Phosphoribosyl modification of poly-ubiquitin chains at the Legionella-containing vacuole prohibiting autophagy adaptor recognition

Ubiquitination is a crucial posttranslational modification in eukaryotes that plays a significant role in the infection of intracellular microbial pathogens, such as Legionella pneumophila, the bacterium responsible for Legionnaires’ disease. While the Legionella-containing vacuole (LCV) is coated with ubiquitin (Ub), it avoids recognition by autophagy adaptors. In this study, we report that the Sdc and Sde families of effectors work together to build ubiquitinated species around the LCV. The Sdc effectors catalyze canonical polyubiquitination directly on host targets or on the phosphoribosyl-Ub (PR-Ub) conjugated to host targets by Sde. Remarkably, the Ub moieties within the poly-Ub chains are either modified with a phosphoribosyl group by Sde and other PDE domain-containing effectors or covalently attached to other host substrates via Sde-mediated PR-ubiquitination. Furthermore, these modifications prevent the recognition by Ub adaptors, such as p62, and therefore exclude host autophagy adaptors from the LCV. Our findings shed light on the nature of the poly-ubiquitinated species present at the surface of the LCV and provide a molecular mechanism for the avoidance of autophagy adaptors by the Ub-decorated LCV.

Introduction substrates (phosphoribosyl-or PR-ubiquitination) or hydrolyzed to phosphoribosyl-Ub (PR-Ub) 81 via its phosphodiesterase (PDE) domain [29][30][31][32][33][34][35] . 82 Surprisingly, although the LCV is enriched with ubiquitinated species, ubiquitin-binding  In this study, we demonstrate the cross-talk between the Sdc and Sde families of effectors. 92 We show that Sdc and Sde work together to build mixed poly-Ub chains on host substrates and  (Fig. 1a, b). The accumulation of ubiquitinated species around the 111 LCV was dependent on the Dot/Icm secretion system as no Ub signals were detected in cells 112 infected with the ΔdotA mutant strain. In agreement with previous results 23, 42 , the enrichment of 113 Ub signals at the LCV was markedly reduced (~8%) in cells challenged with the Δsdc (sidC and 114 sdcA) strain at 1 hpi. However, the percentage of Ub-positive LCVs recovered, albeit to a less 115 extent compared to the WT strain, at later time points of infection (~19% at 2 hpi and ~25% at 6 116 hpi) (Fig. 1a, b). The Ub signals at the LCV were also substantially reduced in cells challenged 117 with the Δsde (sidE, sdeA, sdeB, and sdeC) strain with ~36% of LCVs positive for Ub at 1 hpi. In 118 contrast to the Δsdc strain, the percentage of Ub-positive LCVs further dropped at later infection 119 time points (as low as ~12% at 6 hpi) (Fig. 1a, b) suggesting Sde effectors are important in 120 stabilizing ubiquitinated species at the LCV. Strikingly, the Ub signals were nearly completely 121 abolished at the LCV in cells infected with the Δsde/sdc strain (Fig. 1a, b). These observations It has been shown that some host proteins, such as Rab1 and Rab10, are targeted by both 127 Sdc and Sde families of effectors 23,26,43 . We hypothesized that the accumulation of ubiquitinated 128 species at the LCV is due to direct ubiquitination and PR-ubiquitination on the same host targets 129 by Sdc and Sde, respectively. To test whether Sdc and Sde work together to modify the same host 130 targets, we first analyzed the ubiquitination state of Rab1 in cells after L. pneumophila infection.

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HEK293T cells were transfected with plasmids expressing 3xFlag-Rab1 and FcγRII for 24 hours 132 and were challenged with WT or mutant Legionella strains for 1 hour. Rab1 was then 133 immunoprecipitated and analyzed by Western blot (Fig. 1c,  were substantially reduced after the treatment of either DUB or DupB (Fig. 1e, lanes 3 and 4). 148 Strikingly, ubiquitinated Rab1 was completely abolished after the treatment with both enzymes 149 (Fig. 1e, lane 5). Along this line, ubiquitinated Rab1 prepared from cells infected with Δsde or 150 Δsdc strain was also markedly reduced after DUB or DupB treatment (Fig. 1e, lane 6- we investigated the localization of host substrates and ER membrane markers in infected cells.

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HEK293T cells expressing FcγRII and EGFP-Rab1 were challenged with WT and mutant 166 Legionella stains and the localization of Rab1 was analyzed by confocal microscopy at 1 hpi ( Fig.   167 2a, b). In cells infected with the WT strain, ~61% of the LCVs were detected positive for Rab1, 168 while Rab1-positive LCVs were largely reduced in cells infected with the Δsde (~13%) or Δsdc 169 (~29%) strain. More strikingly, as low as 8% of LCVs were positive for Rab1 in cells infected 170 with the Δsde/sdc strain. Similar patterns were also observed for another confirmed Sdc and Sde 171 target, LULL1 (extended data Fig. 3). These results suggest that the LCV-recruitment of host 172 substrates depends on the activity of both Sdc and Sde families of effectors. We next analyzed the 173 role of Sdc and Sde in the recruitment of ER membranes to the LCV using the ER membrane 174 marker Sec61β (Fig. 2c,  to wonder about the nature of the poly-Ub chains conjugated to host targets by Sdc and Sde. Since 185 the Sde effectors attach mono-PR-Ub to serine residues located at flexible loops of substrate 186 proteins 29-32 , and the Sdc effectors catalyze canonical ubiquitination on lysine residues of 187 substrates or a pre-conjugated Ub molecule 42 , we hypothesized that Sdc and Sde work together to 188 synthesize mixed Ub chains on host proteins (Fig. 3a). In this model, the Sde effectors attach a 189 PR-Ub moiety to serine residues of the substrate while the Sdc effectors conjugate poly-Ub chains 190 either directly to substrate lysine residues or lysine residues of the pre-conjugated PR-Ub moiety.

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Based on this model, we predicted that complete cleavage of the ubiquitinated substrate by a 192 canonical DUB will yield free mono-Ub molecules in the solution while PR-Ub will remain 193 attached to the substrate. On the other hand, treatment with DupB will specifically detach PR-Ub 194 from the substrate and thus will release poly-Ub chains with a variable length while canonical 195 poly-Ub chains will remain conjugated to the substrate (Fig. 3a).

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To test this hypothesis, we performed the DUB/DupB treatment experiments on     Fig. 5). Surprisingly, the 231 ability to suppress p62 recruitment to the LCV was also fully restored when the Δsde strain was 232 introduced to express the SdeAH277A mutant, which was able to ADP-ribosylate Ub but was 233 defective in PR-Ub ligation, but not the SdeAEE/AA mutant, which was defective in its mART 234 activity ( Fig. 4a, b). It is worth noting that cells infected with the Δsde+pSdeAH277A strain exhibited   The phosphoribosyl modification on Ub moieties can be detected by SDS-PAGE followed by Pro-268 Q diamond phosphoprotein stain (Fig. 5a), which specifically stains exposed phosphoryl groups   The Sde family effectors conjugate mono-Ub to a substrate through a two-step reaction, 319 namely PR-ubiquitination. Now we showed that Sde effectors also ADP-ribosylate internal Ubs 320 within the poly-Ub chains. Although some of the ADPR groups can be further processed into PR 321 by Sde or DupA/DupB, it is legitimate that some of the internal Ubs of the poly-Ub chain attached 322 to one substrate can also be conjugated to a second substrate through PR-ubiquitination by Sde. In 323 this way, multiple host targets can be covalently cross-linked to one poly-Ub chain created by Sdc 324 and Sde (Fig. 7a). To test this hypothesis, we infected cells expressing two known Sdc and Sde 325 substrates, 3xFlag-Rab1 and HA-Rab33B, with WT or mutant Legionella strains. Cell lysates were 326 prepared after 2 hpi followed by immunoprecipitation with anti-Flag resins and analyzed by both both Rab1 (Fig. 7b, lane 4) and Rab33B (Fig. 7c, lane 4) were detected in samples from cells 329 infected with the WT strain, but not from uninfected cells or cells infected with ∆sde (Fig. 7b, c, 330 lanes 1 and 2). These high molecular weight bands were markedly reduced in samples from cells 331 infected with the ∆sdc strain (Fig. 7b, c, lane 3). These observations suggest that Rab33B and Rab1 332 were cross-linked to form the high molecular weight species allowing the co-immunoprecipitation infection was also observed between other host targets (for example, between Rab1 and LULL1, 343 Extended data Fig. 8b and c). Together, these results demonstrated that multiple host targets were 344 cross-linked through unconventional Ub chains that were generated by Sde and Sdc at the LCV. substrates can be conjugated to the same poly-Ub chain (Fig. 7d). Thus a "Ub-coat" containing 360 unconventionally modified Ub chains was built around the LCV by the Legionella bacterium.

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An apparent paradox associated with the "Ub-coat" at the LCV is that the Ub signals do our data showed that p62 was excluded from the LCV when host cells were infected with the 371 ∆sde+pSdeAH277A strain, which is unable to PR-ubiquitinate any host targets but only modifies Ub 372 by ADP-ribosylation (Fig. 4a-c). Thus, PR-ubiquitination of host substrates per se does not account 373 for the exclusion of p62, and the true mechanism underlying the avoidance of autophagy adaptors 374 by the LCV remains to be elucidated. Here we provided direct evidence to support the "Ub-coat"  Ub chains generated by Sdc and Sde, the association of ER-derived vesicles with the LCV can be 394 explained by an alternative mechanism. It is possible that the poly-Ub chains synthesized by Sdc 395 and Sde provide multiple anchoring points to covalently crosslink proteins associated with either 396 the LCV or ER vesicles and thus to tether together these two types of membrane bound vesicles.

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To further unravel the physiological role of the Ub-coat at the LCV is certainly an exciting future 398 direction.

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In this study, we reported that the Sdc and Sde families of effectors contribute to the 400 creation of poly-Ub chains at the LCV. Furthermore, we also provided evidence to support that

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In summary, we presented the results to show two distinct family Ub E3 ligases, Sdc and 418 Sde work together to assemble a "Ub-coat" at the LCV during Legionella infection. This "Ub-coat" 419 contains unconventional mixed Ub chains with internal Ub moieties being modified with a 420 phosphoribosyl group. This modification is important for preventing the recognition by p62. Our 421 results elucidate the nature of the "Ub-coat" and provide a molecular mechanism for the avoidance 422 of autophagy adaptors and therefore the protection of the LCV from xenophagy clearance.      The reactions were incubated for 2 hours at 37 °C and reaction products were assessed by 15% 520 SDS-PAGE stained with either Coomassie or Pro-Q Diamond phosphoprotein stain (Invitrogen).

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For co-immunoprecipitation, FCγRII, 3xFlag-Rab1, and HA-p62 transfected HEK293T 561 cells were infected with specified Lp strains for 1 hour. Cells were then lysed in ice-cold lysis 562 buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton, 0.5% deoxycholic acid, 5mM EDTA, 5% 563 glycerol) with protease inhibitor cocktail (Roche) for 30 min. 5% of the supernatant was used as 564 input and the rest was mixed with anti-Flag beads for 2 hours for immunoprecipitation of 3xFlag-   were filtered using the following parameters: the minimum probability of 0.9, minimum peptide 625 length of 7 amino acid residues, accurate mass binning, and restriction to +2, +3, and +4 ion charge     4xFlag-Rab33B was enriched by anti-Flag resins followed by the treatment with DUB and/or 12 DupB. The cleaved products that were released in the supernatant (Sup) and that remained on the 13 beads (Beads) were analyzed by SDS-PAGE followed by anti-Ub Western blot.