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First published online 21 September 2004
doi: 10.1242/jcs.01382

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Early signaling events involved in the entry of Rickettsia conorii into mammalian cells

Institut Pasteur, Unité des Interactions Bactéries-Cellules, INSERM U604, Département de Biologie Cellulaire et Infection, 25 Rue du Dr Roux, 75724 Paris CEDEX 15, France

View larger version (59K): [in a new window]   Fig. 1. Invasion of Rickettsia conorii is dependent on host actin polymerization. (A) In Vero cells infected with R. conorii; F-actin (green, left panel) colocalizes with invading bacteria (red) within 15 minutes of infection, as shown in the merged image (yellow, right panel). (B) Preincubation of Vero cells with cytochalasin D diminished the ability of R. conorii to invade. Bar, 2 µm.

View larger version (53K): [in a new window]   Fig. 2. The Arp2/3 complex is required for efficient bacterial internalization. (A) Extracellular Rickettsia conorii (arrow), but not intracellular bacteria (arrowhead) strongly colocalizes with Arp3, suggesting that Arp2/3 recruitment plays an essential role in the uptake process in Vero cells. (B) Transfection of the WASp-related protein, Scar (Scar FL), and the Arp2/3 binding domain (Scar WA) inhibits the ability of R. conorii to enter Vero cells, but has no effect on bacterial adherence (data not shown). Bar, 2 µm.

View larger version (58K): [in a new window]   Fig. 3. Cdc42 but not Rac1 governs actin rearrangements associated with Rickettsia conorii invasion. (A) Expression of dominant negative N17Cdc42, but not N17Rac1 in Vero cells inhibited R. conorii-mediated invasion when compared to non-transfected (Control) cells as assessed by a fluorescence-based invasion assay (see Materials and Methods). Expression of either construct had no effect on bacterial adherence (data not shown). (B,C) Extracellular R. conorii were found to colocalize with endogenous Cdc42 (arrows) in non-transfected cells within 15 minutes of bacterial infection. (D) Colocalization of Cdc42 with bacteria is not caused by antibody crossreactivity with bacteria (arrowhead). Bar, 4 µm.

View larger version (52K): [in a new window]   Fig. 4. Involvement of host protein tyrosine phosphorylation in the internalization of Rickettsia conorii into Vero cells. (A,B) Immunofluorescence microscopy of infected cells demonstrates that extracellular R. conorii recruits tyrosine phosphoproteins (pTyr) to sites of adherence within 15 minutes of infection (arrows point to bacteria that have unambiguously recruited tyrosine phosphoproteins). (C) Specificity of pTyr protein colocalization with adherent R. conorii is not caused by antibody crossreactivity to bacterial antigens (arrowheads). (D) Pre-incubation of Vero cells with a PTK inhibitor, genistein, blocks R. conorii entry in a concentration-dependent fashion, highlighting a role for PTKs in bacterial entry. (E) Immunoprecipitation and western immunoblot analysis using anti-phosphotyrosine antisera (pTyr) revealed that R. conorii infection of Vero cells induces the phosphorylation of host proteins (arrows) 10 minutes after infection, with strong phosphorylation of a p125/130 protein. Immunoblot analysis of pre-immune precipitated cellular lysates with anti-actin antisera serves as a protein loading control. Bar, 3 µm.

View larger version (30K): [in a new window]   Fig. 5. PI 3-kinase activity is involved in Rickettsia conorii uptake. (A) A specific inhibitor of PI 3-kinase activity (wortmannin) blocks R. conorii invasion, with 64% inhibition at 200 nM compared to levels in control cells. (B) R. conorii infection of Vero cells leads to co-immunoprecipitation of p85 with tyrosine phosphoproteins within minutes of infection (arrow), suggesting that PI 3-kinase activation is an important event in the entry process. Densitometric analysis of scanned blots indicates the relative amount of p85 that co-immunoprecipitates with pTyr proteins during R. conorii infection. Immunoblot analysis of cellular lysates with anti-p85 antibodies.

View larger version (32K): [in a new window]   Fig. 6. Rickettsia conorii infection of Vero cells induces the tyrosine phosphorylation of pp125FAK. (A) Pre-incubation of Vero cells with the indicated inhibitors of bacterial invasion, blocks the induced tyrosine phosphorylation of at least one host protein (p125/130) after 5 minutes of bacterial infection (arrow). (B) Immunoprecipitation of pp125FAK and immunoblot analysis with anti-phosphotyrosine (pTyr) demonstrates an induced phosphorylation of FAK during the infection process, suggesting that this protein may play a key role in the entry process.

View larger version (61K): [in a new window]   Fig. 7. Involvement of c-src and downstream effector, cortactin, in Rickettsia conorii invasion of mammalian cells. (A) Immunofluorescence microscopy reveals that R. conorii colocalizes with endogenous c-src within 15 minutes of infection (arrow). Colocalization is not caused by antibody crossreactivity as not all adherent bacteria recruit c-src (arrowheads). (B) Pharmacological inhibition of Src-family PTKs with protein phosphatase 1 (PP1), diminishes bacterial entry but has no effect on bacterial adherence or cellular viability (data not shown). (C) R. conorii infection induces the co-immunoprecipitation of c-Src with phosphotyrosine proteins, suggesting that this may be a mechanism for activating c-src during the invasion process. Densitometric analysis of scanned immunoblots indicates the relative amount of c-src that co-immunoprecipitates with pTyr proteins in response to bacterial infection. Immunoblot analysis of pre-immune precipitated cellular lysates serves as a loading control. (D) The c-src kinase substrate, cortactin, colocalizes with some (arrows), but not all (arrowheads) extracellular R. conorii within 15 minutes of infection, suggesting that specific cortactin recruitment plays a role in the uptake process. (E) The boxed area in (D) has been enlarged to show detail. Bars in (A,E), 6 µm; (D), 3 µm.

View larger version (19K): [in a new window]   Fig. 8. Model of putative interactions involved in the uptake of Rickettsia conorii in non-phagocytic mammalian cells. Various signaling pathways can activate the Arp2/3 complex independently or in synergy. Internalization of R. conorii most likely involves the interaction of R. conorii surface protein(s) with an unidentified host cell receptor(s) (black rectangle with question mark), which serves to activate multiple pathways involving Cdc42, PI 3-kinase, Src, FAK and cortactin, ultimately leading to Arp2/3-dependent actin cytoskeletal changes that drive bacterial entry (see Discussion). Putative associations of proteins during bacterial entry are indicated by question marks and dotted arrows.