Rab5A forms a “cloud” around the WT LCV in a bacterial effector dependent manner.

(A) Endogenous Rab5A recruitment to the WT or dotA LCV in HeLa FcγR. (B) LCV-associated Rab5A area normalized to bacterial cell area for WT and dotA strains. N=5, 8-30 LCVs measured per strain per replicate. Welch’s two-sample t-test, p = 0.0009. In this and all subsequent jitter plots, small points represent individual measurements, large points are mean values across a given biological replicate, and horizontal bars depict the sample mean across replicates. Color corresponds to biological replicate. (C) Endogenous Rab5A staining in cells overexpressing EGFP or indicated fusion constructs. (D) Mean endosome area per cell in samples overexpressing indicated construct. N=3, 20-40 cells scored per replicate. ANOVA, Tukey Kramer post hoc test, ** p<0.005, *** p<0.0005. (E) Manual scoring of endogenous Rab5 recruitment to WT or dotA LCVs in cells overexpressing indicated construct. N=3, 50-130 LCVs scored per replicate. G test, Bonferroni adj. p value = 0.0125, ***p<0.000125. (F) LCV-associated Rab5A area as in panel B in cells expressing indicated construct. N = 3, 10-30 LCVs scored per replicate. ANOVA, Tukey Kramer post hoc test, n.s. p>0.05. For all infection experiments, cells were fixed at 1hpi.

The SidE family is required for Rab5 cloud formation at the LCV.

(A) Endogenous Rab5A recruitment to the LCV for the indicated strains in HeLa FcγR. (B) LCV-associated Rab5 area normalized to bacterial cell size for the indicated strains. N=3, 5-60 LCVs scored per strain per replicate. One way ANOVA followed by Tukey-Kramer post hoc test, n.s. p>0.05, * p<0.05, **p<0.005. (C) Immunoprecipitation of Flag-Rab5A from cells expressing HA ubiquitin ΔGG and indicated myc-tagged SidE family effector, or vector control. (D) Immunoprecipitation of Flag-Rab5A from cells expressing HA ubiquitin ΔGG and infected with indicated L.p. strainFor C and D, * indicates non-specific band, most likely light chain. For all infection experiments, cells were fixed or lysed at 1hpi.

Rab5 is detergent resistant when associated with the WT LCV.

(A) Saponin vs SDS permeabilized uninfected cells stained for endogenous Rab5A. (B) Quantification of normalized total Rab5 fluorescence in cells permeabilized as in panel A. N = 3, 50 cells scored per condition per replicate. Welch’s two-sample t-test, p = 0.00069.(C) Endogenous Rab5A immunofluorescence in infected cells after either saponin or SDS permeabilization. (D) Quantification of experiment described in panel C. N = 3, 80-250 LCVs scored per condition per replicate. Welch’s ANOVA followed by Games-Howell post hoc test, n.s. p>0.05, *p<0.05. For all infection experiments, cells were fixed at 1hpi.

The SidE family of effectors regulates ubiquitin morphology at the LCV.

(A) Manual scoring immunofluorescence imaging of endogenous ubiquitin recruitment to the indicated L.p. strain. N = 3, 60-120 LCVs scored per strain per replicate. G test, Bonferroni adj. p value = .01. *** p<0.0001. (B) Representative images of endogenous ubiquitin recruitment to indicated strain in experiment described in panel A. (C) Quantification of LCV-associated ubiquitin area normalized to bacterial cell area for indicated strain for experiment described in A. N = 4, 20-120 LCVs scored per strain per replicate. Welch’s ANOVA followed by Games-Howell post hoc test, n.s. p>0.05, *p<0.05.

Ubiquitin at the WT LCV resists detergent washout in a SidE family dependent manner.

(A) Endogenous ubiquitin immunofluorescence in cells infected with the indicated strain and permeabilized with either saponin or SDS. (B) Quantification of normalized ubiquitin fluorescence at the LCV for experiment described in A. N=3, 87-240 LCVs scored per strain per replicate. ANOVA, Tukey Kramer post hoc test, n.s. p>0.05. (C) Background-normalized cytosolic ubiquitin intensity in infected or uninfected cells after SDS washout. 17-72 cells analyzed per condition per replicate. Welch’s two-sample t-test, p = 0.09334. (D)-(E) Immunoblot analysis of total ubiquitin in the (D) soluble or (E) insoluble fraction of cells infected with the indicated L.p. strain from the ΔsidE/sdeABC panel, heat shocked, or left untreated, with total protein (StainFree, BioRad) shown as the loading control. (F) Quantification of normalized high molecular weight ubiquitin in experiments described for D and E. Total endogenous ubiquitin intensity was measured at 150 kDa and above and normalized to total protein and the fold change over the dotA infected control was calculated. N=3, ANOVA, Tukey Kramer post hoc test when applicable, n.s. p>0.05, * p<0.05, ** p<0.005. (G)-(I) Western blot analysis and quantification of soluble-insoluble fractionation carried out on cells infected with the L.p. ΔsidC/sdcA strain panel as described for D-F.

LCV-associated ubiquitin stability decreases as infection progresses.

(A) Immunofluorescence analysis of endogenous ubiquitin localization to the WT LCV at the indicated timepoint post-infection after permeabilization with either saponin or SDS. (B) Normalized ubiquitin intensity quantified for the experiment described in A. Bacteria per LCV were approximated by dividing the area of each LCV by the mean LCV area for the 1hpi timepoint for each experiment and permeabilization condition. Intensity data was subjected to one way ANOVA, followed by a Tukey-Kramer post hoc test, n.s p>0.05, *** p<0.0005. (C)-(D) Immunoblot analysis of endogenous ubiquitin in cells infected with either L.p. WT or dotA for the indicated length of time and fractionated by solubility. Total protein (StainFree) is shown as a loading control. (E) Quantification of normalized high molecular weight ubiquitin in experiments described for C and E. Total endogenous ubiquitin intensity was measured at 150 kDa and above and normalized to total protein, and the fold change over the dotA infected control was calculated. N=3, ANOVA, Tukey Kramer post hoc test, n.s. p>0.05, ** p<0.005, *** p<0.0005. (F) Quantification of normalized ubiquitin intensity at the WT LCV at 1-hpi after saponin or SDS permeabilization in host cells expressing either EGFP or EGFP DupA. Points represent mean values for each biological replicate for a given condition, and replicates are indicated by color. (G) Analysis of the distribution of the normalized LCV-associated ubiquitin intensity values for the experiment described in panel E.

Ubiquitin expands outwards from the WT LCV during early infection and is co-recruited with Rab5.

(A) Insets from live imaging time courses (see movies S1 and S2) of cells expressing EGFP ubiquitin infected with indicated L.p. strain labeled using the HaloTag system. (B) LCV-associated ubiquitin area normalized to bacterial cell area for LCVs shown in panel A. (C) Percentage of LCVs falling into “compact” or “expansive category. “Compact” defined as normalized ubiquitin area at T30/T0≤1.5, “expansive” indicates normalized ubiquitin area at T30/T0>1.5. Data was pooled across 3 biological replicates, 49 LCVs total for L.p. WT, 41 for L.p. ΔsidE/sdeABC. (D) Normalized LCV-associated ubiquitin area across imaging time course for indicated L.p. strains. Violin plots show distribution of pooled data, and dots indicate mean values for each biological replicate. N=3, 6-30 LCVs scored per replicate. ANOVA, Tukey Kramer post hoc test, n.s. p>0.05, *p<0.05, **p<0.005. (E)-(F) Insets from timelapse imaging in infected cells expressing mCherry Rab5 and EGFP ubiquitin, infected with either L.p. WT or ΔsidE/sdeABC (see movies S3 and S4). Graphs in F are intensity measurements for each FP tagged protein for these insets. (G) Visual scoring of ubiquitin and Rab5 recruitment patterns across all replicates. R5 is Rab5, Ub is ubiquitin. Percentages are calculated for all pooled data. 164 LCVs were score in total for L.p. WT, and 175 for L.p. ΔsidE/sdeABC. (H) Manual approximation of Rab5 association time with either the WT or ΔsidE/sdeABC LCV across 3 biological replicates. Each point represents a single LCV. LCVs that transition from Rab5 positive to negative are labeled as + to -, whereas LCVs that remain Rab5 positive at the end of the time course are marked as + only. 26 LCVs were scored for L.p. WT, and 24 for L.p. ΔsidE/sdeABC. (I) Comparison of the timepoints at which the intensity maximum occurs for Rab5 and ubiquitin for each LCV across three biological replicates. 3-9 LCVs were scored per strain per replicate. Welch’s two-sample t-test, p = 0.00473.

SdeB EE/AA is unable to catalyze PR-ubiquitination.

Western blot analysis of HA-Ub βGG conjugation in whole cell lysates prepared from cells infected with the indicated L.p. strain for 1 hour.

Validation of dual-stain method for detection of extracellular bacteria.

(A) Representative images of cells permeabilized either before or after Bap31 primary antibody and GAR-405 staining. (B) Pearson correlation of GAR-633 and GAR-405 signal in cells prepared as in flowchart shown in A. N=2, 50 cells analyzed per replicate per condition. Welch’s two-sample t-test, p = 0.006. (C) Representative images of cells infected with L.p. WT and processed using dual stain method for the L.p. opsonization antibody, as well as immunofluorescence analysis of endogenous Rab5. Double stained (extracellular) bacteria are indicated by blue arrows, whereas single stained (intracellular) bacteria are indicated by magenta arrows. (D) Quantification of normalized Rab5 intensity at either double (405+) or single (405−) stained bacteria. N = 4, ∼30-90 bacteria scored per replicate per condition. Welch’s two sample t-test, p = 0.0127. (E) Measurement of normalized GAR405 intensity at bacterial cell bodies for same dataset described in D. Welch’s t-test, p = 0.0002. (F) Distribution of Rab5 intensity values for dataset described in D. For each replicate, data was pooled, and the percentage of 405+ versus 405-bacteria falling into each quartile was tabulated. (G) Normalized GAR405 intensity vs. normalized Rab5 intensity for dataset described in D. Note that very few datapoints show strong signal for both Rab5 and GAR405. Color represents biological replicate (N=4), 418 total bacteria scored.

Extended representative images of ubiquitin detergent resistance.

Endogenous ubiquitin staining in cells infected with the indicated strain and permeabilized with either saponin or SDS at 1hpi.

Ectopically expressed EGFP-DupA is active against PR-ubiquitin conjugates.

(A) Western blot analysis of lysates from cells transfected with HA-ubiquitin βGG and the indicated construct (LotC is a canonical ubiquitin ligase effector), and either left uninfected or infected with L.p. WT. (B) Representative images of endogenous ubiquitin staining in cells transfected with either EGFP alone or EGFP-DupA, infected with L.p. WT, and permeabilized with either sapoinin or SDS. For all infection experiments, cells were fixed or lysed at 1hpi.

Uncommon LCV-associated ubiquitin morphology patterns observed during live imaging.

Example cases of (A) diffuse ubiquitin localization throughout imaging at WT LCV, (B) compact ubiquitin localization at WT LCV, and (C) highly dynamic compact-to-expansive ubiquitin morphology at the SidE family knockout strain LCV.