Canonical and phosphoribosyl ubiquitination coordinate to stabilize a proteinaceous structure surrounding the Legionella-containing vacuole

Protein ubiquitination is one of the most versatile signaling mechanisms in eukaryotic cells. Ubiquitin, a small, highly conserved globular protein, can be covalently linked to a target protein, serving as a post-translational modification. Most frequently, this linkage is an isopeptide bond between the carboxy terminus of ubiquitin and a lysine side chain or amino terminal amine group on the target protein. Ubiquitin itself can be ubiquitinated, resulting in polyubiquitin chains, and the identity of the residues used to form these intermolecular linkages dictates downstream signaling outcomes (Komander and Rape, 2012). The modularity of the ubiquitin conjugation system, in which attachment site, chain length, and chain linkage pattern affect signaling and target protein activity, confers the versatility required to regulate a broad range of cellular processes. Examples include cell division and differentiation, protein homeostasis, autophagy, immune signaling, and membrane trafficking (Yau and Rape, 2016; Jacomin and Dikic, 2024). Given the critical role and flexibility of the ‘ubiquitin code’, it is unsurprising that many pathogens have evolved to subvert this signaling system as part of their infection strategy (Ribet and Cossart, 2018).

Legionella pneumophila (L.p.) is an intracellular bacterial pathogen of diverse eukaryotic cells. In humans, L.p. infection of alveolar macrophages can cause a severe pneumonia known as Legionnaires’ disease; however, humans are a dead-end host for the bacterium. The natural hosts for L.p. are free-living protozoa across multiple phyla, demonstrating the bacterium’s remarkable ability to establish a protected niche within evolutionarily divergent eukaryotic cells (Romanov and O’Connor, 2024). Upon phagocytic uptake, L.p. uses a type IV secretion system (T4SS) to translocate over 300 bacterial proteins, known as effectors, across the phagosome membrane and into the host cell. These effectors regulate host processes on a global scale, such as protein translation (Moss et al., 2019; Subramanian et al., 2023) and gene expression (Rolando et al., 2013), while simultaneously remodeling the phagosomal membrane surrounding the bacterium to create a specialized compartment known as the Legionella-containing vacuole (LCV) (Dong et al., 2016; Sherwood and Roy, 2016). Isogenic strains lacking a functional T4SS are efficiently trafficked to the lysosome after internalization, underscoring the essential role of bacterial effectors in L.p. pathogenesis (Berger and Isberg, 1993; Roy et al., 1998).

Mounting evidence suggests that hijacking the host’s ubiquitin signaling network is an important element of L.p. pathogenesis (Tomaskovic et al., 2022). Ubiquitinated proteins begin to accumulate around the LCV during early infection (Dorer et al., 2006; Ivanov and Roy, 2009), and over 20 bacterial effectors have been shown to act on the ubiquitin system in various ways (Luo et al., 2021). In eukaryotic cells, ubiquitination is a highly regulated process involving a three-enzyme cascade: an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase, which, in coordination with the E2, facilitates the covalent attachment of ubiquitin to a target protein. This process is reversible, as cells express deubiquitinating enzymes (DUBs) that hydrolyze the isopeptide bonds linking ubiquitin to substrates (Komander and Rape, 2012). L.p. secretes multiple effectors that act as E3 enzymes and DUBs (Luo et al., 2021). Furthermore, recent studies have shown that four homologous L.p. effectors known as the SidE family can bypass the canonical eukaryotic E1-E2-E3 ubiquitination cascade. These effectors instead catalyze the formation of a phosphoribosyl (PR) linkage between an arginine residue on ubiquitin and a serine residue on the target protein, a process termed PR-ubiquitination (Qiu et al., 2016; Bhogaraju et al., 2016). The activity of the SidE family of effectors has been implicated in the manipulation of host endoplasmic reticulum (ER) proteins (Kotewicz et al., 2017; Shin et al., 2020), alterations in host metabolism (De Leon et al., 2017), and evasion of autophagy (Omotade and Roy, 2020; Kotewicz et al., 2024; Wan et al., 2024; Mukherjee et al., 2025) during infection.

Observations from multiple studies suggest that the SidE family and the SidC/SdcA family, effectors that catalyze canonical ubiquitination (Hsu et al., 2014), regulate closely related processes during infection. Both effector families are essential for recruitment of ER membrane to the LCV shortly after formation, a hallmark of LCV maturation (Kotewicz et al., 2017; Hsu et al., 2014; Horenkamp et al., 2014). Furthermore, both families ubiquitinate multiple small GTPases in the Rab family, as demonstrated by our lab (Steinbach et al., 2024; Jeng et al., 2019) and others (Qiu et al., 2016; Ma et al., 2024; Kubori et al., 2024). A recent study provides evidence that these small GTPases are modified with mixed canonical and PR-linked ubiquitin chains during infection through the combined activities of the SidC and SidE effector families, and that the SidE family of effectors may crosslink these chains (Wan et al., 2024). This hypothesis aligns with the intriguing proposal that ER membrane surrounding the LCV during infection is stabilized by SidE family activity, forming a protective barrier around the vacuole (Kim and Isberg, 2023).

In this work, we seek to place the biochemical activities of the SidC and SidE effector families within the context of cell biological events at the LCV membrane. Using Rab5 as a model substrate—previously shown by our lab to require both the SidC and SidE effectors for maximal ubiquitination and LCV recruitment (Steinbach et al., 2024)—we demonstrate that the morphology of the Rab5-positive regions surrounding the LCV depends on SidE activity. Further, we observe that Rab5 forms a detergent-resistant structure around wild-type (WT) LCVs during early infection. We identify a similar pattern for ubiquitin itself, where SidE activity is required to establish the distinctive, cloud-like morphology and detergent resistance of ubiquitin associated with the LCV. We find that this hyperstable structure is disassembled as infection progresses, indicating that the ubiquitin shell may provide protection shortly after internalization. We propose a model in which the SidC family initiates a wave of canonical ubiquitination around the LCV early in infection, forming a tight, detergent-sensitive ubiquitin coat. This coat is subsequently modified by the SidE family, likely in tandem with further ubiquitin chain synthesis by SidC/SdcA (Wan et al., 2024), creating an expansive, detergent-resistant structure. Live imaging of ubiquitin during infection supports this hypothesis, as ubiquitin surrounding WT LCVs expands rapidly outward from the bacterial cell, while in the SidE knockout strain, ubiquitin remains tightly LCV-associated. Further, timelapse imaging of both Rab5 and ubiquitin during infection demonstrates that LCV recruitment of these two host proteins is temporally coupled by the activity of the SidE family of effectors. Our findings highlight the complex interplay between the SidE and SidC effector families and provide a foundation for future studies on the physical and cell biological properties of the enigmatic proteinaceous structure surrounding the LCV.

To survive within the host cell, intravacuolar bacterial pathogens must efficiently remodel the organelle within which they reside to support replication, evade detection, and withstand host attacks. The mechanisms contributing to each of these three missions are often inseparable, and as such gaining understanding in one realm informs all three. In this study, we explore the unique properties and formation of a hyperstable proteinaceous structure assembled rapidly around the LCV during early L.p. infection. Multiple host proteins, including the small GTPase Rab5A and polyubiquitin conjugates, are incorporated into this structure, adopting a distinctive ‘cloud’ morphology around the LCV that resists harsh detergent washout. We find that the SidE family of bacterial effectors, which catalyzes non-canonical PR-ubiquitination, is required for the formation of this structure. This structure is disassembled as infection progresses on a timeline that correlates with vacuolar expansion, demonstrating that L.p. dynamically reshapes the properties of the proteins associated with the LCV as the bacterium’s needs shift during its intracellular life cycle.

What advantage might an expansive, stable ‘cloud’ of protein around the LCV confer to L.p.? One model pertains to the vacuole guard hypothesis, the idea that intravacuolar pathogens secrete effectors that maintain the integrity of the vacuole by preventing membrane disruption (Anand et al., 2020). L.p. effector SdhA (Choi et al., 2021), and, more recently, the SidE family of effectors (Kim and Isberg, 2023), have been identified as vacuole guards. In the latter study, it was proposed that PR-ubiquitination of Rtn4, an ER structural protein, stabilizes ER tubules around the LCV to exclude unwanted host factors. Our experiments on Rab5 and ubiquitin suggest that many host proteins beyond Rtn4 may be incorporated into such a barrier. Further, while to our knowledge ‘cloud’ morphology of LCV-associated proteins has heretofore not been quantified, published images of a variety of host cell types reveal expansive morphology of LCV-associated Rab6A and Rab33B (Kawabata et al., 2021), Rtn4 (Kotewicz et al., 2017), and ubiquitin (Omotade and Roy, 2020). In addition to building a physical barrier, the SidE family protects the LCV by camouflaging ubiquitin conjugates. Phosphoribosylation of polyubiquitin chains prevents binding of the host autophagy adaptor p62 in vitro (Kotewicz et al., 2024; Wan et al., 2024), and the SidE family knockout LCV is targeted by p62 (Omotade and Roy, 2020; Kotewicz et al., 2024; Wan et al., 2024). The SidE family of effectors appears to be a versatile player in vacuolar protection during infection, and the extent to which barrier formation and camouflage might be coupled during infection remains to be determined.

How might PR-ubiquitination induce detergent resistance and the expansive morphology we observe for host proteins around the LCV? The unique chemistry of PR-ubiquitination, in coordination with canonical ubiquitination, opens the possibility of complex intermolecular linkages between modified target proteins. A recent publication provides evidence that host proteins can be crosslinked during infection by forming PR-ubiquitin bridges between ubiquitinated substrates (Wan et al., 2024). These crosslinks might result in a macromolecular meshwork around the LCV that could be maintained in the absence of an anchoring membrane. The expansive morphology of such a structure could be a result of disruption of molecular packing of polyubiquitin chains, and/or crosslinking to nearby cellular structures. The broader implications of this hyperstable structure are intriguing. Whether incorporation of host proteins is selective or indiscriminate remains unclear. Integration into such a crosslinked network likely impacts the activity of host proteins such as GTPases that play key roles in LCV maturation (Jeng et al., 2019; Nagai et al., 2002; Neunuebel and Machner, 2012). Additionally, understanding how these structures influence membrane dynamics, derived from both ER tubules and the LCV, remains an exciting avenue for future research.

How is the ubiquitin ‘cloud’ around the LCV constructed? We provide evidence that this structure may be built in two phases: an initial wave of canonical ubiquitination, followed by co-modification by both the SidE and SidC/SdcA effector families. First, we demonstrate that the ΔsidE/sdeABC LCV is coated in compact, detergent-sensitive ubiquitin shortly after infection, in contrast to the expansive, detergent resistant ubiquitin surrounding the WT LCV. Using live cell imaging, we observe that ubiquitin tightly surrounds the WT LCV shortly after infection, then expands outward as infection progresses. In contrast, ubiquitin surrounding the SidE family knockout strain remains tightly LCV-associated. Further, both the SidE and SidC/SdcA families are required for maximal accumulation of detergent insoluble polyubiquitin conjugates during infection. Elegant biochemical experiments performed by Wan et al., 2024 demonstrated that the SidE and SidC/SdcA effector families co-modify Rab33B, resulting in mixed canonical and PR-linked ubiquitin chains. Our data is consistent with a model in which canonically linked ubiquitin accumulates around the LCV in a SidC/SdcA dependent manner shortly after infection, at which point the ubiquitin coat is detergent-sensitive and compact. Subsequently, PR modification and crosslinking by the SidE family of effectors, as well as further canonical ubiquitination by SidC/SdcA as suggested by Wan et al. generates the expansive, detergent resistant ‘cloud’ of polyubiquitin conjugates around the LCV. While we suspect that, in the presence of both the SidE and SidC/SdcA effector families, substrate proteins are rapidly co-modified upon LCV association, the accumulation of ubiquitin around LCVs harboring L.p.ΔsidE/sdeABC, but not L.p. ΔsidC/sdcA (Horenkamp et al., 2014; Kubori et al., 2024), points to the essentiality of canonical ubiquitination driven by SidC/SdcA in initiating construction of the hyperstable polyubiquitin structure during infection. We emphasize that we do not argue that canonical ubiquitination must proceed PR-ubiquitination biochemically, but rather that, during infection, the activity of canonical ubiquitin ligase effectors is required for initial ubiquitin accumulation around the LCV, providing a properly localized substrate for SidE family modification. The role of additional regulatory mechanisms in constructing the ubiquitin cloud, such as the SidE family’s N-terminal DUB domain implicated in ubiquitin (Sheedlo et al., 2015) and ER tubule (Kotewicz et al., 2024) dynamics at the LCV surface, remains an open question.

The complex interplay between ubiquitination and LCV membrane association is illustrated by the phenotypic differences in Rab5 recruitment relative to ubiquitin accumulation around the LCV for L.p. WT and ΔsidE/sdeABC. While LCV recruitment of Rab5 and ubiquitin appears tightly coordinated in the L.p. WT case, Rab5 association with the ΔsidE/sdeABC LCV, when captured in our imaging, is transient and precedes ubiquitin accumulation. Previously, our lab demonstrated that Rab5 is mono-ubiquitinated during infection with the ΔsidE/sdeABC strain without the accumulation of exceptionally high molecular weight species (Steinbach et al., 2024). While Rab5 ubiquitination is largely dependent on SidC/SdcA during infection (Steinbach et al., 2024; Ma et al., 2024), a recent report provides evidence that another effector, lpg2525, can also ubiquitinate Rab5, and that in the absence of this effector Rab5 accumulates at the LCV surface at high levels (Tanaka et al., 2025). Intriguingly, we observe the opposite phenotype with the ΔsidC/sdcA strain, in which loss of Rab5 ubiquitination correlates with loss of LCV association (Steinbach et al., 2024). This apparent conflict may be explained by tight coordination between SidC/SdcA and the SidE family, in which co-modification by both effector families locks Rab5 into place at the LCV surface, as previously discussed. Further, we have not eliminated the possibility that SidC/SdcA recruits Rab5 to the LCV through means other than direct ubiquitination. Taken together, we hypothesize that, in the ΔsidE/sdeABC infection case, Rab5 is recruited to the LCV via the activity of either host or bacterial proteins, and then canonically ubiquitinated, which, in the absence of the SidE family, drives dissociation from the membrane. Why Rab5 recruitment consistently precedes ubiquitin accumulation at the ΔsidE/sdeABC LCV remains an open question, and further investigation will likely yield insights into the intricate web of bacterial effectors regulating the relationship between small GTPase LCV association and ubiquitination.

While Rab5 recruitment to endocytic vesicles within eukaryotic cells usually confers ‘early endosome’-like properties, data from our lab and others indicate that L.p. effectors divert the vacuole from ‘normal’ progression through the endolysosomal system. Previously, we found that the Rab5 binding partner EEA1, required for homotypic fusion, is excluded from the WT LCV membrane in the same time window as peak Rab5 recruitment (Steinbach et al., 2024), and it has been demonstrated that the WT LCV resists fusion with early endosomes (Joshi et al., 2001; Choi et al., 2021). Further, it is well established that L.p. effectors remodel the phosphoinositide composition of the vacuole membrane from endosome-like (PI(3)P+or PI(3,5)P₂+) to predominately PI(4)P+during early infection (Dong et al., 2016; Weber et al., 2014; Li et al., 2021). Interestingly, the Rab5 GEF Rabex5 binds ubiquitin; this is thought to be one mechanism by which Rab5 is targeted to membranes (Mattera and Bonifacino, 2008). It is possible that Rabex5 binding to ubiquitin contributes to Rab5 LCV recruitment, although if this were the case, one might predict Rabex5 overexpression to increase Rab5 recruitment to the WT LCV, which we do not observe, and for Rab5 association with the LCV to trail behind ubiquitin recruitment. Instead, we found that ubiquitin and Rab5 were recruited simultaneously to the WT LCV, and that Rab5 recruitment preceded ubiquitin association with the ΔsidE/sdeABC LCV. Finally, it has been reported that late endosomal GTPase Rab7 is recruited to the LCV (Hoffmann, 2014), and that this recruitment may be driven by SUMOylation (Li et al., 2024). While there is evidence that the WT LCV has characteristics of late endosomes/lysosomes in the end stages of infection (Sturgill-Koszycki and Swanson, 2000), the WT LCV resists fusion with lysosomes throughout early infection (Roy et al., 1998; Clemens et al., 2000). Our data is not consistent with a model in which the LCV undergoes classical endosome maturation from Rab5+ to Rab7+driven by host regulators; while it is fascinating that both host GTPases are recruited to the WT LCV, this association seems to be regulated by post-translational modification of these proteins by bacterial effectors rather than the activity of host regulators.

Ubiquitin tagging of invading bacterial cells or vacuoles often corresponds to targeting by host autophagy machinery and ultimate trafficking to the lysosome, and accordingly many pathogens evade ubiquitination as part of their virulence programs (Fujita and Yoshimori, 2011). It appears that L.p. has taken an entirely different approach and integrated ubiquitin recruitment into the maturation of its vacuole. As proposed here and elsewhere (Kotewicz et al., 2024; Wan et al., 2024; Kim and Isberg, 2023), ubiquitin may serve as a glue that holds together a physical barrier around the LCV to protect the membrane from unwanted vesicular fusion or targeting by autophagy. Ubiquitin localization may also function as a molecular timer, coordinating stages of LCV maturation; a recent paper links late-stage LCV expansion to a bacterial effector phospholipase that is activated by ubiquitin (Li et al., 2022). Our finding that ubiquitin hyperstability at the LCV is lost as infection progresses further suggests that ubiquitin dynamics are tightly coordinated with stages of the L.p. life cycle within the host cell. Considering the astounding evolutionary breadth of eukaryotic cells that support L.p. replication (Romanov and O’Connor, 2024), embracing rather than escaping ubiquitin tagging of the LCV may allow L.p. to rapidly protect and remodel its vacuole in a wide variety of cell types, as ubiquitin is extremely highly conserved across eukaryotes (Zuin et al., 2014). In conclusion, our study highlights a novel role for ubiquitin in L.p. pathogenesis. By leveraging both canonical and noncanonical ubiquitination, L.p. constructs a hyperstable structure surrounding the LCV that may protect against host defenses. These findings deepen our understanding of ubiquitin’s versatility and suggest that further exploration of L.p.’s co-opting of host signaling pathways will reveal fundamental insights into host-pathogen interactions.

CellProfiler pipelines, ImageJ macros, and custom code used to process CellProfiler output, generate plots, and run statistical analyses are available on GitHub (https://github.com/adysteinbach/legionella-ubiquitin-code copy archived at Steinbach, 2026). Source data used to generate all figures has been included along with the manuscript.

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Author details

1. Adriana Steinbach

   1. G.W. Hooper Foundation, University of California, San Francisco, San Francisco, United States
   2. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, United States

   Contribution

   Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5073-7180

2. Chetan Mokkapati

   1. G.W. Hooper Foundation, University of California, San Francisco, San Francisco, United States
   2. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Puspangana Singh

   1. G.W. Hooper Foundation, University of California, San Francisco, San Francisco, United States
   2. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4395-4221

4. Shaeri Mukherjee

   1. G.W. Hooper Foundation, University of California, San Francisco, San Francisco, United States
   2. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, United States
   3. Chan Zuckerberg Biohub, San Francisco, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – review and editing

   For correspondence

   Shaeri.Mukherjee@ucsf.edu

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0003-3820-0174

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