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Evasion of autophagy mediated by Rickettsia surface protein OmpB is critical for virulence

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Abstract

Rickettsia are obligate intracellular bacteria that evade antimicrobial autophagy in the host cell cytosol by unknown mechanisms. Other cytosolic pathogens block different steps of autophagy targeting, including the initial step of polyubiquitin-coat formation. One mechanism of evasion is to mobilize actin to the bacterial surface. Here, we show that actin mobilization is insufficient to block autophagy recognition of the pathogen Rickettsia parkeri. Instead, R. parkeri employs outer membrane protein B (OmpB) to block ubiquitylation of the bacterial surface proteins, including OmpA, and subsequent recognition by autophagy receptors. OmpB is also required for the formation of a capsule-like layer. Although OmpB is dispensable for bacterial growth in endothelial cells, it is essential for R. parkeri to block autophagy in macrophages and to colonize mice because of its ability to promote autophagy evasion in immune cells. Our results indicate that OmpB acts as a protective shield to obstruct autophagy recognition, thereby revealing a distinctive bacterial mechanism to evade antimicrobial autophagy.

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Fig. 1: The R. parkeri surface protein OmpB is required for polyubiquitylation avoidance in HMECs.
Fig. 2: OmpB acts locally on R. parkeri to promote polyubiquitin avoidance and is required for bacterial growth in BMDMs but not in HMECs.
Fig. 3: OmpB is required for the formation of an electron-lucent halo in host cells.
Fig. 4: OmpB protects OmpA against ubiquitylation in diverse host cell lines.
Fig. 5: OmpB is required to avoid both the recruitment of autophagy receptors and LC3.
Fig. 6: OmpB interferes with autophagy and promotes R. parkeri growth in macrophages.

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Data availability

The datasets generated and/or analysed during the current study are available from the corresponding authors on request. The genome sequences of bacterial strains are available at the Sequence Read Archive as accession no. SRP154218 (WT, SRX4401164; ompB::tn, SRX4401163; ompBSTOP::tn, SRX4401167; ompBPROM::tn, SRX4401166 and MC1_RS02370STOP, ompB::tn, SRX4401165).

Change history

  • 24 January 2020

    In the version of this Article originally published, Supplementary Table 1 and the Source Data for Figs. 1–6 and Extended Data Fig. 1 were linked to the wrong files; this has now been amended.

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Acknowledgements

We thank the labs of D. Portnoy and R. Vance (University of California, Berkeley), T. Hackstadt (NIH Rocky Mountain Laboratories) and D. Green (St. Jude Children’s Research Hospital) for providing us with equipment, reagents and discussions. We thank J. Choe and V. Ahyong, other current and former members of the Welch lab, the lab of M.R., A. Roberts and members of the lab of J.S.C., R. Zalpuri, and K. McDonald and other members of the UC Berkeley Electron Microscopy facility for their technical assistance and fruitful discussions. P.E. was supported by postdoctoral fellowships from the Foundation Olle Engkvist Byggmästare, the Swedish Society of Medical Research and the Sweden–America Foundation. M.D.W. is supported by NIH/NIAID grants R01 AI109044 and R21 AI138550. G.M. was supported by fellowships from Fonds de recherche du Québec—Nature et technologies, Fonds de recherche du Québec—Santé, and the Natural Sciences and Engineering Research Council of Canada. J.S.C. was supported by NIH grants P01 AI063302 and R01 AI120694. K.G.M was supported by a Ruth L. Kirschstein National Research Service Award (grant no. F32 GM120956). The mass spectrometer used in this study was purchased with support from the NIH (grant no. 1S10 OD020062-01). N.I. was supported by the UC Berkeley Amgen Scholars Program and the Amgen Foundation. M.R. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

Authors

Contributions

P.E. and M.D.W. conceived the study. P.E. performed all of the work with infections of tissue culture cells and imaging, with assistance from N.I. P.E. and T.P.B. performed the mouse studies. P.E., T.P.B. and G.M. generated the BMDMs. G.G. bred the mice. M.D.W., G.M., K.G.M., M.R. and J.S.C. provided resources and protocol assistance. A.T.I. conducted the mass-spectrometry analysis. P.E. and M.D.W. drafted the initial manuscript and all authors provided editorial feedback. P.E., M.D.W., G.M., A.T.I., K.G.M., M.R. and J.S.C. obtained funding.

Corresponding authors

Correspondence to Patrik Engström or Matthew D. Welch.

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Extended data

Extended Data Fig. 1 Isolation and characterization of suppressors of the parental ompB::tn strain.

(a) A graphic depiction of the scheme for the isolation of suppressor mutations. The parental ompB::tn strain was subjected to 10 serial passages in Vero cells (step 1). The mixed suppressor population was then tested for growth in HMECs (step 2) and compared with the parental ompB::tn strain. Clonal strains that appeared to have larger plaque phenotypes than parental ompB::tn were isolated from the two independent mixed suppressor populations, propagated in Vero cells, and deep sequenced (step 3). (b) Growth curves in HMECs of WT, parental ompB::tn, and ompB::tn suppressor population (passage 10, selection-1) (starting MOI: 0.002) as measured by genomic equivalents using qPCR. Data are means (n = 2). (c) Diagram of the ompB genomic regions and other relevant genes, showing the location of the transposon insertions (tn) in: the parental ompB::tn strain (at base pair (bp) position 1045462 in the R. parkeri genome); ompBSTOP::tn (with an additional 23-bp deletion at position 1045835-1045857); ompBPROM::tn (with an additional 17-bp insertion in the promoter region of ompB at position 1046683); and MC1_RS02370STOP, ompB::tn (with an additional two deletions, of 112 at position 461273-461384 and 125 bp at position 461425-461549 in a gene encoding a predicted glycosyltransferase (MC1_RS02370), leading to a premature stop codon). (d) Western blot of 106 30%-purified WT, ompB::tn and ompB suppressor strains probed for OmpB (anti-OmpB antibody). Reduced levels of truncated OmpB products were observed in the suppressor strains (n =2). (e) Quantification of the percentage of bacteria that co-localize with polyubiquitin at 72 h.p.i. Data are mean ± s.e.m. (WT, n = 3; ompBSTOP::tn, n = 2; parental ompB::tn, n = 3; MC1_RS02370STOP, ompB::tn, n = 2; ompBPROM::tn, n = 2; ≥136 bacteria were counted for each strain and infection). (f) Western blot of 106 30%-purified WT, ompBSTOP::tn and ompA::tn strains probed for OmpB (anti-OmpB antibody) and OmpA (13-3 antibody) (n = 1).

Source Data

Extended Data Fig. 2 The host ubiquitin machinery is necessary to label the ompB mutant with ubiquitin chains.

(a) Immunofluorescence micrographs of infected HMEC cells treated with the E1-inhibitor PYR-41 (50 μM) or DMSO control for 6 h (66-72 h.p.i.) and stained as in (Fig. 1a). Scale bars, 5 μm. (b) Quantification of the percentage of polyubiquitin-positive bacteria. Data are mean ± s.e.m. (n = 3; statistical comparisons between PYR-41 treated and control ompBSTOP::tn and WT infections were determined by the unpaired Student’s t-test (two-sided); ***, p <0.001; * p <0.05; ≥229 bacteria were counted for each infection). (c) Quantification of the percentage of bacteria that showed rim-like surface localization of the indicated ubiquitin- chain at 72 h.p.i. in HMECs based on staining with ubiquitin linkage-specific antibodies. Data are mean ± s.e.m. (n = 4; statistical comparisons between the ompBSTOP::tn and WT bacteria were determined by the unpaired Student’s t-test (two-sided); ***, p < 0.001; ≥164 bacteria were counted per strain, ubiquitin-chain stain and experiment).

Extended Data Fig. 3 In HMECs, OmpA/Sca2/17- kDa-antigen are not required for R. parkeri invasion, and OmpB is not required for actin mobilization or bacterial membrane integrity.

(a) Quantification of the percentage of internalization into HMECs at 15 m.p.i., visualized by differential staining of extracellular versus total R. parkeri. Data are mean ± s.e.m. (WT, n = 3; ompA::tn, n = 2; sca2::tn, n = 3; hrtA::tn, n = 3; ≥180 bacteria were counted for each strain and infection; means were not significantly different between WT, sca2::tn and hrtA::tn by a one-way ANOVA with Tukey’s post hoc-test). (b and c) Quantification of the percentage of bacteria with (b) early actin clouds or (c) actin tails. The delayed recruitment of actin can be explained by slower internalization of ompBSTOP::tn bacteria. Data are mean (n = 2; ≥84 bacteria were counted for each strain and infection for each time point). (d) Representative immunofluorescence micrographs of bacteria (blue; anti-Rickettsia 14-13 antibody) with late actin tails (green; Alexa 488 phalloidin). (e) Quantification of the percentage of WT and ompBSTOP::tn bacteria with late actin tails that started from the bacterial pole (green bar) or the side of the bacteria (brown bar). (n = 2; ≥274 bacteria were counted for each strain and infection). (f) Fluorescence micrographs of bacteria attached to HMECs prior to internalization, first stained with propidium iodide (PI; red) prior to fixation to label dead bacteria, and next fixed and stained for total bacteria (green; anti-Rickettsia 14-13 antibody). The bottom panels are a positive control in which 50 µg/ml digitonin was included to permeabilize bacteria and HMECs prior PI staining. Scale bars, 2 μm. (g) Quantification of the bacterial viability (membrane integrity) as determined by PI staining. Data are means (n = 2; ≥182 bacteria were counted for each strain and infection).

Extended Data Fig. 4 OmpB is critical to block ubiquitylation shortly after internalization into HMECs and BMDMs.

(a) Quantification of the percentage of internalization into BMDMs from 0-90 m.p.i., visualized by differential staining of extracellular versus total R. parkeri. Data are mean ± s.e.m. (0, 15, 30, 60, 90 min, n = 4; 5, 10, 20 min, n = 3; ≥80 bacteria were counted for each strain, infection and time-point; statistical comparisons with WT for each time point were made by the unpaired Student’s t-test (two-sided); *, p < 0.05). (b) Quantification of the percentage of WT and ompBSTOP::tn mutant bacteria that co-localize with polyubiquitin from 0-60 m.p.i. Data are mean ± s.e.m. (0, 5, 10, 20, 30, 60 min: n = 3; 15 min: n = 5); ≥80 bacteria were counted for each strain, infection, and time point; statistical comparisons with WT for each time point were made by the unpaired Student’s t-test (two-sided); *, p < 0.05; **, p < 0.01; ***, p < 0.001). (c) Immunofluorescence micrographs of BMDMs infected with WT and ompBSTOP::tn mutant at 60 m.p.i., stained for extracellular R. parkeri (blue; anti-Rickettsia I7205 antibody), total R. parkeri (green; anti-Rickettsia I7205 antibody), polyubiquitin (red; FK1 antibody), and a merged image. Arrows indicate an intracellular bacterium that is positive for polyubiquitin. The stars indicate an extracellular bacterium that is polyubiquitin-negative in BMDMs. Scale bars, 5 µm. (d) Quantification of the percentage of intracellular and extracellular WT and ompBSTOP::tn mutant bacteria that co-localize with polyubiquitin. Data are mean ± s.e.m. (n = 3; ≥122 bacteria were counted for each strain and infection; statistical comparisons were by an unpaired Student’s t-test (two-sided); ***, p < 0.01). (e) Immunofluorescence micrographs of HMECs infected with WT and ompBSTOP::tn mutant at 60 m.p.i., stained as in (c). Arrows indicate an intracellular bacterium that is polyubiquitin- positive. Scale bars, 5 µm. (f) Quantification of the percentage of intracellular and extracellular WT and ompBSTOP::tn mutant bacteria that co-localize with polyubiquitin in HMECs. Data are mean ± s.e.m. (WT, n = 2; ompBSTOP::tn, n = 3; ≥79 bacteria were counted for each strain and infection).

Extended Data Fig. 5 OmpB is required for R. parkeri to proliferate in BMDMs.

(a) Fluorescence micrographs of BMDMs infected with WT and ompBSTOP::tn bacteria at 48 h.p.i. Left panels show cellular and bacterial genomic DNA (blue; Hoechst). Right panels show bacteria (green; anti-Rickettsia antibody I7205), cellular and bacterial genomic DNA (blue, Hoechst), and actin (red; Alexa 568 phalloidin). Scale bars, 20 μm. (b) Quantification of bacteria per BMDM cell for WT, ompBSTOP::tn, and MC1_RS05535::tn bacteria, using Hoechst to count the number of cell nuclei and the number of bacteria. Data are mean (n = 2; 5 fields of view per infection and strain were used to count the number of bacteria per cell). (c) Fluorescence micrographs of BMDMs infected with WT and ompBSTOP::tn bacteria at 96 h.p.i., stained as in (a). Scale bars, 20 μm. (d-f) Quantification of (d) the mean number of bacteria per BMDM cell as in (b) (n = 2), (e) mean number of host cells per field of view (n = 2; 5 fields of view per infection and strain were used to count the number of host cell per field of view), and (f) the percentage LDH release, normalized to Triton-X100-lysed cells, all determined for WT, ompBSTOP::tn, and uninfected cells. Data are mean ± s.e.m. (n = 3; statistical comparisons between WT and ompBSTOP::tn infected and uninfected cell were performed using a one-way ANOVA with Tukey’s post hoc-test; **, p < 0.01; ***, p < 0.001).

Extended Data Fig. 6 In BMDMs, neither OmpA nor the gene downstream of ompB is required for halo formation.

(a) TEM images of ompA::tn and MC1_RS05535::tn mutant bacteria in BMDMs at 1 h.p.i. Scale bars, 1 μm. (b) Quantification of halo thickness for WT (n = 79), ompA::tn (n = 12) and MC1_RS05535::tn (n = 17) bacteria. All data points are presented, and the lines indicate the means (statistical comparisons were performed using a Mann-Whitney rank-sum test (two-sided)). Data for WT are the same as that shown in Fig. 3.

Extended Data Fig. 7 Ubiquitylation of the ompB mutant is a prerequisite for LC3 recruitment and activation of autophagy does not increase LC3 recruitment to ompB mutant bacteria.

(a) Immunofluorescence micrographs showing cellular LC3 puncta (red; rabbit anti-LC3 antibody) that do not co-localize with bacteria (blue; Hoechst) in infected BMDMs. Stars indicate cellular LC3 puncta. (b) Quantification of cellular LC3 puncta per host cell at 2.5 h.p.i. in BMDMs. Data are mean (n = 2; 5 fields of view per infection and strain were used to count the number of cellular puncta). (c) Quantification of the percentage of bacteria co-localizing with pUb in HMECs treated with 500 nM rapamycin (Rapa), 20 µM MG132 or DMSO control for 3 h (from 1 h.p.i. to 4 h.p.i.) and fixed at 4 h.p.i. Data are mean (n = 2; ≥87 ompBSTOP::tn bacteria were counted for each infection and treatment). (d) Quantification of the percentage of bacteria co-localizing with LC3 in HMECs treated with 500 nM rapamycin (Rapa), 20 µM MG132 or DMSO control for 3 h (1-4 h.p.i.) and fixed at 4 h.p.i. Data are means (n = 2; ≥87 ompBSTOP::tn bacteria were counted for each infection and treatment). (e) Immunofluorescence micrographs of BMDMs infected with WT and ompBSTOP::tn mutant at 2.5 h.p.i., stained for R. parkeri (blue; Hoechst), polyubiquitin (green; FK1 antibody), LC3 (red; rabbit anti-LC3 antibody), and a merged image. Arrows indicate bacteria that are positive for both polyubiquitin and LC3. Scale bars, 5 μm. Related to Fig. 5k. (f, g) Quantification of the percentage of (f) polyubiquitin-positive bacteria and (g) bacteria with both LC3 and polyubiquitin, after 4 h treatment (1-5 h.p.i.) with PYR-41. Data are means (n = 2; ≥110 ompBSTOP::tn bacteria were counted per infection and experiment).

Extended Data Fig. 8 Autophagy is the primary mechanism that restricts the growth of ompB mutant bacteria in macrophages, and OmpB protects R. parkeri from ubiquitylation in autophagy- deficient cells.

(a) Immunofluorescence micrographs of Atg5+/+ or Atg5-/- BMDMs infected with WT or ompBSTOP::tn bacteria at 72 h.p.i., stained for bacteria (green), cellular and bacterial genomic DNA (blue; Hoechst), polyubiquitin (red; FK1 antibody). Scale bars, 5 μm. (b) Quantification of the percentage of bacteria that co-localize with polyubiquitin in Atg5+/+, Atg5-/-, or Becn1-/- BMDMs at 72 h.p.i. (nd indicates that too few ompBSTOP::tn bacteria were visualized in Atg5+/+ cells to quantify a percentage). Data are mean (n = 2; ≥250 bacteria were counted for each strain and infection). (c) Quantification of the percentage of bacteria that co-localize with polyubiquitin in C57BL/6 or Becn1-/- BMDMs at 1 h.p.i. Data are mean (n = 2; ≥84 bacteria were counted for each strain and infection). (d) Combined growth curves of WT and ompBSTOP::tn in Atg5+/+ or Atg5-/- BMDMs as measured by genomic equivalents using qPCR. Data are the same as that shown in Fig. 6a, b and are mean ± s.e.m. (n = 4). (e) Growth curves of WT and ompBSTOP::tn in Rubicon-/- or Rubicon+/+ BMDMs as measured in (d). Data are mean (n = 2). (f) Quantification of the percentage LC3 and polyubiquitin-positive bacteria in C57BL/6 or Becn1-/- BMDMs at 1 h.p.i. Data are mean (n = 2; ≥84 bacteria were counted for each strain and infection). (g) Quantification of the percentage LC3-positive bacteria at 1, 2.5 and 4 h.p.i. in BMDMs treated with 300 nM bafilomycin A (Baf) or corresponding amount of DMSO, starting at 1 h.p.i. Data are means (n = 2; ≥111 ompBSTOP::tn bacteria were counted per infection, experiment and time-point). (h,i) Quantification of the percentage (h) LC3-positive ompBSTOP::tn that also co-localize with polyubiquitin at 4 h.p.i., and (i) polyubiquitin-positive ompBSTOP::tn, after bafilomycin A treatment as in (g). Data are mean (n = 2; ≥119 ompBSTOP::tn bacteria were counted per infection and experiment).

Extended Data Fig. 9 In HMECs, OmpB is required for a subpopulation of R. parkeri to avoid trafficking to a LAMP1-positive compartment.

(a) Immunofluorescence micrographs of HMECs infected with WT or ompBSTOP::tn mutant at 2.5 h.p.i., and stained for R. parkeri (green; anti-Rickettsia I7205 antibody), cellular and bacterial genomic DNA (blue; Hoechst), and LAMP1 (red; anti-LAMP1 antibody). Scale bars, 5 μm. (b) Quantification of the percentage bacteria that co-localize with LAMP1 at 2.5 and 4 h.p.i. in HMECs. Data are mean (n = 2; ≥106 bacteria were counted for each strain, infection and time point).

Extended Data Fig. 10 WT R. parkeri establishes an infection of mouse organs but is eventually cleared.

(a) C57BL/6 mice were infected with 107 PFUs of 30%-purified WT R. parkeri. At 1-7 d post infection, organs were harvested at the indicated time points and homogenized, and PFUs were counted. Medians are indicated as bars (1 d, n = 4; 2 d, n = 8; 3 d, n = 8; 4 d, n = 3; 6 d, n = 3; 7 d, n = 5). (b) Relative PFU counts for WT or ompBSTOP::tn bacteria following incubation in blood from uninfected mice at 37 °C for the indicated times. PFUs were normalized to 0 m.p.i. Data are mean ± s.e.m. (n = 2 for all time points, except 0 and 20 min, n = 3; statistical comparisons were by an unpaired Student’s t-test (two-sided) and no statistical difference was observed between WT and ompBSTOP::tn at 20 min; p = 0.46).

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

Lys-diGly proteomics identify OmpA as a candidate ubiquitylated protein on ompBSTOP::tn bacteria. Related to Fig. 4.

Source data

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Source Data Extended Data Fig. 1

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Engström, P., Burke, T.P., Mitchell, G. et al. Evasion of autophagy mediated by Rickettsia surface protein OmpB is critical for virulence. Nat Microbiol 4, 2538–2551 (2019). https://doi.org/10.1038/s41564-019-0583-6

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