key: cord-0004404-5f079o3h authors: Engström, Patrik; Burke, Thomas P.; Mitchell, Gabriel; Ingabire, Nadia; Mark, Kevin G.; Golovkine, Guillaume; Iavarone, Anthony T.; Rape, Michael; Cox, Jeffery S.; Welch, Matthew D. title: Evasion of autophagy mediated by Rickettsia surface protein OmpB is critical for virulence date: 2019-10-14 journal: Nat Microbiol DOI: 10.1038/s41564-019-0583-6 sha: 10218d6165dcbaa34d75f2135348cbac1ffc9ec9 doc_id: 4404 cord_uid: 5f079o3h 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 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, revealing a distinctive bacterial mechanism to evade antimicrobial autophagy. autophagy. Our results suggest that cytosolic pathogens have evolved distinctive mechanisms to shield themselves from polyubiquitin coat formation to avoid destruction via autophagy. To identify surface proteins that may protect Rickettsia from autophagic recognition, we assessed whether R. parkeri transposon mutants lacking known surface proteins were polyubiquitylated. In particular, we assessed strains with transposon (tn) mutations in ompA, ompB, sca2, and hrtA (encoding the 17-kDa antigen) 33, 38 . The ompB mutant (ompB STOP ::tn), obtained via serial-passaging the parental ompB::tn strain, contains a suppressor mutation in the form of a stop codon upstream of the transposon that prevents expression of a truncated OmpB product, which appears to be toxic ( Fig. 1b ; Extended Data Fig. 1a-e) . The mutants were analyzed for co-localization with polyubiquitin at 72 hours post infection (hpi) in human dermal microvascular endothelial cells (HMECs) (Fig. 1a , c; Extended Data Fig. 1e ). Wild type (WT), ompA::tn, and hrtA::tn bacteria were polyubiquitin-negative. Notably, the sca2::tn mutant, which is defective in actin polymerization at this time point 33 , was also polyubiquitin-negative. However, more than 75% of the ompB STOP ::tn (and ompB PROM ::tn, an independent mutant; Extended Data Fig. 1a -d) bacteria co-localized with polyubiquitin. To confirm that polyubiquitylation of ompB STOP ::tn bacteria was due to mutations in ompB and not to polar effects on surrounding genes, we tested R. parkeri mutants with insertions in the genes directly upstream or downstream of ompB (Fig. 1b) . We also tested a mutant with an insertion in the gene rickCE encoding for a rickettsial deubiquitylase 39 . Mutants with insertions in the genes directly flanking ompB, or in rickCE, were all polyubiquitin-negative (Fig. 1a, c) . Thus, polyubiquitylation of ompB STOP ::tn bacteria is caused by mutations in ompB. Furthermore, purified ompB STOP ::tn bacteria were heavily polyubiquitylated while WT bacteria showed little polyubiquitylation (Fig. 1d) , suggesting that bacterial proteins or tightly associated host components are ubiquitylated. These data demonstrate that R. parkeri requires OmpB, but not Sca2-mediated actin mobilization, for avoidance of polyubiquitylation. To confirm the involvement of the host ubiquitin machinery in polyubiquitylation of ompB STOP ::tn bacteria, we treated infected cells with PYR-41, an inhibitor of host ubiquitin activating enzyme E1. PYR-41 treatment caused a 9-fold reduction in the number of polyubiquitin-positive ompB STOP ::tn bacteria compared with a vehicle control (Extended Data Fig. 2a, b) . We next examined the nature of ubiquitin chains in the coat by identifying which of the seven lysine or the N-terminal methionine residue(s) are linked, using ubiquitin linkage-specific antibodies. We observed that ~90% of ompB STOP ::tn bacteria were positive for M1, K63, or K48-linked ubiquitin chains, whereas WT bacteria were polyubiquitinnegative (Extended Data Fig. 2c ). This demonstrates a role for the host ubiquitin machinery in forming the polyubiquitin coat and shows that the coat may consist of heterotypic ubiquitin conjugates, as observed for mitochondria during mitophagy 40 . In HMECs, OmpB blocks polyubiquitylation following host cell entry but is dispensable for bacterial growth To assess the timing of bacterial polyubiquitylation, we first established internalization rates of WT and ompB STOP ::tn bacteria, and observed that WT bacteria reached maximum internalization (internalization max ) at 20 min post infection (mpi), whereas the ompB STOP ::tn reached internalization max at 30 mpi (Fig. 2a) . The internalization of ompA::tn, sca2::tn, or hrtA::tn mutants, in contrast, were not impaired (Extended Data Fig. 3a ). Thus, OmpB is important for optimal internalization into HMECs. Nevertheless, internalization was rapid enough to determine the kinetics of polyubiquitin coat formation. Some purified ompB STOP ::tn bacteria in the initial inoculum had a polyubiquitin coat, and there was increased co-localization with polyubiquitin over time, corresponding with internalization (Fig. 2b) . We also observed that the majority of intracellular ompB STOP ::tn bacteria were polyubiquitin-positive whereas the majority of extracellular bacteria were polyubiquitin-negative at 60 mpi (Extended Data Fig. 4e, f) . Collectively, these data suggest that the host ubiquitin machinery targets ompB STOP ::tn bacteria after they enter HMECs. To ascertain whether OmpB protects against polyubiquitylation by acting locally on the bacterial surface or globally by inhibiting the host ubiquitin machinery, HMECs were coinfected with WT and ompB STOP ::tn. We confirmed co-infection by staining DNA to label all bacteria and OmpB to distinguish between WT and ompB STOP ::tn bacteria by immunofluorescence microscopy (Fig. 2c, d) . We found that the ompB STOP ::tn bacteria were polyubiquitylated at similar frequencies in singly-infected and co-infected cells (Fig. 2c, e) . Furthermore, WT bacteria in co-infected cells remained polyubiquitin-negative (Fig. 2c, e) . These data suggest that OmpB acts locally on the bacterial surface and not globally to suppress ubiquitylation. We next assessed if OmpB is important for R. parkeri growth in HMECs. We observed that the growth of ompB STOP ::tn bacteria was indistinguishable from WT over 96 h (Fig. 2f) . Consistent, ompB STOP ::tn and WT bacteria had similar maximum frequencies of actin recruitment, actin tail formation, and viability (Extended Data Fig. 3b-g) . Together, these data demonstrate that OmpB is essential for avoidance of polyubiquitylation but dispensable for R. parkeri growth and actin mobilization in endothelial cells. Because SFG Rickettsia also grow in macrophages in vitro8 and in vivo7, we next assessed internalization and polyubiquitylation kinetics of the ompB STOP ::tn mutant in mouse bone marrow-derived macrophages (BMDMs). In contrast with their slower internalization into HMECs, ompB STOP ::tn reached internalization max into BMDMs at rates similar to WT bacteria (Extended Data Fig. 4a ), suggesting that OmpB does not impact the internalization of Rickettsia into macrophages. The ompB STOP ::tn mutant also showed increased colocalization with polyubiquitin over time, corresponding with internalization, and polyubiquitylated bacteria were primarily intracellular at 60 mpi (Extended Data Fig. 4b-d) . These data demonstrate that the host ubiquitin machinery targets ompB STOP ::tn bacteria after their phagocytosis by BMDMs. We also measured bacterial growth over 96 h. In contrast with their robust growth in HMECs, the ompB STOP ::tn bacteria were unable to grow in BMDMs, while WT increased in number ~50-fold (Fig. 2g ). Failure to grow was also observed using immunofluorescence microscopy, which revealed that ompB STOP ::tn bacteria were rare whereas WT bacteria were abundant in infected cells (Extended Data Fig. 5a-d) . In addition, failure of the ompB STOP ::tn to grow in BMDMs was not due to host cell death (Extended Data Fig. 5c-f ). Thus, OmpB is critical for the growth of R. parkeri in macrophages. We next assessed the role of OmpB in intracellular trafficking or morphology of R. parkeri using transmission electron microscopy (TEM). At 1 hpi, the majority of WT and ompB STOP ::tn bacteria had escaped from the phagosome and were in the host cytosol of HMECs and BMDMs (Fig. 3a-d) . However, in HMECs, there was a reduction in the percentage of ompB STOP ::tn bacteria in the cytosol, and a corresponding increase in bacteria associated with single-membrane vacuoles, in comparison with WT bacteria (Fig. 3a, b) , which likely was a consequence of a delayed invasion (Fig. 2a) . No differences in the percentage of cytosolic bacteria between ompB STOP ::tn and WT bacteria were observed in BMDMs (Fig. 3c, d) . Collectively, these data further suggest that the restriction of growth of the ompB STOP ::tn bacteria in BMDMs occurs following escape into the cytosol. Once in the cytosol, ompB STOP ::tn bacteria exhibited increased association with membranes resembling phagophores that localized near one bacterial pole or partly surrounding bacteria ( Fig. 3a-d) . However, ompB STOP ::tn and WT bacteria were comparable in their relatively rare localization within double-membrane vacuoles or electron-dense lysosomal compartments at 1 hpi. At 72 hpi in HMECs, the majority of both ompB STOP ::tn and WT bacteria were free of associated membranes (Fig. 3e) . These observations suggest that OmpB inhibits association of cytosolic R. parkeri with host membranes shortly after infection. The size of ompB STOP ::tn and WT bacteria was indistinguishable in HMECs, whereas the ompB STOP ::tn mutant was slightly smaller than WT in BMDMs at 1 hpi (Fig. 3g) . A striking difference, however, was that ompB STOP ::tn bacteria lacked an electron-lucent capsular-like halo that surrounded WT ( Fig. 3a-d) , ompA::tn and MC1_RS05535::tn R. parkeri (Extended Data Fig. 6a, b) , as well as R. rickettsii 41, 42 . For WT bacteria, the size of the electron-lucent halo at 1 hpi was of 100 ± 40 nm (mean ± SD) in BMDMs and 84 ± 60 nm in HMECs (Fig. 3f) . At 72 hpi in HMECs, the mean thickness decreased to 22 ± 21 nm and many WT bacteria lacked a visible halo (Fig. 3e, f) . These data demonstrate that R. parkeri is surrounded by an electron-lucent halo hat decreases in thickness during the time course of an infection, and that OmpB is required for the formation of this halo, but not for other basic structural properties of the bacteria. We next sought to identify proteins that are substrates of ubiquitylation on ompB STOP ::tn bacteria. We developed an approach that combined polyubiquitin enrichment from the surface fraction of bacteria, followed by mass spectrometry (MS) to detect Lys-diGly (diGly) remnants, a signature for ubiquitin (Fig. 4a) . We did not identify any host protein candidate that was reproducibly enriched over 5-fold in fractions from ompB STOP ::tn in comparison with WT bacteria Supplementary Table 1) . We did, however, identify a bacterial peptide of OmpA with diGly remnants that was consistently enriched in fractions from ompB STOP ::tn (Fig. 4b) . The ubiquitylated lysine identified on OmpA is predicted to be exposed to the host cytosol (Fig. 4c) , and OmpA was shifted towards a higher molecular mass in the ompB STOP ::tn compared with WT bacteria after ubiquitin enrichment (Fig. 4d ). To validate that OmpA is ubiquitylated, we overexpressed 6x-His-tagged ubiquitin in Vero, HMEC and A549 cells, and then assessed the association of His-tagged ubiquitin with bacteria by immunofluorescence microscopy or by affinity purification of His-ubiquitin followed by western blotting. His-ubiquitin co-localized with ompB STOP ::tn but not WT bacteria during infection ( Fig. 4e ; data from Vero cells). Furthermore, OmpA was detected in His-ubiquitin-enriched fractions from ompB STOP ::tn but not WT bacteria with a size shift that reflects ubiquitin chains of different lengths (Fig. 4f) . These data demonstrate that OmpB protects OmpA from ubiquitylation in diverse host cells. Based on the ubiquitylation of ompB STOP ::tn bacteria, we hypothesized that OmpB is important for avoidance of autophagic recognition. To test this, we first infected both HMECs and BMDMs and stained for the autophagic receptors p62 and NDP52 (Fig. 5a-d) . More than 30% of ompB STOP ::tn bacteria co-localized with p62 and NDP52 at 1 hpi in both cell types and at 72 hpi in HMECs, whereas fewer than 5% of WT bacteria were associated with these proteins (Fig. 5g,h) . Thus, OmpB is crucial for avoiding association with p62 and NDP52. We also assessed recruitment of LC3, a common marker for autophagic membranes. A subpopulation of ompB STOP ::tn bacteria was associated with LC3 in both BMDMs and HMECs, with a peak in LC3 association at 2 hpi and diminishing association thereafter, whereas WT bacteria showed little LC3 association (Fig. 5e , f, i and j; no difference in the numbers of cellular LC3-puncta was observed in cells infected with WT versus ompB STOP ::tn (Extended Data Fig.7a , b), and treatment with the proteasome inhibitor MG132 or the mTORC1 inhibitor rapamycin, which activate autophagy, did not increase the number of LC3-positive or pUb-positive ompB STOP ::tn (Extended Data Fig. 7c, d) ). Furthermore, almost all LC3-positive ompB STOP ::tn were polyubiquitin-positive ( This indicates that ubiquitylation is required for LC3 recruitment. Collectively, these data demonstrate that OmpB blocks ubiquitylation of R. parkeri to evade autophagic recognition in diverse host cells. To directly test if OmpB is critical to evade antimicrobial autophagy in macrophages, we employed BMDMs from mice genetically lacking the Atg5 or Becn1 genes in myeloid cells. (LAP) 43 . BMDMs were infected with ompB STOP ::tn and WT R. parkeri, and bacterial genomic DNA was quantified to assess growth during a 96-h infection. In control Atg5 +/+ (also known as Atg5 flox/flox ) 44 macrophages, ompB STOP ::tn was unable to grow whereas WT bacteria grew ~10-fold (Fig. 6a ). In contrast, in Atg5 −/− (Atg5 flox/flox -LysMcre + ) 45 macrophages, ompB STOP ::tn bacteria grew similarly to WT during the first 72 hpi (Fig. 6b) . The growth of ompB STOP ::tn was also rescued in Becn1 −/− (Beclin1 flox/flox -LysMcre + ) 46 macrophages (Fig. 6c) . Although the growth of the ompB STOP ::tn mutant was rescued in Atg5 −/− and Becn1 −/− cells, the mutant was polyubiquitylated (Extended Data Fig. 8a-c) , demonstrating that polyubiquitylation does not restrict bacterial growth when the autophagy cascade is prevented. We also tested for bacterial growth in Rubicon −/− macrophages 43 , which are specifically defective in LAP 43 , and growth of the ompB STOP ::tn mutant was not rescued (Extended Data Fig. 8d, e) . These results suggest that the growth of the ompB STOP ::tn mutant is restricted by autophagy but not by LAP. 17, 18 . Together, these results demonstrate that OmpB is critical for R. parkeri to evade destruction by autophagy in macrophages. OmpB is important for R. parkeri to colonize mouse organs due to its ability to promote autophagy evasion in immune cells We next sought to determine whether and how OmpB may contribute to R. parkeri virulence in vivo. We used C57BL/6 mice for these studies because of the availability of mutants in this strain carrying mutations in immunity genes. We injected WT R. parkeri intravenously into the tail-vein of mice which were then sacrificed each day over a 7 d period, and the number of plaque-forming units (PFU) was determined in spleen, lung, liver, and kidney. PFUs were recovered from all these organs, although the mice were asymptomatic and bacteria were cleared between days 4 and 7 (Extended Data Fig. 10a ). Next, we infected mice with WT or ompB STOP ::tn bacteria and we were unable to recover any ompB STOP ::tn from any organ within the window of 2 to 72 hpi (Fig. 6g) . Because R. conorii OmpB had been implicated in serum resistance 47 , we also tested if the ompB STOP ::tn mutant could survive in blood ex vivo over a 6 h time course. The ompB STOP ::tn bacteria remained as infectious as WT over this time (Extended Data Fig. 10b ), suggesting that OmpB does not by itself impact the survival of R. parkeri in blood. These results show that OmpB is a R. parkeri virulence factor that is critical for establishing an infection in vivo. We next examined if OmpB enables autophagy evasion in vivo, similar to its role in cultured macrophages in vitro. To test this, we infected Atg5 −/− (Atg5 flox/flox -LysMcre + ) mice, which only lack Atg5 in myeloid cells including macrophages, as well as control Atg5 +/+ (Atg5 flox/flox ) and C57BL/6 mice, with WT and ompB STOP ::tn bacteria. We observed a 5-16-fold rescue of the number of ompB STOP ::tn bacteria in the spleens and livers of Atg5 −/− mice compared to Atg5 +/+ and C57BL/6 mice, and the mutant reached levels that were not significantly different from those of WT (Fig. 6h) . These findings demonstrate that OmpB plays a major role in R. parkeri colonization of mice because of its ability to promote autophagy evasion in immune cells. Bacterial pathogens that grow in the cytosol of host cells often avoid recognition by innate immune pathways, including antimicrobial autophagy. Here, we demonstrate that R. parkeri surface protein OmpB is critical for protecting against autophagic recognition by blocking the formation of a polyubiquitin coat. We further show that OmpB is critical for R. parkeri growth in cultured macrophages via its role in autophagy evasion. Finally, our data suggest that OmpB is critical for R. parkeri to colonize mouse organs due to its ability to promote autophagy evasion in vivo. Together, these data indicate that OmpB acts as a protective shield to inhibit antimicrobial autophagy and as a critical virulence factor in Rickettsia pathogenesis. Our work also demonstrates that OmpB protects the bacterial surface protein OmpA from ubiquitylation, establishing OmpA as a specific bacterial target of the ubiquitylation machinery. Additional rickettsial surface proteins such as OmpW are also likely ubiquitylated targets that contribute to ubiquitin coat formation. Interestingly, OmpA is a conserved outer membrane protein in Gram-negative bacteria, and it was previously identified as a candidate ubiquitylated protein on Salmonella enterica Typhimurium 48 . Thus, we hypothesize that OmpA may be a common bacterial target for the host ubiquitin machinery. We further show that OmpB protects R. parkeri from polyubiquitylation by acting locally on the bacterial surface, rather than globally on the host ubiquitylation machinery. However, it remains unclear exactly how OmpB exerts its protective activity, and the mechanisms could be multifaceted. For example, it is possible that OmpB has deubiquitylase activity. Alternatively, OmpB may camouflage the bacterial surface, either directly and/or via its role in formation of a capsule-like halo. In support of these hypotheses, OmpB makes up more than 10% of the cellular protein mass 35 and is thus abundant enough for a camouflaging role. Moreover, a role for the OmpB-dependent capsule-like halo would be consistent with speculation that the capsule O-antigen shields Francisella tularensis from ubiquitylation 23 . A third possibility is that OmpB recruits host proteins that camouflage the bacterial surface or cleave polyubiquitin chains. A fourth possibility is that OmpB possesses an enzymatic activity that alters properties of the bacterial surface required for recognition by the host ubiquitin machinery. Our data suggest that OmpB is required for R. parkeri to evade autophagy in immune cells during initial colonization of mice. However, it remains unclear whether OmpB protects R. parkeri from autophagy during long-term infections in vivo. Although we find that OmpB is not required for autophagy avoidance in endothelial cell lines in vitro, it was previously observed that rickettsiae can be found in autolysosomes of cytokine-treated mouse endothelial cells 49 , suggesting that bacteria could be targeted by autophagy during long-term infection following release of inflammatory cytokines 50 . OmpB appears to act differently from other pathogen proteins that protect against polyubiquitylation. Form example, the L.monocytogenes surface protein ActA blocks polyubiquitylation by mobilizing the host actin machinery to the bacterial surface, which is required to protect the bacterium from autophagic recognition 11, 25, 51 . In contrast, OmpB has no effect on host actin mobilization by R. parkeri. In addition, a sub-population of WT L. monocytogenes is polyubiquitylated during intracellular growth 11, 22, 52 , whereas nearly all R. parkeri are polyubiquitin-negative, suggesting that OmpB is a more robust shield, perhaps because its mechanism may not involve recruitment of host components such as the actin machinery. Thus, although cytosolic pathogens have evolved a common strategy of using abundant surface proteins to block host polyubiquitylation, the mechanism by which each protein acts as a shield is distinct. Understanding the commonalities and differences in how pathogen proteins protect against polyubiquitylation and autophagy will enhance our understanding of pathogenic strategies for avoidance of innate immunity, and might be coopted as a therapeutic strategy for treating infectious disease. HMEC-1, A549, and Vero cells were purchased from the UC Berkeley Cell Culture Facility and were authenticated by short-tandem repeat analysis. All cells were grown at 37°C and 5% CO 2 . HMECs were grown in MCDB 131 media containing 10 mM L-glutamine (Sigma, M8537), supplemented with 10% heat-inactivated fetal bovine serum (Hi-FBS, Hyclone), 10 ng/mL epidermal growth factor (EGF) (Fisher Scientific, CB40001; Corning, 354001), 1 μg/mL hydrocortisone (Spectrum Chemical, CO137), and 1.18 mg/mL sodium bicarbonate. Fresh HMEC media was prepared every 1-2 months, and aliquoted and stored at 4°C prior to experiments or to passaging cells to minimize degradation of EGF. A549 cells were grown in DMEM (Gibco, 11965-092) with high glucose (4.5 g/L) and 10% Hi-FBS (Atlas Biologicals). Vero cells were grown in DMEM (Gibco, 11965-092) with high glucose (4.5 g/L) and 2% Hi-FBS (Benchmark). All cell lines were confirmed to be mycoplasmanegative by DAPI staining and fluorescence microscopy screening at the UC Berkeley Cell Culture Facility. Wild-type BMDMs were generated from the femurs of female C57BL/6 mice (Charles River Laboratories, Wilmington, MA) according to standard protocols 54 . Briefly, flesh was removed and bones were washed in 70% ethanol and then with BMDM media (DMEM high glucose; Gibco, 11965-092) containing 20% Hi-FBS (Hyclone), 10% supernatant from NIH 3T3 fibroblasts that produced macrophage colony-stimulating factor (M-CSF), 1% sodium pyruvate (Gibco, 11360-070), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco), and 0.1% β-mercaptoethanol (Gibco, 21985-023). Next, bones were crushed with a mortar and pestle and cells were filtered through a 70-μm nylon Corning Falcon cell strainer (Thermo Fisher Scientific, 08-771-2) to remove particulates. Cells were centrifuged for 8 min at 290 x g, 4°C, and the cell pellet was resuspended in BMDM media and incubated at 37°C for 7 d prior to harvesting in media lacking antibiotics. The percentage of CD11b(+)F4/80(+) BMDMs was determined by blocking cells with antimouse CD16/32 (Biolegend, 101320) and then staining with PE-Cyanine7 anti-mouse F4/80 antigen antibody (Tonbo Bioscience, 604801, dilution 1:200), APS anti-mouse CD11b (Tonbo Bioscience, 200112, dilution 1:250) and DAPI (dilution 1:1000). Samples were subjected to flow cytometry using a BD LSRFortessa 2010 Cell Analyzer (BD Biosciences) and data was analyzed using FlowJo software (FlowJo, LLC). Single viable DAPI-negative cells were gated and the percentage of CD11b(+)F4/80(+) macrophages was determined to be 99.4% ( Supplementary Fig. 1 For R. parkeri propagation, five T175 flasks of Vero cells growing in DMEM plus 2% Hi-FBS were infected with a multiplicity of infection (MOI) of ~0.01 and incubated until 50-75% of the 740 cells were infected (after 5-7 days (d). Cells were scraped into the growth media, collected in cold centrifuge bottles, and centrifuged at 12,000 x g for 20 min at 4°C. The cell pellet was resuspended in cold K-36 buffer (0.05 M KH 2 PO 4 , 0.05 M K 2 HPO 4 , pH 7, 100 mM KCl, 15 mM NaCl) and transferred to a cold dounce homogenizer (tight fit). To release bacteria, cells were dounce-homogenized for 60 strokes, and the homogenate was centrifuged at 200 x g for 5 min at 4°C to remove cell debris. To yield "30%-purified" bacteria, the supernatant was overlaid onto cold 30% MD-76R (Mallinckrodt Inc., St Louis, 1317-07), diluted in K-36 buffer, and centrifuged at 58,300 x g for 20 min at 4°C in an SW-28 swinging bucket rotor. The pellet was resuspended in cold brain heart infusion (BHI) (BD Difco, 237500) broth (0.5 mL per infected T175 flask) and subsequently aliquoted and frozen at −80°C. For further purification, to yield "gradient-purified" bacteria, bacteria were overlaid on a 40/44/54% MD-76R step gradient and centrifuged at 58,300 x g for 30 min at 4°C using a SW-28 swinging bucket rotor. Bacteria were collected from the 44-54% interface and subsequently pelleted by centrifugation at 12,000 x g for 15 min at 4°C. The pellet was resuspended in BHI and subsequently aliquoted and frozen at −80°C. In all experiments, fresh vials of bacteria were used to avoid experimental variations due to loss of bacterial infectivity upon freezing-thawing. Stocks of WT and ompB STOP ::tn mutant bacteria were prepared every ~6 months, and side-by-side experimental comparisons were made between stocks prepared at similar times. To enumerate infectious bacteria, purified bacterial strains were serial-diluted in DMEM with 2% Hi-FBS and used to infect Vero cells grown in 6-well plates by centrifugation at 300 x g for 5 min, at room temperature. Infections were allowed to proceed at 33°C overnight (~16 h) and then cells were overlaid in DMEM with 10% Hi-FBS plus 0.5% (w/v) UltraPure agarose (Invitrogen, 16-500). At 5-7 d post infection, when plaques could be visualized, cells were further overlaid with PBS plus 0.5% UltraPure agarose containing 0.0125% (v/v) neutral red (Sigma, N6264) and incubated at 33°C. The next day plaques were counted to determine the number of plaque-forming units (PFU) per mL. For bacterial growth curves, 30%-purified bacteria were diluted in the appropriate media to an MOI of 0.005-0.01 for infection of HMECs and 0.25-0.5 for infection of BMDMs (the higher MOI for BMDMs was to compensate for the reduced bacterial growth rate in these cells). R. parkeri infections were carried out in 24-well plates. Bacteria were centrifuged onto cells at 300 x g for 5 min at room temperature, then incubated at 33°C for 1 h, at which point the culture media was exchanged. At various times post infection, including the 1 h time point, cells were scraped off into the media (to include both intracellular and extracellular bacteria), centrifuged 20,000 x g for 5 min at room temperature, and pellets were stored at −20°C until DNA was purified using DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's instruction for Gram-negative bacteria. R. parkeri genome equivalents were quantified by quantitative PCR (pPCR) using primer 5'-CGCCATTCTACGTTACTACC-'3 and 5'-GCATTACTTGGAGTTCT-'3 to the hrtA gene, relative to a standard curve of hrtA gene DNA 55 . Growth was normalized to values at 1 h for each strain. R. parkeri ompA::tn, hrtA::tn, sca2::tn and ompB::tn mutant strains were previously isolated in a screen for small plaque mutants 33, 38, 56 . The ompA::tn mutant lacked full-length OmpA as determined by western blotting using the mouse anti-OmpA 13-3 antibody (kindly provided by Dr. Ted Hackstadt) 29,57 (Extended Data Fig. 1f ). The MC1_RS05535::tn, MC1_RS05545::tn and rickCE::tn strains were isolated in an independent screen in which mutants were isolated without regard for plaque size. The genomic locations of transposon insertion sites for all mutants were determined by semi-random nested PCR and Sanger sequencing at the UC Berkeley DNA Sequencing Facility, as previously described 58 . Expanded strains were either verified by semi-random nested PCR or by PCR reactions that amplified the transposon insertion site using primers for flanking regions. The ompB STOP ::tn, MC1_RS02370 STOP ,ompB::tn and ompB PROM ::tn strains were isolated in a screen for mutations that suppress the slow-growth phenotype of ompB::tn. To obtain strains carrying suppressor mutations, two independent selections were carried out in which the ompB::tn strain was subjected to 10 serial passages in Vero cells growing in T75 flasks in DMEM with 2% Hi-FBS, with the length of each passage varying between 5 to 7 d, depending on the rate of bacterial growth. For each passage, cells were lysed by addition of 1 mL cold sterile water per 796 T75 flask for 1-2 min at room temperature. Subsequently, 1 mL of cold 2 x BHI was added to the lysed cells and ~10% of the suspension was added to DMEM plus 2% Hi-FBS, and this was used 798 to infect a fresh T75 flask of Vero cells. The remaining bacteria were frozen at −80°C. To map suppressor mutations in the mixed population, clonal strains were isolated and larger plaques were selected and expanded in Vero cells, as described above. The genome sequences of 3 strains from each suppressor screen, which had been passaged 4 to 5 times, were determined (NCBI Trace and Short-Read Archive; Sequence Read Archive (SRA) as accession number SRP154218), as was the sequence of the parental ompB::tn (individual accession number SRX4401163) and WT R. parkeri strains (SRX4401164), as described below. The strains were isogenic with the exception of the suppressor mutations in the following locations: the MC1_RS02370 gene, encoding a putative glycosyltransferase (the same mutations in 4 out of 6 clonal strains, SRX4401165); the promoter region of ompB (ompB PROM ::tn) (1 clonal strain, SRX4401166); and upstream of the transposon in the coding sequence of ompB, leading to a premature stop codon (ompB STOP ::tn) (1 clonal strain, SRX4401167) (See also Extended Data Fig. 1a-c) . Rickettsia genomic DNA was isolated from frozen bacterial stocks (~10 8 PFUs, stored in BHI) that were centrifuged at 16,000 x g for 5 min at room temperature and resuspended in Dulbecco's PBS (DPBS) with calcium and magnesium (Invitrogen, 14040) containing DNase reaction buffer and 100 U of recombinant RNase-free DNase 1 (Roche, 04716728001) to reduce host genomic DNA in the final sample, as described previously 59 . Intact bacteria were incubated at 37°C for 30 min and thereafter washed 2 x with DPBS. Subsequently, rickettsial genomic DNA was purified using the DNeasy Blood and Tissue kit (Qiagen), according to manufacturer's instruction for Gram-negative bacteria. To confirm that Rickettsia genomic DNA was largely intact, we verified that DNA fragments were mostly > 20,000 bp by agarose gel electrophoresis. The genome sequences of R. parkeri strains (3 strains from each suppressor screen, as well as parental ompB::tn and WT strains) were determined at the UC Berkeley Functional Genomics Laboratory, using single end 50 bp read lengths on the Illumina Hiseq2000 sequencing platform. Genome assembly as well as single nucleotide variant and indel identification were performed with CLC Genomics Workbench (QIAGEN) and Integrated Genomics Viewer (IGV) 60 . The R. parkeri str. Portsmouth genome (GenBank accession no. NC_017044.1) was used as the reference sequence. Mutations identified by genome sequencing were confirmed by PCR followed by Sanger sequencing at the UC Berkeley DNA Sequencing Facility. The sequence encoding amino acids 36-218 of OmpB (ompB aa36-218 ), a protein that lacks he signal peptide and stops at the same position as the fragment encoded in the ompB STOP ::tn suppressor strain (Extended Data Fig. 1c) , was amplified by PCR from R. parkeri genomic DNA. ompB aa36-218 was cloned into plasmid pSMT3, which encodes Nterminal 6xHis and SUMO tags 61 . 6xHis-SUMO-OmpB aa36-218 was expressed in E. coli strain BL21 codon plus RIL-Camr (DE3) (QB3 Macrolab, UC Berkeley) by induction with 0.1 mM isopropyl-β-D-thio-galactoside (IPTG) for 16 h at 16°C. Bacterial pellets were resuspended in lysis buffer (50 mM NaH 2 PO 4 , pH 8.0, 300 mM NaCl, 10 mM imidazole) and stored at −80°C. For protein purification, bacteria were thawed, lysozyme was added to 1 mg/mL (Sigma, L4919), and lysis was carried out by sonication. Lysate containing 6xHis-SUMO-OmpB 36-218 was incubated with Ni-NTA resin (Qiagen, 1018244), and bound proteins were eluted with a step gradient of 100, 250 and 500 mM imidazole (in lysis buffer, pH adjusted to 8.0). Fractions were analyzed by SDS-PAGE and those with the highest concentrations of 6xHis-SUMO-OmpB were pooled prior to desalting, using PD-10 desalting columns (GE Healthcare, 17085101) and equilibrated with lysis buffer lacking imidazole. To generate rabbit antibodies against OmpB, 0.8mg of purified 6xHis-SUMO-OmpB protein was sent to Pocono Rabbit Farm and Laboratory (Canadensis, PA), and immunization was carried out according to their 91-d protocol. To affinity-purify anti-OmpB antibodies, ompB aa36-218 was subcloned into plasmid pET1M, which encodes N-terminal 6xHis and maltose-binding 850 protein (MBP) tags. 6xHis-MBP-OmpB aa36-218 was expressed and induced as above for His-SUMO-OmpB aa36-218 . Bacterial pellets were resuspended in column buffer (50 mM NaH 2 PO 4 , pH 852 8.0, 300 mM NaCl, 1 mM EDTA and 1 mM DTT), and the fusion protein was purified by affinity chromatography on amylose resin (New England Biolabs, E8021L). Bound proteins were eluted n column buffer lacking EDTA and containing 10 mM maltose. 500μg of purified protein was coupled to NHSactivated Sepharose 4 Fast Flow resin (GE-Healthcare, 17090601) in ligand-coupling buffer (200 mM NaHCO 3 pH 8.3, 500 mM NaCl) overnight at 4°C. Resin was incubated with 10 mL of anti-OmpB serum diluted in binding buffer (20 mM Tris, pH 7.5), which was loaded on the resin and incubated for 3 h at 4°C and eluted with 100 mM glycine pH 2.5 and neutralized 62 . For western blotting of endogenous OmpB, OmpA, RickA, and polyubiquitin, 30%-purified or gradient-purified bacteria were boiled in 1 x SDS loading buffer (150 mM Tris pH 6.8, 6% SDS, 0.3% bromophenol blue, 30% glycerol, 15% β-mercaptoethanol) for 10 min. Then, 1 to 5 × 10 6 PFUs were resolved on an 8-12% SDS-PAGE gel and transferred to an Immobilon-FL PVDF membrane (Millipore, IPEL00010). Membranes were probed for 30-60 min at room temperature with antibodies as follows: affinity-purified rabbit anti-OmpB antibody (this study) diluted 1:10,000 in TBS-T (20 mM Tris, 150 mM NaCl, pH 8.0, 0.05% Tween 20 (Sigma, P9416)) plus 5% dry milk (Apex, 20-241); mouse monoclonal anti-OmpA 13-3 antibody diluted 1:20,000 in TBS-T plus 5% dry milk; affinity-purified rabbit anti-RickA antibody 63 diluted 1:1,000 in TBS-T plus 5% dry milk; or mouse monoclonal FK1 anti-polyubiquitin antibody (Enzo Life Sciences, BML-PW8805-0500) diluted 1:2,500 in TBS-T plus 2% BSA. Secondary antibodies were: mouse anti-rabbit horseradish peroxidase (HRP) (Santa Cruz Biotechnology, sc-2357), or goat anti-mouse HRP (Santa Cruz Biotechnology, sc-2005), both diluted 1:2,500 in TBS-T plus 5% dry milk. Secondary antibodies were detected with ECL Western Blotting Detection Reagents (GE, Healthcare, RPN2106) for 1 min at room temperature and developed using Biomax Light Film (Carestream, 178-8207). R. parkeri infections were carried out in 24-well plates with sterile circle 12-mm coverslips (Thermo Fisher Scientific, 12-545-80). For infecting HMECs, 2×10 5 cells were seeded into each well, and then cells were infected after 2 d. For infecting BMDMs, 4-5×10 5 cells were seeded into each well and infection was conducted after 16-20 h. To initiate infection, 30%purified bacteria were diluted in cell culture media at room temperature to an MOI of 0.005-0.01 for HMECs and of 0.25-0.5 for BMDMs. The higher MOI for the macrophage infection was employed due to the reduced bacterial growth rate in these cells. Bacteria were centrifuged onto cells at 300 x g for 5 min at room temperature and subsequently incubated at For immunofluorescence microscopy, infected cells were fixed for 10 min at room temperature in pre-warmed (37°C) 4% paraformaldehyde (Ted Pella Inc., 18505) diluted in PBS, pH 7.4, then washed 3 x with PBS. Primary antibodies were the following: for staining with the guinea pig polyclonal anti-p62 antibody (Fitzgerald, 20R-PP001; 1:500 dilution), mouse polyclonal anti-NDP52 antibody (Novus Biologicals, H00010241-B01P; 1:100 dilution), rabbit monoclonal anti-M1 (linear) polyubiquitin linkage-specific antibody (Millipore, MABS199, 1:250 dilution), rabbit monoclonal anti-K63 polyubiquitin linkagespecific antibody (Millipore, 05-1308, 1:250 dilution), human anti-K48 polyubiquitin linkage-specific antibody, clone 5B6 64 (final concentration 0.2 μg/mL), or anti-polyubiquitin FK1 antibody (1:250 dilution), cells were permeabilized with 0.5% Triton-X100 for 5 min prior to staining. For staining with the mouse monoclonal anti-LC3 antibody (NanoTools, 0260-100, clone 2G6; 1:500 dilution), the rabbit polyclonal anti-LC3 antibody (Novus Biologicals, NB100-2220SS, 1:250 dilution), the rat anti-mouse-LAMP1 antibody (Biolegend, 121609, dilution 1:250), the mouse anti-human-LAMP1 antibody (555801, BD Bioscience, dilution 1:25) or the rabbit polyclonal anti-OmpB antibody (this study; 1:1000 dilution), cells were post-fixed in 100% methanol for 5 min at room temperature. Cells were washed 3 x with PBS and incubated with the primary antibody diluted as indicated in PBS with 2% BSA for 30 min at room temperature. To detect the primary antibodies, secondary goat anti-rabbit Alexa-568 (Invitrogen, A11036), goat anti-mouse Alexa-568 (Invitrogen, A11004), goat anti-human Alexa-488 (Invitrogen, A11013), goat anti-rat Alexa-568 (Invitrogen, A11077) or goat anti-guinea pig Alexa-568 (Invitrogen, A11075) antibodies were incubated at room temperature for 30 min (all 1:500 in PBS with 2% BSA). Next, a rabbit anti-Rickettsia I7205 antibody (gift from Ted Hackstadt) 57 or a mouse monoclonal anti-Rickettsia 14-13 antibody (gift from Ted Hackstadt) 57 , both diluted 1:500, was used to detect R. parkeri. Secondary antibodies used were: Alexa 488 anti-rabbit antibody (Invitrogen, A11008; 1:500 dilution), Alexa 405 anti-rabbit antibody (Invitrogen, A31556), Alexa 488 anti-mouse antibody (Invitrogen, A11001; 1:500 dilution), or an Alexa 405 antimouse antibody (Invitrogen, A31553; 1:150 dilution). Actin filaments were visualized in samples fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton-X100 using Alexa 488 phalloidin (Life Technologies, P3457; 1:500 dilution) or Alexa 568 phalloidin (Life Technologies, A12380; 1:500 dilution). Images were captured as z-stacks (0.1-μm step size) on a Nikon Ti Eclipse microscope with a Yokogawa CSU-XI spinning disc confocal with 60X and 100X (1.4 NA) Plan Apo objectives, and a Clara Interline CCD Camera (Andor Technology) using MetaMorph software (Molecular Devices). Rendered Z-stacks were used for quantifications. Images were processed using ImageJ 65 and assembled in Adobe Photoshop. Image adjustments of each color were applied equally for all bacterial strains within each experiment. To prepare representative images, we used a single optical section, in which most or all bacteria were in that focal plane. The only exceptions were for representative images of LAMP1 or LC3/pUb staining, for which we used z stack average maximum intensity projections. For quantification of polyubiquitin, polyubiquitin linkages, p62 and NDP52 staining, only bacteria that co-localized with rim-like patterns of the respective marker were scored as positive for staining. For quantification of LC3 staining, bacteria that co-localized with either LC3 clouds or puncta were considered positive for staining. For macrophage infections that extended beyond 60 min, ompB STOP ::tn bacteria were occasionally not stained adequately with anti-Rickettsia antibodies, and therefore we included Hoechst (Sigma, B2261) staining in all experiments to detect bacterial genomic DNA. To determine bacterial internalization frequencies, infections were carried out as described above except that bacteria were diluted in media at 4°C to a MOI of 5 in 500 μL of cell culture media, which was added to each well, and centrifuged at 300 x g for 5 min at 4°C. For the 0-min time point, coverslips were transferred to 4% paraformaldehyde immediately after the centrifugation step, fixed for 10 min and washed 3 times with 1 x PBS, pH 7.4. To initiate invasion/internalization, 500 μL media at 37°C was added to each well and the plate was transferred to 33°C. At 5, 10, 15, 20, 30, 60, and 90 min, cells were fixed, as described above. For immunofluorescence staining of extracellular bacteria, rabbit anti-Rickettsia I7205 antibody (or rabbit anti-Rickettsia R2868 antibody (a gift from Ted Hackstadt) for the ompA::tn mutant) and a secondary Alexa 405-conjugated anti-rabbit antibody (Invitrogen, A31556) were used. To subsequently stain all bacteria (intracellular and extracellular), cells were permeabilized with 0.5% Triton-X100 (Sigma, T9284) for 5 min and the anti-Rickettsia I7205 antibody (or rabbit anti-Rickettsia R2868 antibody to detect the ompA::tn mutant) and a secondary Alexa 488-conjugated goat anti-rabbit antibody (Invitrogen, A11008) were used. Coverslips were mounted with Prolong Gold anti-fade (Invitrogen, P36930) and stored at 4°C prior to imaging. Imaging was carried out as described above, and the percentage of internalized bacteria was calculated by first dividing the number of extracellular bacteria by the total number of bacteria, multiplying by 100, and then subtracting this number from 100% to get the percentage of intracellular bacteria. For TEM experiments, infection of HMECs and BMDMs was performed as described above for internalization assays. At 1 hpi, media was removed and fixative (1.5% paraformaldehyde, 2.0% glutaraldehyde, 0.03% CaCl 2 , in 0.05 M cacodylate buffer pH 7.2) was added to each well for 45 min at room temperature, as previously described 66 . Samples were embedded in 2% low-melting agarose and incubated overnight at 4°C in 2.0% glutaraldehyde, 0.1 M cacodylate buffer, pH 7.2. The next day, samples were post-fixed in 1% osmium tetraoxide and 1.6% potassium ferricyanide, dehydrated in a graded series of ethanol concentrations, and embedded in EPON 812 resin (11.75 g of Eponate 12 (18005, Ted Pella Inc., Redding, CA), 6.25 g DDSA (18022, Ted Pella Inc., Redding, CA), 7 g NMA (18032, Ted Pella, Redding Inc., CA) were mixed prior addition of 0.375 ml of the embedding accelerator BMDA (18241, Ted Pella Inc., Redding, CA). Next, samples were stained with 2% uranyl acetate and lead citrate. Images were obtained on a FEI Tecnai 12 transmission electron microscope. The radius of the halo (r halo or halo thickness) was calculated by measuring the area of the bacterium plus halo (A total ) and bacterium minus halo (A bacterium ), assuming circularity and calculating the radius of each as r = √A/π, and then calculating r halo = r total − r bacterium 67 . To enrich for polyubiquitylated proteins, 4 × 10 8 PFUs of 30%-purified or 4 × 107 PFUs gradient-purified WT and ompB STOP ::tn bacteria were centrifuged at 20,000 x g for 3 min at room temperature. Next, to release the surface protein fraction, the bacterial pellets were resuspended in lysis buffer (50 mM Tris-HCI, pH 7.5, 150 mM NaCl, 1 mM EDTA and 10% glycerol), supplemented with 0.0031% v/v lysonase (Millipore, 71230), the deubiquitylase inhibitor PR619 at a final concentration of 20 μM (Life Sensor, SI9619) and 0.8% w/v octyl β-D-glucopyranoside (Sigma, O8001), and incubated on ice for 10 min. Subsequently, the lysate was cleared bycentrifugation at 14,000 x g at 4°C for 5 min and incubated with equilibrated agarose TUBE-1 (Life Sensor, UM401) for 4 h at 4°C. After binding of polyubiquitylated proteins to TUBE-1, agarose beads were washed 3 times with TBS (no Tween) and centrifuged at 5,000 x g for 5 min. To prepare samples for mass spectrometry (MS) analysis, enriched proteins were digested at 37°C overnight on agarose beads in RapiGest SF solution (Waters, 186001861) supplemented with ~0.5 μg trypsin (Promega, V511A). The reaction was stopped using 1% trifluoroacetic acid (Sigma, T6508). Octyl β-D-glucopyranoside was extracted using water-saturated ethyl acetate, as previously described 68 . Prior to submission of samples for MS analysis, samples were desalted using C18 OMIX tips (Agilent Technologies, A57003100), according to the manufacturer's instructions. Samples of proteolytically digested proteins were analyzed using a Synapt G2-Si mass spectrometer that was equipped with a nanoelectrospray ionization source (Waters). The mass spectrometer was connected in line with an Acquity M-class ultra-performance liquid chromatography system that was equipped with trapping (Symmetry C18, inner diameter: 180 μm, length: 20 mm, particle size: 5 μm) and analytical (HSS T3, inner diameter: 75 μm, length: 250 mm, particle size: 1.8 μm) columns (Waters). Data-independent, ion mobilityenabled, high definition mass spectra and tandem mass spectra were acquired in the positive ion mode [69] [70] [71] . Data acquisition was controlled using MassLynx software (version 4.1), and tryptic peptide identification and relative quantification using a label-free approach 72 databases to identify tryptic peptides, allowing for up to two missed proteolytic cleavages, with carbamidomethylcysteine as a fixed post-translational modification and methionine sulfoxide and diglycine-modified lysine (i.e., ubiquitylation remnant) as variable posttranslational modifications. To assess localization of 6xHis-ubiquitin during infection, confluent Vero cells grown in 24well plates with coverslips were transfected with ~2 μg of pCS2-6xHis-ubiquitin plasmid DNA using Lipofectamine 2000 (Invitrogen, 11668-019) for 6 h in Opti-MEM (Gibco, 31985-070). Subsequently, media was exchanged to media without transfection reagent, and cells were incubated overnight at 37°C, 5% CO 2 . The following day (~16 h after transfection), transfected cells were infected with WT or ompB STOP ::tn bacteria at a MOI of 1. At 24 hpi, infected cells were fixed with 4% paraformaldehyde (Ted Pella Inc., 18505) diluted in PBS, pH 7.4 for 10 min, then washed 3 x with PBS. Primary anti-6xHis monoclonal mouse antibody (Clontech, 631212, diluted 1:1,000) was used to detect 6xHisubiquitin in samples permeabilized with 0.5% Triton-X100, and a goat anti-mouse Alexa-568 (Invitrogen, A11004) to detect the primary 6xHis antibody. Hoechst (Thermo Scientific, 62249, diluted 1:2500) was used to detect bacterial DNA. Samples were imaged as already described. To validate OmpA as a polyubiquitylated substrate on ompB STOP ::tn bacteria, confluent Vero, A549 and HMEC cells grown in 6-well plates were transfected and infected as described above with the exception that HMECs and A549s were transfected for only 60 min. At 26 hpi, cells were washed once with 1 x PBS (this step was excluded for HMECs), pH 7.4, and subsequently lysed in urea lysis buffer (8 M urea, 50 mM Tris-HCI, pH 8.0, 300 mM NaCI, 50 mM Na 2 HPO 4 , 0.5% Igepal CA-630 (Sigma, I8896)) for 20 min at room temperature (for HMECs, the volume of lysis buffer was reduced 2x). Subsequently, samples were sonicated, and lysate was cleared by centrifugation at 15,000 x g for 15 min at room temperature. Prior to incubation with Ni-NTA resin, an aliquot was saved for the input sample. 6xHis-ubiquitin conjugates were purified by incubation and rotation with Ni-NTA resin for 4 h at room temperature. Beads were washed 3 x with urea lysis buffer and 1 x with urea lysis buffer lacking Igepal CA-630. Ubiquitin conjugates were eluted at 65°C for 15 min in 2 x Laemmli buffer containing 200 mM imidazole and 5% 2-mercaptoetanol (Sigma, M6250), vortexed for 1 min, and centrifugation at 5,000 x g for 3 min. Eluted and input proteins were detected by SDS-PAGE followed by western blotting, as described above. To assess bacterial membrane integrity, confluent HMEC cells were infected with 30%purified WT and ompB STOP ::tn bacteria, as described above, for invasion/internalization experiments. After centrifugation at 300 x g for 5 min at 4°C (considered the 0 min time point), cells were washed once in KHM buffer (110 mM CH 3 CO 2 K, 2 mM Mg(CH3COO) 2 and 20 mM HEPES, pH 7.2), then incubated in KHM buffer with or without 50 μg/ml digitonin (positive control) for 1 min, and subsequently incubated in 2.6 μM propidium iodide (Molecular Probes, P-3560) diluted in KHM buffer for 10 min at room temperature, as previously described 23, 74 . Cells were then washed once in KHM buffer, fixed in 4% paraformaldehyde, and stained for bacteria using the anti-Rickettsia 14-13 antibody and a secondary goat-anti-mouse Alexa-488 antibody (Invitrogen, A11004), as described above. Supernatants from infected or uninfected BMDMs were collected at 96 hpi and then mixed with 2x LDH buffer (100 μg/ml tetrazolium salt (Sigma I18377, in PBS), 150 μg/ml βnicotineaminde adenine dinucleotide hydrate (Sigma, N3014), 0.675 units/ml diaphorase (Sigma, D5540), 0.06% sucrose, 12 mg/ml lithium lactate (Sigma L2250, resuspended in 10 mM Tris HCl pH 8.5 to 36 mg/ml), 0.25% BSA in PBS). Supernatant from uninfected cells lysed with 1% Triton-X100 was used to calculate 100% lysis. Reactions were incubated at room temperature for 20 min prior reading at 490 nm absorbance using an Infinite F200 Pro plate reader (Tecan). Values were divided by the difference of Triton-X100 lysed cells and the experimental supernatants, and multiplied by 100, to calculate percent lysis. Female C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA), and 8-10-week-old littermates were used for each experiment. Groups that received different bacterial strains were randomly assigned. C57BL/6 Atg5 flox/flox mice were from Dr. N. Mizushima (Tokyo Medical and Dental University, Japan) 44 and C57BL/6 Atg5 flox/flox -LysMcre + were a generous gift from Dr. H. Virgin (University of Washington) 75 . Mice were genotyped as previously described 45 using primers exon3-1, short2, and check2 for detection of the Atg5 gene and primers cre1 and cre2 for detection of the cre gene. Randomly assigned 8-10-week-old male and female C57BL/6, Atg5 flox/flox , and Atg5 flox/flox -LysMcre+ mice were used in the experiments comparing these genotypes. No data blinding was performed. The sample size for mice experiments was chosen based on the results between two groups after the first experiment, in which 2-3 mice per group were used. Mice were housed in specific pathogen-free conditions and euthanized according to standard protocols approved by UC Berkeley Animal Care and Use Committee. Mice were infected with 10 7 PFU of WT or ompB STOP ::tn diluted into 200 μL of 1 x PBS, pH 7.4 (Gibco, 1001023) via injection into the lateral tail vein. Immediately after injection, to confirm the infectivity and titer of the bacterial strains, bacteria were enumerated using a plaque assay, as described above. At 2, 24, 48, and 72 hpi, mice were sacrificed, doused with ethanol, laid on the right side, and the spleen, liver, lung, and kidney were removed and transferred into cold sterile PBS. Organs were disrupted in a homogenizer (Polytron) for ~10 seconds and centrifuged at 200 x g for 5 min. Supernatants were collected to assess the PFUs per organ using a plaque assay, as described above (Fig. 6g) . Due to moderate toxicity of the organ supernatants on Vero cells (particular from the liver), we optimized the protocol as follows. Instead of centrifuging the homogenized organs at 200 x g for 5 min, we employed 300 x g for 5 min. Subsequently, instead of centrifugation of supernatants at 300 x g for 5 min, which we normally use for R. parkeri infections, supernatants were centrifuged at 230 x g for 5 min. These minor changes reduced the toxic effects on Vero cells, leading to the detection of ompB STOP ::tn PFUs and increased WT PFUs (Fig. 6h) . To determine bacteria viability in mouse blood, we collected blood from the diaphragm cavity of female mice upon dissection. Next, 2 × 10 5 PFUs of WT or the ompB STOP ::tn strain were added to 40 μL of fresh blood and incubated at 37°C. At 0, 20, 60, 120, 180, and 360 min, samples were resuspended and 5 μL was taken for PFU determination using the plaque assay, as described above. PFUs were normalized to 0 min for each strain. Statistical parameters and significance are reported in the figure legends. Data were considered to be statistically significant when p < 0.05, as determined by unpaired Student's t-test, Mann-Whitney rank-sum test, or a one-way ANOVA with Tukey's post-test. Statistical analysis was performed using PRISM 6 software (GraphPad Software). The datasets generated during and/or analysed during the current study are available from the corresponding authors upon request. The genome sequences of bacterial strains are available at Sequence Read Archive (SRA) as accession number SRP154218 (WT: SRX4401164; ompB::tn: SRX4401163; ompB STOP ::tn: SRX4401167; ompB PROM ::tn; SRX4401166; MC1_RS02370 STOP , ompB::tn: SRX4401165). Engström et al. 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, probed for OmpA or polyubiquitin (FK1). The asterisks* indicate OmpA from 6xHisubiquitin-expressing cells infected with ompB STOP ::tn exhibits higher molecular weights than endogenous OmpA, consistent with ubiquitylation (Vero, n = 4; HMEC, n = 2; A549, n = 2). (a-f) Immunofluorescence micrographs of (a, c, e) HMECs or (b, d, f) BMDMs infected with WT (upper panels) or ompB STOP ::tn mutant (lower panels) at 1 hpi, and stained for R. parkeri (green; anti-Rickettsia I7205 antibody), cellular and bacterial genomic DNA (blue; Hoechst), and (a, b) p62 (red; anti-p62 antibody), (c, d) NDP52 (red; anti-NDP52 antibody), or (e, f) LC3 (red; anti-LC3 antibody). Right panels show merged images. Arrows indicate ompB STOP ::tn bacteria positive for p62, NDP52, or LC3. The star in (e) indicates LC3 puncta that co-localize with ompB STOP ::tn. Scale bars, 5 μm. Image adjustments of each autophagic marker, R. parkeri, and DNA, were applied equally for both bacterial strains and cell types (exception, LC3, between HMECs and BMDMs). (g, h) Quantification of the percentage of WT or ompB STOP ::tn mutant bacteria that localized with (g) p62 or (h) Natural history of tick-borne spotted fever in the USA. Susceptibility of small mammals to virulent Rickettsia rickettsii Rickettsia parkeri Transmission to Amblyomma americanum by Cofeeding with Amblyomma maculatum 491(Acari: Ixodidae) and The rice rat Euryoryzomys russatus, a competent amplifying host of Rickettsia parkeri strain Atlantic rainforest for the tick Amblyomma ovale Emerging and re-emerging rickettsioses: endothelial cell infection and early disease events Rickettsia parkeri rickettsiosis and its clinical distinction from Rocky Mountain spotted fever Differential interaction of dendritic cells with Rickettsia conorii: impact on host susceptibility to murine spotted fever rickettsiosis Nonselective Persistence of a Rickettsia conorii Extrachromosomal Plasmid during Mammalian Infection Differences in Intracellular Fate of Two Spotted Fever Group Rickettsia in Macrophage-Like Cells Self and nonself: how autophagy targets mitochondria and bacteria Molecular definitions of autophagy and related processes Recognition of bacteria in the cytosol of Mammalian cells by the ubiquitin system Fluorescence-based sensors to monitor localization and functions of linear and K63-linked ubiquitin chains incells Linear ubiquitination of cytosolic Salmonella Typhimurium activates NF-kappaB and restricts bacterialproliferation The adaptor protein p62/SQSTM1 targets invading bacteria to the autophagy pathway p62 and NDP52 proteins target intracytosolic Shigella and Listeria to different autophagy pathways The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria Induction of autophagy and inhibition of tumorigenesis by beclin 1 Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network A ubiquitin-like system mediates protein lipidation The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy Bacteria-autophagy interplay: a battle for survival Listeria monocytogenes ActA-mediated escape from autophagic recognition The Francisella O-antigen mediates survival in the macrophage cytosol via autophagy avoidance Escape of intracellular Shigella from autophagy Actin-based motility allows Listeria monocytogenes to avoid autophagy in the macrophage cytosol Restriction of the growth of a nonpathogenic spotted fever group rickettsia Atg5 Supports Rickettsia australis Infection in Macrophages In Vitro and In Vivo Characterization of monoclonal antibodies protecting mice against Rickettsia rickettsii Targeted knockout of the Rickettsia rickettsia OmpA surface antigen does not diminish virulence in a mammalian model system Evidence for proteolytic cleavage of the 120-kilodalton outer membrane protein of rickettsiae: identification of an avirulent mutant deficient in processing Disruption of the Rickettsia rickettsii Sca2 autotransporter inhibits actin-based motility Rickettsia Sca2 is a bacterial forminlike mediator of actin-based motility Rickettsia actin-based motility occurs in distinct phases mediated by different actin nucleators The 17-kilodalton protein antigens of spotted fever and typhus group rickettsiae Partial purification and characterization of the major species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii identified by rocket immunoelectrophoresis Differential activity of Rickettsia rickettsii ompA and ompB promoter regions in a heterologous reporter gene system Evaluation of changes to the Rickettsia rickettsii transcriptome during mammalian infection A streamlined method for transposon mutagenesis of Rickettsia parkeri yields numerous mutations that impact infection The Molecular Basis for Ubiquitin and Ubiquitin-like Specificities in Bacterial Effector Proteases Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis Comparative ultrastructural study on the cell envelopes of Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia tsutsugamushi External layers of Rickettsia prowazekii and Rickettsia rickettsii: occurrence of a slime layer Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice Coronavirus replication does not require the autophagy gene ATG5 Autophagy is a cell survival program for female germ cells in the murine ovary The rickettsial OmpB beta-peptide of Rickettsia conorii is sufficient to facilitate factor H-mediated serum resistance Global Analysis of Host and Bacterial Ubiquitinome in Response to Salmonella Typhimurium Infection Cytokine-induced, nitric oxide-dependent, intracellular antirickettsial activity of mouse endothelial cells Immune response against rickettsiae: lessons from murine infection models Listeria monocytogenes ActA is a key player in evading autophagic recognition Strain-Specific Interactions of Listeria monocytogenes with the Autophagy System in Host Cells TLR signaling is required for Salmonella typhimurium virulence Avoidance of autophagy mediated by PlcA or ActA is required for Listeria monocytogenes growth in macrophages Susceptibility of inbred mice to Rickettsia parkeri Rickettsia Sca4 Reduces Vinculin-Mediated Intercellular Tension to Promote Spread Reactivity of monoclonal antibodies to Rickettsia rickettsii with spotted fever and typhus group rickettsiae Expression of an epitope-tagged virulence protein in Rickettsia parkeri using transposon insertion Genome sequence of the endosymbiont Rickettsia peacockii and comparison with virulent Rickettsia rickettsii: identification of virulence factors Integrative Genomics Viewer (IGV): highperformance genomics data visualization and exploration The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase Purification of antibodies on an antigen column A Rickettsia WASP-like protein activates the Arp2/3 complex and mediates actinbased motility Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control NIH Image to ImageJ: 25 years of image analysis Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of Sao Paulo, Brazil, where Brazilian spotted fever is endemic Capsule growth in Cryptococcus neoformans is coordinated with cell cycle progression Rapid detergent removal from peptide samples with ethyl 666 acetate for mass spectrometry analysis Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics UPLC/MS(E); a new approach for generating molecular fragment information for biomarker structure elucidation Effects of traveling wave ion mobility separation on data independent acquisition in proteomics studies Tools for label-free peptide quantification Less label, more free: approaches in label-free quantitative mass spectrometry Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication Autophagosome-independent essential function for the autophagy protein Atg5 in cellular immunity to intracellular pathogens the halo) in HEMCs at 1 hpi and 72 hpi, and in BMDMs at 1 hpi. All data points are presented, and the lines indicate the means of Author manuscript; available in PMC Data are mean (n = 2; ≥91 bacteria were counted for the 1 hpi time point for each experiment; ≥350 bacteria were counted for the 72 hpi time point for each experiment). (i, j) Quantification of the percentage of WT or ompB STOP ::tn mutant bacteria that localized with LC3 in (i) BMDMs or (j) HMECs at the indicated times post infection HMECs 2 h, n = 2; 20 min and 6 h, n = 3strain, infection and time-point 001). (k) Quantification of the percentage LC3-postive ompB STOP ::tn mutant bacteria that co-localize with polyubiquitin at 2.5 and 4 hpi in BMDMs and HMECs. Data are mean ± SEM (BMDMs, n = 3; ≥74 bacteria were counted for each strain All data points are shown, and medians are indicated as bars (for WT infected mice at 2 h, n = 6 Note that the limit of detection was different between liver and the other organs due to the fact that concentrated liver homogenate interfered with PFU determination All data points are shown, and medians are indicated as bars Refer to Web version on PubMed Central for supplementary material.