Fibroblast growth factor receptor-1 mediates internalization of pathogenic spotted fever rickettsiae into host endothelium

<div><p>Rickettsial infections continue to cause serious morbidity and mortality in severe human cases around the world. Host cell adhesion and invasion is an essential requisite for intracellular growth, replication, and subsequent dissemination of pathogenic rickettsiae. Heparan sulfate proteoglycans [HSPGs] facilitate the interactions between fibroblast growth factor(s) and their tyrosine kinase receptors resulting in receptor dimerization/activation and have been implicated in bacterial adhesion to target host cells. In the present study, we have investigated the contributions of fibroblast growth factor receptors [FGFRs] in rickettsial entry into the host cells. Inhibition of HSPGs by heparinase and FGFRs by AZD4547 (a selective small-molecule inhibitor) results in significant reduction in rickettsial internalization into cultured human microvascular endothelial cells (ECs), which represent the primary targets of pathogenic rickettsiae during human infections. Administration of AZD4547 during <i>R</i>. <i>conorii</i> infection in a murine model of endothelial-target spotted fever rickettsiosis also diminishes pulmonary rickettsial burden in comparison to mock-treated controls. Silencing of FGFR1 expression using a small interfering RNA also leads to similar inhibition of <i>R</i>. <i>rickettsii</i> invasion into ECs. Consistent with these findings, <i>R</i>. <i>rickettsii</i> infection of ECs also results in phosphorylation of tyrosine 653/654, suggesting activation of FGFR1. Using isobaric tag for relative and absolute quantitation [iTRAQ]-based proteomics approach, we further demonstrate association of β-peptide of rickettsial outer membrane protein OmpA with FGFR1. Mechanistically, FGFR1 binds to caveolin-1 and mediates bacterial entry via caveolin-1 dependent endocytosis. Together, these results identify host cell FGFR1 and rickettsial OmpA as another novel receptor-ligand pair contributing to the internalization of pathogenic rickettsiae into host endothelial cells and the potential application of FGFR-inhibitor drugs as adjunct therapeutics against spotted fever rickettsioses.</p></div

FGFR1 interacts with β-peptide of OmpA.

<p>Confluent ECs were infected with <i>R</i>. <i>rickettsii</i> and FGFR1 was immunoprecipitated from the total protein lysates. The samples were subjected to mass spectroscopic analysis using isobaric tag for relative and absolute quantitation [iTRAQ] method [described in materials and methods]. (A): The peptides interacting with FGFR1 were identified as peptide 1 and 2. (B): The location of the peptide 1 within the beta peptide sequence of OmpA is shown in red. (C): FGFR1 was immunoprecipitated (IP) from <i>R</i>. <i>rickettsii-</i>infected ECs and samples were subjected to SDS-PAGE and Western blotting using rickettsial OmpA antibody. Mouse IgG was used as the control. The blot was also probed with an FGFR1 antibody to demonstrate that the immunoprecipiatation was successful. A representative blot from three independent experiments is shown.</p

Additional file 4: of Anticoagulated patient’s perception of their illness, their beliefs about the anticoagulant therapy prescribed and the relationship with adherence: impact of novel oral anticoagulant therapy – study protocol for The Switching Study: a prospective cohort study

Questionnaire packs administered to patients prescribed rivaroxaban*. (PDF 277 kb

Effect of FGFR/HSPG inhibition on rickettsial internalization <i>in vitro and in vivo</i>.

<p>(A): ECs were incubated with heparinase (1U/ml), FGFR inhibitor AZD4547 (100 nM), or the vehicle alone (mock-treated) for 1 hour prior to infection with <i>R</i>. <i>rickettsii</i>. At 6 hours post-infection, total DNA was extracted and rickettsial copy number was determined using a standard curve. The asterisks represent a significant change (p ≤ 0.001) compared to mock treatment and the data represent mean ± Standard Error (SE) from a minimum of three independent experiments. (B): AZD4547 was dissolved in a vehicle containing DMSO and 1% (v/v) Tween-80 and orally administered (25 mg/kg/day) to 6–8 weeks old C3H/HeN mice infected with <i>R</i>. <i>conorii</i> (2.25×10⁵ pfu). The control group of animals (infected but mock-treated) received injection of <i>R</i>. <i>conorii</i> and vehicle (DMSO + Tween-80). Mice were euthanized on day 3 post-infection and rickettsial copy number was determined using DNA from the lungs. The data are presented as the mean ± SE of three independent observations.</p

FGFR1 interactions with caveolin-1 and caveolin-2.

<p>(A): Confluent ECs were infected with <i>R</i>. <i>rickettsii</i>. At 1 hour post-infection, the cell lysates were prepared, FGFR1 was then immuno-precipitated using an FGFR1-specific antibody. Mouse IgG was used as a negative control. Samples were subjected to SDS-PAGE and Western blotting using antibodies against caveolin-1, caveolin-2 and FGFR1. (B): ECs were transfected with either control, caveolin-1 or caveolin-2 siRNA for 72 hours and then infected with <i>R</i>. <i>rickettsii</i> for 1 hour. Rickettsial copy number was measured by q-PCR using the OmpA primer pair. The asterisks represent a significant change (p≤ 0.001). The data are presented as the mean ± SE of three independent experiments. (C): caveolin-1 (cav-1) and caveolin-2 (cav-2) expression were measured by Western blotting to demonstrate the functionality of siRNAs used in our experiments.</p

Additional file 3: of Anticoagulated patient’s perception of their illness, their beliefs about the anticoagulant therapy prescribed and the relationship with adherence: impact of novel oral anticoagulant therapy – study protocol for The Switching Study: a prospective cohort study

Questionnaire packs administered to patients prescribed dabigatran. (PDF 276 kb

<i>Rickettsia</i> infection activates FGFR1.

<p>ECs were infected with <i>R</i>. <i>rickettsii</i> for 30 minutes and the levels of FGFR1 phosphorylation (tyrosine 653/654) (A), total FGFR1 (B), and α—tubulin as the loading control (C) were measured by immunoblotting and probing of the blots using specific antibodies. Quantitation of FGFR1 phosphorylation from three separate experiments is also presented as the mean ± standard error (D).</p

Flagellin was not internalized by siTLR5 Caco-2BBe cells or IEC in which TLR5 was blocked.

<p><b>A</b>. Immunoblotting for TLR5 via Western blot showed that TLR5 expression is dramatically reduced in siTLR5 Caco-2BBe cells compared to wild type Caco-2BBe cells. Further, wild type Caco-2BBe cells infected with <i>S. Typhimurium</i> (MOI 500) did not change the expression of TLR5 2 h post-infection. <b>B.</b> Transverse image of polarized siTLR5 Caco-2BBe cells constructed from optical z-stacks taken by confocal microscopy, showing flagellin (green) was localized to the AP surface at thirty minutes post-exposure. Cell nuclei were stained with DAPI (blue). <b>C</b>. The majority of flagellin remained on the apical surface of siTLR5 Caco-2BBe cells and wild type Caco-2BBe cells pre-treated with TLR5 blocker antibody following 1 h incubation with 5 µg of flagellin. Whereas, the predominant quantity of flagellin was found in the cell layer of wild type Caco-2BBe controls. Flagellin was not detected in the BL supernatants of any of the tested cell samples. These results represent 1 of 3 repeated experiments with similar results.</p

Flagellin co-localizes with endosomal and lysosomal markers.

<p><b>A</b>. Following AP exposure to Caco-2BBe cells, GFP-labeled flagellin (green) was internalized and, within 15 min, co-localized with an antibody that recognized an early endosomal surface marker (EEA1) and secondary antibody (red) as shown in the middle z-sections by confocal microscopy. Lower right panel shows merged images and indicates co-localization of flagellin with EEA1.Cell nuclei were stained with DAPI (blue). <b>B</b>. Within 1 h flagellin (green) co-localized with the lysosome marker LAMP-1 (red). The confocal images shown are representative of 3 independent experiments. <b>C</b>–<b>F</b>. 6HIS-Flagellin was added to the AP or BL side of polarized Caco-2BBE cells at 19°C allowing traffic up to (but not beyond) the recycling endosome. In some experiments biotinylated transferrin also was added to the apical surface as a marker for the endosomal pathway. Cells then were either fractionated (see methods) without allowing further traffic or warmed to 37°C for the times indicated prior to fractionation. NOTE: White arrows indicate flagellin protein in A and B. <b>C</b>. Intracellular localization of internalized flagellin protein (normalized for total cell-associated protein) following incubation at 19°C. <b>D</b>. Western blots of endosomal fractionation as described in C and in the methods. <b>E</b>. Intracellular localization of 6HIS-Flagellin when internalization of protein at 19°C was followed by incubation at 37°C allowing traffic beyond the recycling endosome. <b>F-G</b>. Western blots of fractionation as described in E and in the methods.</p

IL-8 secretion decreased from TLR5 silenced (siRNA) Caco-2BBe cells exposed to flagellin.

<p><b>A. </b><i>S. dublin</i> flagellin was added to the AP or BL surface of polarized Caco-2BBe cells where TLR5 was knocked down using siRNA. Parallel wild type Caco-2BBe cells were treated as above. Six hours post-flagellin exposure, AP and BL supernatants were collected to quantitate IL-8 secretion. IL-8 secretion was significantly decreased from siRNA Caco-2BBe cells stimulated with <i>S. dublin</i> flagellin regardless of AP or BL exposure compared to wild type Caco-2BBe cells. <b>B</b>. IL-8 secretion was significantly decreased in siTLR5 Caco-2BBe cells apically exposed to flagellin from <i>S. dublin</i>, ECO83, <i>E. coli</i> K12, and ND1/2ECHCD2/1 compared to wild type cells. Data is expressed as mean ± SD. *p<0.05 vs. all groups.</p