Multi-tiered actions of Legionella effectors to modulate host Rab10 dynamics

Reviewer #1 (Public Review):

In this manuscript, Kubori and colleagues characterized the manipulation of the host cell GTPase Rab10 by several Legionella effector proteins, specifically members of the SidE and SidC family. They show that Rab10 undergoes both conventional ubiquitination and noncanonical phosphoribose-ubiquitination, and that this posttranslational modification contributes to the retention of Rab10 around Legionella vacuoles.

Strengths
Legionella is an emerging pathogen of increasing importance, and dissecting its virulence mechanisms allows us to better prevent and treat infections with this organism. How Legionella and related pathogens exploit the function of host cell vesicle transport GTPases of the Rab family is a topic of great interest to the microbial pathogenesis field. This manuscript investigates the molecular processes underlying Rab10 GTPase manipulation by several Legionella effector proteins, most notably members of the SidE and SidC families. The finding that MavC conjugates ubiquitin to SdcB to regulate its function is novel, and sheds further light into the complex network of ubiquitin-related effectors from Lp. The manuscript is well written, and the experiments were performed carefully and examined meticulously.

Weaknesses
Unfortunately, in its current form this manuscript offers only little additional insight into the role of effector-mediated ubiquitination during Lp infection beyond what has already been published. The enzymatic activities of the SidC and SidE family members were already known prior to this study, as was the importance of Rab10 for optimal Lp virulence. Likewise, it had previously been shown that SidE and SidC family members ubiquitinate various host Rab GTPases, like Rab33 and Rab1. The main contribution of this study is to show that Rab10 is also a substrate of the SidE and SidC family of effectors. What remains unclear is if Rab10 is indeed the main biological target of SdcB (not just 'a' target), and how exactly Rab10 modification with ubiquitin benefits Lp infection.

Reviewer #2 (Public Review):

This manuscript explores the interplay between Legionella Dot/Icm effectors that modulate ubiquitination of the host GTPase Rab10. Rab10 undergoes phosphoribosyl-ubiquitination (PR-Ub) by the SidE family of effectors which is required for its recruitment to the Legionella containing vacuole (LCV). Through a series of elegant experiments using effector gene knockouts, co-transfection studies and careful biochemistry, Kubori et al further demonstrate that:

1. The SidC family member SdcB contributes to the polyubiquitination (poly-Ub) of Rab10 and its retention at the LCV membrane.
2. The transglutaminase effector, MavC acts as an inhibitor of SdcB by crosslinking ubiquitin at Gln41 to lysine residues in SdcB.

Some further comments and questions are provided below.

1. From the data in Figure 1, it appears that the PR-Ub of Rab10 precedes and in fact is a prerequisite for poly-Ub of Rab10. The authors imply this but there's no explicit statement but isn't this the case?
2. The complex interplay of Legionella effectors and their meta-effectors targeting a single host protein (as shown previously for Rab1) suggests the timing and duration of Rab10 activity on the LCV is tightly regulated. How does the association of Rab10 with the LCV early during infection and then its loss from the LCV at later time points impact LCV biogenesis or stability? This could be clearer in the manuscript and the summary figure does not illustrate this aspect.
3. How do the activities of the SidE and SidC effectors influence the amount of active Rab10 on the LCV (not just its localisation and ubiquitination)
4. What is the fate of PR-Ub and then poly-Ub Rab10? How does poly-Ub of Rab10 result in its persistence at the LCV membrane rather than its degradation by the proteosome?
5. Mutation of Lys518, the amino acid in SdcB identified by mass spec as modified by MavC, did not abrogate SdcB Ub-crosslinking, which leaves open the question of how MavC does inhibit SdcB. Is there any evidence of MavC mediated modification to the active site of SdcB?
6. I found it difficult to understand the role of the ubiquitin glycine residues and the transglutaminase activity of MavC on the inhibition of SdcB function. Is structural modelling using Alphafold for example helpful to explain this?
7. Are the lys mutants of SdbB still active in poly-Ub of Rab10?