cdu1-null mutant in C. trachomatis and used proteomics to analyse ubiquitinated proteins in cells infected with Cdu1-producing and Cdu1-deficient chlamydiae, by comparison to mock-infected cells. It was found that among the four proteins specifically ubiquitinated after infection with Cdu1-deficient chlamydiae there were three other C. trachomatis effectors (InaC, IpaM and CTL0480). These three proteins are part of a large family of Chlamydia effectors, known as Incs, that insert in the inclusion membrane.

Based on these observations, the authors then focused in understanding how Cdu1 protects InaC, IpaM and CTL0480 from ubiquitination, and what are the consequences of this protection for the protein levels of these Incs and for their functions during infection. It is shown that Cdu1 can bind InaC, IpaM and CTL0480, and protects these Incs and itself from ubiquitination and proteasomal degradation. This protective function of Cdu1 depends on its acetylation, but not on its deubiquitinating activity, and host cells infected by the cdu1 null mutant show defects that phenocopy those of cells infected by inaC, ipaM or ctl0480 null-mutants.

Finally, it was previously shown that CLT0480 controls/inhibits a pathway of chlamydial egress from host cells involving extrusion of the entire inclusion. The authors show that InaC and IpaM also control/promote extrusion of C. trachomatis inclusion and that the cdu1 null mutant also shows a defect in this process. This leads to the conclusion stated in the title that Cdu1 regulates chlamydial exit from host cells by protecting specific C. trachomatis effectors from degradation.

This is an excellent and impressive work, both from technical and conceptual perspectives, which accomplishes the goal of providing mechanistic insights on the mode of action of Cdu1. Overall, the data provides solid evidence for the proposed model by which the acetylation activity of Cdu1 protects itself and three Incs (InaC, IpaM and CTL0480) from degradation.

I agree that (all together) the data provides a solid support for the idea that the multiple phenotypes displayed by cells infected with the cdu1 null mutant are related to the decreased levels of InaC, IpaM and CTL0480. However, to some extent, these Incs can still be detected in cells infected with the cdu1 null mutant and it cannot be formally excluded that Cdu1 directly promotes assembly of F-actin and Golgi repositioning around the inclusion, MYPT1 recruitment to the inclusion, and extrusion of the inclusion.

Still, I think the major significance of this work comes from the combined use of proteomics and chlamydial genetics to disclose a unique a mechanism by which one effector controls the levels of other effectors. This further emphasizes that for a single bacterium injecting dozens of effectors into host cells, the function of one bacterial effector can control, and be controlled by other effectors.

Reviewer #2 (Public Review):

The manuscript describes the detailed characterization of the C. trachomatis protein Cdu1. Previous work that laid the foundation identified two enzymatic activities associated with Cdu1 - deubiquitinase and transacetylase. This work advances current knowledge by identifying Cdu1 targets for stabilization, and establishing the relationship between the two activities of Cdu1. Furthermore, the authors determined that Cdu1 is subject to autostabilization. In addition to the novelty of the findings, the strength of this report is its scientific rigor, with several experimental evidence independently confirmed using a variety of approaches, including the creation of mutants that decoupled deubiquitination from transacetylase activity. Another strength is the direct demonstration of transacetylation of the targets, which increased the relevance of the reported colocalization and interaction of Cdu1 with the targets.

The authors also made a convincing case for the basis of Cdu1 modification of each of the effector targets by linking loss of acetylation with decreased stability. An unexpected result, at least to this reviewer is the requirement for the three effectors in chlamydial egress by extrusion of the inclusion. Cdu1 regulating all three effectors underscores the importance of the timing and efficiency of inclusion extrusion. Additional insights into how the three effectors interact functionally could be obtained by specifically monitoring the timing of extrusion. Data for CTL0480 points to a negative regulator of extrusion, which could be at the level of timing, in addition to efficiency.

Overall, the work is rigorous, and makes important contribution to our understanding of the significance of Cdu1 function in in vitro infection.

Reviewer #3 (Public Review):

In this article by Bastidas et al. the authors examine the functions of the Chlamydia deubiquitinating enzyme 1 (Cdu1) during infections of human cells. First, a mutant lacking Cdu1 but not Cdu2 was constructed using targetron and quantitative proteomics was used to identify differences in ubiquitinated proteins (both host and bacterial) during infection. While they found minimal changes in host protein ubiquitination, they identified three Chlamydia effector proteins, IpaM, InaC and CTL0480 were all ubiquitinated in the absence of Cdu1. Microscopy and immunoprecipitations found Cdu1 directly interacts with these Chlamydia effectors and confirmed that Cdu1 mediates the stabilization of these effectors at the inclusion membrane during late infection time points. Surprisingly rather than deubiquitination driving this stabilization, the acetylation function of Cdu1 was required, and acetylation on lysine residues prevented degradative ubiquitination of Cdu1, IpaM, InaC and CTL0480. In line with this observation the authors show that loss of Cdu1 phenocopies the loss of single effector mutants of InaC, IpaM and CTL0480, including golgi stack formation and the recruitment of MYPT1 to the inclusion. The aggregation of changes to the Chlamydia inclusion does not alter growth but controls extrusion of chlamydia from cells with reduced extrusion in Cdu1 mutant Chlamydia infections. The strengths of the manuscript are the range of assays used to convincingly examine the biochemical and cellular biology underlying Cdu1 functions. The finding that acetylation of lysine residues is a mechanisms for bacterial effectors to block degradative ubiqutination is impactful and will open new investigations into this mechanism for many intracellular pathogens. There are a few weaknesses that temper enthusiasm for the manuscript in its current form. These include caveats related to the timing of proteomics, the lack of an effect of Cdu1 directly on bacterial growth, and discussion of previous studies. Altogether this is an important series of findings that help to understand the mechanisms underpinning Chlamydia pathogenesis using orthologous methods with a few caveats that lower the overall impact.