Rickettsial outer-membrane protein B (rOmpB) mediates bacterial invasion through Ku70 in an actin, c-Cbl, clathrin and caveolin 2-dependent manner

Abstract

Rickettsia conorii, an obligate intracellular tick-borne pathogen and the causative agent of Mediterranean spotted fever, binds to and invades non-phagocytic mammalian cells. Previous work identified Ku70 as a mammalian receptor involved in the invasion process and identified the rickettsial autotransporter protein, rOmpB, as a ligand; however, little is known about the role of Ku70-rOmpB interactions in the bacterial invasion process. Using an Escherichia coli heterologous expression system, we show here that rOmpB mediates attachment to mammalian cells and entry in a Ku70-dependent process. A purified recombinant peptide corresponding to the rOmpB passenger domain interacts with Ku70 and serves as a competitive inhibitor of adherence. We observe that rOmpB-mediated infection culminates in actin recruitment at the bacterial foci, and that this entry process relies in part on actin polymerization likely imparted through protein tyrosine kinase and phosphoinositide 3-kinase-dependent activities and microtubule stability. Small-interfering RNA studies targeting components of the endocytic pathway reveal that entry by rOmpB is dependent on c-Cbl, clathrin and caveolin-2. Together, these results illustrate that rOmpB is sufficient to mediate Ku70-dependent invasion of mammalian cells and that clathrin- and caveolin-dependent endocytic events likely contribute to the internalization process.

Figure 1. Surface expression of recombinant epitope tagged rOmpB in E. coli

A. Schematic of the pET-22b expression vector showing the relevant features on the 5′ and 3′ ends of the ompB gene. The vector encodes an N-terminal E. coli PelB signal sequence to direct fusion proteins through the Sec secretion pathway, as well as a C-terminal 6xHis tag. B. Western immunoblot analysis of (1) whole cell bacterial lysate, (2) soluble and inner membrane and (3) outer membrane fractions using anti-6xHis rabbit sera (top panel) or an anti-R. conorii rabbit hyper-immune sera (αRc7, bottom panel). The plasmid pYC9 encodes the full-length rOmpB from R. conorii, while pET22-RJPOB encodes the full-length rOmpB from R. japonica. Arrows denote the full-length rOmpB species. C. Immunofluorescence staining for surface-exposed rOmpB in uninduced (-IPTG) or induced (+IPTG) E. coli using an anti-R. conorii rabbit hyper-immune sera (αRc7, bottom panels). Top row panels show phase images of the E. coli. Propidium iodide staining (PI, middle row panels) was used to confirm that positive αRc7-staining was not a result of increased bacterial membrane permeabilization. Scale bars = 1 μm.

Figure 2. Expression of rOmpB in E. coli sufficiently mediates association to cultured mammalian cells

A. Fluorescence micrographs of monolayers of HeLa cells infected with E. coli BL21(DE3) expressing the empty vector (pET-22b) and full-length rOmpB proteins from R. conorii (pYC9) or R. japonica (pET22-RJPOB). HeLa cells seeded at 90% confluency were infected for 20 min at 37°C, then washed repeatedly with PBS and processed for immunofluorescence. Scale bars = 10 μm. B. Colony forming unit-based quantification of bacterial adherence on host epithelial cells. Confluent monolayers of HeLa cells were infected for 20 min at 37°C with induced cultures of E. coli BL21(DE3) as in (A). Cell-associated bacteria were extracted from host cells by detergent lysis, and plated for colony forming units (cfu). Association was determined as the percent cfu of cell-associated bacteria from the initial bacterial inoculums. P-values were determined by a two-tailed Student’s t-test, *P < 0.05.

Figure 3. rOmpB expression in E. coli mediates invasion of cultured mammalian cells

A. Scanning electron microscopy (SEM) examining the surface interaction of rOmpB-expressing E. coli with HeLa cells. HeLa cell monolayers on glass coverslips were infected with for 1 h, and then processed for SEM. White arrowheads highlight possible rOmpB-mediated cellular membrane rearrangements. Scale bars = 500 nm. B. Transmission electron microscopy (TEM) of internalized rOmpB-expressing E. coli in HeLa cells. HeLa cell monolayers were infected for 2 h with E. coli BL21(DE3) expressing rOmpB, then processed and visualized by TEM. Inset: enlargement of the internalized E. coli. Scale bars are as noted (2 _m, 0.5 _m [inset]). C. Bacterial invasion of HeLa cells. HeLa cell monolayers were infected for 1 h with E. coli BL21(DE3) expressing the empty vector (pET-22b) or rOmpB (pYC9 or pET22-RJPOB) and assessed for invasion using the gentamicin protection assay. Invasion is presented as the percent of bacteria recovered after the gentamicin challenge out of the inoculums. Actual percentages varied from assay to assay (ranging from 0.1 to .5%) depending on the passage number of mammalian cells used and the expression of rOmpB on the E. coli outer membrane. P-values were determined by a two-tailed Student’s t-test, *P < 0.05.

Figure 4. rOmpB-mediated invasion of mammalian cells is dependent on Ku70 expression

A. Effect of Ku70 siRNA-depletion on rOmpB-mediated cellular association. HeLa cells transfected with irrelevant control (black bars) or Ku70 (gray bars) siRNAs were infected with E. coli BL21(DE3) expressing the empty vector (pET-22b) or alleles of full-length rOmpB (pJJM104 or pET22-RJPOB) and assayed for bacterial association. Dashed dark gray line shows non-specific levels of association (as a percent of rOmpB-mediated association in control-siRNA transfected cells) seen with E. coli expressing the empty vector. Inset: immunoblot analysis of Ku70 protein levels in (1) control and (2) Ku70 siRNA-transfected HeLa cells. The anti-β-actin immunoblot serves as a protein loading control. B. Effect of Ku70 siRNA-depletion on rOmpB-mediated cellular invasion. HeLa cells transfected with an irrelevant control (black bars) or Ku70 (gray bars) siRNAs were infected with E. coli BL21(DE3) expressing the empty vector (pET-22b) or alleles of full-length rOmpB (pJJM104 or pET22-RJPOB) and assayed for bacterial invasion. Dashed dark gray line shows non-specific levels of invasion (as a percent of rOmpB-mediated invasion in control-siRNA transfected cells) seen with E. coli expressing the empty vector. Inset: Immunoblot analysis of Ku70 protein levels in (1) control and (2) Ku70 siRNA-transfected HeLa cells. The anti-β-actin immunoblot serves as a protein loading control.

Figure 5. The purified recombinant rOmpB passenger domain, GST-OmpB_36–1334 competitively inhibits rOmpB-mediated adherence and interacts with Ku70

A. Coomassie-stained SDS-PAGE of GST-rOmpB_36–1334 and GST proteins expressed and purified under native conditions from the IPTG-inducible pGEX expression system in E. coli TOP10. Purifications were performed from bacteria harboring the empty vector pGEX-2TKP or the gene encoding the R. conorii passenger domain, pGEX-ompB_106–4001. Fractions shown include the cleared bacterial high-speed supernatant (HSS), flow-throw (FT), washes (W) and elutions (E1, E2 and E3). B. Phase contrast and fluorescence micrographs of HeLa cells incubated with purified GST-rOmpB_36–1334 (top panels) or GST alone (bottom panels). Cell nuclei were stained with DAPI, and GST-tagged proteins with rabbit anti-GST and AlexaFluor 488-conjugated goat anti-rabbit IgG. C. rOmpB-mediated adherence in the presence of exogenous GST-rOmpB_36–1334. Confluent monolayers of HeLa cells pre-incubated for 15 min with PBS, or 100 μg/ml of GST or GST-rOmpB_36–1334 in serum-free media were infected with E. coli BL21(DE3) expressing the R. conorii or R. japonica rOmpB allele (pYC9 and pET22-RJPOB, respectively) then assayed for bacterial association. P-values were determined using a two-tailed Student’s t-test, *P < 0.05. D. GST or GST-rOmpB_36–1334 coupled to glutathione-agarose beads were incubated with detergent-extracted HeLa cell lysates, washed, pelleted and resolved by SDS-PAGE for analysis of Ku70 association by immunoblotting using anti-Ku70 sera. E. Purified 10xHis-Ku70_1–609 (1) or GST (2) and GST-rOmpB_36–1334 (3) coupled to glutathione-sepharose beads were resolved by SDS-PAGE and assessed for relative purity by coomassie staining (left panel). GST and GST-rOmpB_36–1334 coupled to glutathione-sepharose beads were incubated with 5 _g of purified 10xHis-Ku70_1–609, then washed, pelleted and resolved by SDS-PAGE. Ku70 association was determined by immunoblot analysis using anti-Ku70 mouse sera (right panel). *Denotes full-length recombinant Ku70 construct. Smaller species are all αHis-reactive Ku70 C-terminal truncations.

Figure 6. Actin involvement in rOmpB-mediated invasion

HeLa cell monolayers on glass coverslips were infected for 1 h with E. coli BL21(DE3) expressing R. conorii rOmpB (pYC9) in A and E. coli BL21(DE3) expressing R. japonica rOmpB (pET22-RJPOB) in B. Cells were washed thoroughly with PBS, fixed, and then processed for immunofluorescence staining of extracellular bacteria and cellular actin. Images represent an individual confocal slice from a Z-stack capture. White arrowheads depict sites of actin recruitment around rOmpB-expressing bacteria. C. Confluent monolayers of HeLa cells pre-treated for 30 min with DMSO or inhibitors of the actin cytoskeleton (cytochalasin D), phospho-tyrosine kinases (genistein), microtubules (nocodazole) and phosphoinositide kinases (wortmannin) at the indicated concentrations were infected with E. coli BL21(DE3) expressing the R. conorii rOmpB (pJJM104), and subsequently assessed for bacterial invasion using the gentamicin protection assay. Levels of invasion are displayed as a percent of the rOmpB-mediated invasion seen in DMSO-treated cells. Red dashed line denotes background levels of invasion seen in vector control infection. D. HeLa cells were infected with E. coli BL21(DE3) expressing the R. japonica rOmpB (pET22-RJPOB) as described in (C). Levels of invasion are displayed as a percent of the rOmpB-mediated invasion seen in DMSO-treated cells. Red dashed line denotes invasion background seen in vector control infection.

Figure 7. rOmpB-mediated invasion of mammalian cells is dependent on c-Cbl, clathrin, and caveolin-2

A. Effect of c-Cbl siRNA-depletion on rOmpB-mediated invasion. HeLa cells transfected with control (black bars), Ku70 (gray bars), or c-Cbl (white bars) siRNAs were infected with E. coli BL21(DE3) harboring the empty vector (pET-22b) or rOmpB (pYC9 or pET22-RJPOB) and assayed for bacterial invasion by the gentamicin protection assay. Invasion levels are displayed as percent of rOmpB-mediated invasion in control siRNA-transfected cells. Dark gray dashed line represents background invasion levels seen in E. coli expressing the empty vector. Inset: immunoblot analysis of Ku70 (left-top panel) or c-Cbl (right-top panel) protein levels in cells transfected with (1) control or (2) gene-specific siRNAs. Anti-β-actin immunoblots (bottom panels) were used as a protein loading control. B. Effect of caveolin-1, caveolin-2, and clathrin heavy chain siRNA-depletion on rOmpB-mediated invasion. HeLa cells transfected with control (black bars), Ku70 (dark gray bars), caveolin-1 (Cav1, light gray bars), caveolin-2 (Cav2, white bars) or clathrin heavy chain (CLTC, cross-hatched bars) siRNAs were infected with E. coli BL21(DE3) harboring the empty vector or rOmpB (pYC9 or pET22-RJPOB) and assayed for bacterial invasion by the gentamicin protection assay as described above. Inset: immunoblot analysis of Ku70 (first-top panel), Cav1 (second-top panel), Cav2 (third-top panel) and CLTC (fourth-top panel) protein levels in cells transfected with (1) control or (2) gene-specific siRNAs. Anti-β-actin immunoblots (bottom panels) were used as a protein loading control.