Antibodies against recombinant proteins recognize <i>A</i>. <i>phagocytophilum</i> in infected tick cells and ticks by immunofluorescence.

<p>(A) Uninfected and <i>A</i>. <i>phagocytophilum</i> (NY18)-infected ISE6 tick cells were characterized by immunofluorescence in (a, c, e, g) uninfected and (b, d, f, h) infected cells. Representative immunofluorescence images are shown for tick cells stained with rabbit preimmune (control) or anti-<i>A</i>. <i>phagocytophilum</i> protein antibodies (green, FITC; blue, DAPI). Arrows show the localization of <i>A</i>. <i>phagocytophilum</i> proteins in infected cells. Bars, 5 μm. (B) Sections were made from <i>I</i>. <i>scapularis</i> female ticks after feeding on an uninfected (a, c, e) or <i>A</i>. <i>phagocytophilum</i> (NY18)-infected (b, d, f) sheep. Representative immunofluorescence images are shown for salivary gland sections stained with rabbit preimmune (control) or anti-<i>A</i>. <i>phagocytophilum</i> protein antibodies (green, FITC). Arrows show the localization of <i>A</i>. <i>phagocytophilum</i> proteins in infected cells. Bars, 10 μm. (C) IDE8 tick cells were collected in low and high percentage <i>A</i>. <i>phagocytophilum</i> (L610)-infected cells and representative immunofluorescence images are shown. (a, b) Bright-field images of Giemsa-stained (a) low percentage and (b) high percentage infected tick cells. Bacteria stain purple (arrows) and host nuclei stain pink. (c) Low percentage and (d) high percentage infected tick cells were stained with rabbit anti-<i>A</i>. <i>phagocytophilum</i> HSP70 protein antibodies (green, FITC). Arrows show the localization of <i>A</i>. <i>phagocytophilum</i> proteins in infected cells. Bar, 5 μm. (D) Flow cytometry profile showing MFI values determined using a FITC-conjugated secondary antibody. <i>A</i>. <i>phagocytophilum</i> (NY18)-infected and uninfected control ISE6 tick cells were washed, fixed, permeabilized and incubated with primary unlabeled antibody (preimmune IgG isotype control, MSP4, SOD, HSP70 and GroEL), washed in PBS and incubated with FITC-goat anti-rabbit IgG. MFI was calculated as the MFI of the test-labeled sample minus the MFI of the isotype control, shown as Ave+SD and compared between infected and uninfected tick cells by Student's t-test (*P<0.05) (N = 3).</p

Characterization of the mRNA levels for selected genes encoding for <i>A</i>. <i>phagocytophilum</i> over-represented proteins.

<p>The mRNA levels for <i>groEL</i>, <i>msp4</i> and <i>hsp70</i> were determined by real-time RT-PCR in low and high percentage infected ISE6 tick cells. Amplification efficiencies were normalized against tick <i>16S rRNA</i> and mRNA levels expressed in arbitrary units. The ratio of normalized mRNA levels in high to low percentage-infected cells was represented as Ave+SD. Normalized Ct values were compared between low and high percentage infected tick cells by Student's t-test (*P<0.05) (N = 5).</p

Inhibition of <i>A</i>. <i>phagocytophilum</i> infection by antibodies against over-represented proteins.

<p>The antibodies against surface-exposed proteins, GroEL, HSP70 and MSP4, were used to characterize the inhibition of pathogen infection in ISE6 tick cells. Tick cells were treated with different rabbit antibodies and then infected with <i>A</i>. <i>phagocytophilum</i> (NY18). Treatments included rabbit pre-immune serum, anti-<i>A</i>. <i>phagocytophilum</i> GroEL, HSP70 and MSP4 protein antibodies and anti-tick Porin antibodies. Untreated cells were left uninfected or infected with <i>A</i>. <i>phagocytophilum</i> (NY18). <i>A</i>. <i>phagocytophilum</i> infection levels were determined by <i>16S rDNA</i> and <i>msp4</i> PCR and normalized against tick <i>16S</i> mitochondrial <i>rDNA</i> with similar results. Normalized <i>msp4</i> levels are shown in arbitrary units as Ave+S.D and were compared between infected and uninfected untreated cells and between infected cells treated with the pre-immune serum and antigen-specific antibodies by Student’s t-test with unequal variance (P<0.05; N = 4 replicates per treatment).</p

Characterization of <i>A</i>. <i>phagocytophilum</i> protein adhesion to tick cells.

<p>(A) Schematic representation of GroEL and HSP70 functional domains in full-length and short-length proteins produced in <i>E</i>. <i>coli</i> and used for binding experiments to tick cells. (B) <i>E</i>. <i>coli</i> strains were grown and induced for the production of recombinant proteins. <i>E</i>. <i>coli</i> strains with expression vector alone producing recombinant Thioredoxin and <i>A</i>. <i>marginale</i> MSP1a were used as negative and positive control, respectively. Adhesive bacteria were quantitated as the number of colony forming units (CFU) recovered from each test, shown as fold increase and compared to the Thioredoxin (Trx) control values by Student’s t-test for paired samples (*P<0.05; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137237#pone.0137237.t001" target="_blank">Table 1</a>).</p

Specificity of antibodies produced in rabbits against <i>A</i>. <i>phagocytophilum</i> recombinant proteins.

<p>(A) Western blot analysis of 10 μg of recombinant <i>A</i>. <i>phagocytophilum</i> proteins produced in <i>E</i>. <i>coli</i> (red dots) demonstrates the specificity of the antibodies produced in rabbits. <i>E</i>. <i>coli</i> cell proteins were included as negative control. (B) <i>A</i>. <i>phagocytophilum</i> (NY18) purified from infected ISE6 tick cells and recombinant <i>E</i>. <i>coli</i> were mock treated (-) or surface digested with trypsin (+) and 10 μg protein loaded onto polyacrylamide gels for Western blot analysis using rabbit antibodies produced against recombinant proteins. (C) Immunofluorescence assay of <i>E</i>. <i>coli</i> producing recombinant <i>A</i>. <i>phagocytophilum</i> MSP4, HSP70 and GroEL proteins and reacted with (a) control pre-immune IgGs which gave similar results for all recombinant <i>E</i>. <i>coli</i> or purified antibodies against (c) MSP4, (e) HSP70, and (g) GroEL (green, FITC) or (b, d, f, h) stained with DAPI (blue). Bars, 10 μm. (D) Immunofluorescence assay of control <i>E</i>. <i>coli</i> producing recombinant tick Porin and reacted with purified antibodies against (a) MSP4, (b) GroEL, and (c) HSP70 (green, FITC) or (d-f) stained with DAPI to rule out cross-reaction of antibodies against <i>A</i>. <i>phagocytophilum</i> proteins with <i>E</i>. <i>coli</i> proteins. Bars, 10 μm. The <i>E</i>.<i>coli</i> induced for the production of recombinant <i>A</i>. <i>phagocytophilum</i> proteins were fixed with 4% paraformaldehyde and used for immunofluorescence. <i>E</i>. <i>coli</i> cells producing recombinant tick Porin were used as control.</p

Adhesion to cultured tick cells by recombinant <i>E</i>. <i>coli</i> producing surface-exposed <i>A</i>. <i>phagocytophilum</i> proteins.

<p><i>E</i>. <i>coli</i> strains were grown and induced for the production of recombinant proteins. <i>E</i>. <i>coli</i> strains with expression vector alone producing recombinant Thioredoxin and <i>A</i>. <i>marginale</i> MSP1a were used as negative and positive control, respectively. Adhesive bacteria were quantitated as the number of colony forming units (CFU) recovered from each test and compared to the Thioredoxin control values by Student’s t-test for paired samples (*P<0.05). GroEL and HSP70 were produced as full-length and short-length (amino acids 275–475 and 262–460 for GroEL and HSP70, respectively) proteins.</p><p>Adhesion to cultured tick cells by recombinant <i>E</i>. <i>coli</i> producing surface-exposed <i>A</i>. <i>phagocytophilum</i> proteins.</p

Characterization of <i>A</i>. <i>phagocytophilum</i> protein-protein interactions.

<p>(A) Protein-protein interactions were characterized <i>in silico</i> using STRING 8.3 (<a href="http://string-db.org" target="_blank">http://string-db.org</a>). The STRING score value is shown, defined as threshold of significance to include the interaction (maximum value = 1) computed by combining the probabilities from the different evidence channels, correcting for the probability of randomly observing an interaction. (B) Protein-protein interactions were characterized <i>in vitro</i> using <i>A</i>. <i>phagocytophilum</i> HSP70 (red arrow) and GroEL (blue arrow) recombinant proteins and tick Porin as control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137237#pone.0137237.ref015" target="_blank">15</a>]. The proteins were mixed in equimolar amounts and immunoprecipitated using anti-GroEL or anti-HSP70 antibodies and Protein G Dynabeads. The purified proteins were eluted using Laemmli sample buffer and loaded onto a 12% SDS-PAGE gel for Western blot analysis using anti-HSP70, anti-GroEL or anti-Porin antibodies. (C) Protein-protein interactions were characterized <i>in vitro</i> using <i>A</i>. <i>phagocytophilum</i> protein extracts, recombinant HSP70 (red arrow) and GroEL (blue arrow) proteins and tick Porin as control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137237#pone.0137237.ref015" target="_blank">15</a>]. Protein G Dynabeads were incubated with purified anti-HSP70, anti-GroEL or anti-Porin antibodies and then 130 μg of <i>A</i>. <i>phagocytophilum</i> proteins were added. Unbound proteins were removed and the beads were washed three times with PBS with addition of 0.1% Triton X-100, resuspended in Laemmli sample buffer and loaded onto a 12% SDS-PAGE gel for Western blot analysis using anti-HSP70 or anti-GroEL antibodies. (D) Protein-protein interactions were characterized <i>in vitro</i> using <i>A</i>. <i>phagocytophilum</i> HSP70 (red arrow), GroEL (blue arrow) and MSP4 (green arrows) recombinant proteins and tick Porin as control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137237#pone.0137237.ref015" target="_blank">15</a>]. Nickel beads were covered with histidine-tagged MSP4, washed and incubated with GroEL or HSP70, MSP4 or Porin as control. After incubation, beads were washed and proteins eluted in Laemmli sample buffer and loaded onto a 15% SDS-PAGE gel.</p

Electron micrographs of ISE6 tick cells reacted with recombinant <i>E</i>. <i>coli</i> producing <i>A</i>. <i>phagocytophilum</i> recombinant proteins.

<p>Representative images of recombinant <i>E</i>. <i>coli</i> shown by arrowheads adhered to tick cells. (a, b) <i>A</i>. <i>marginale</i> MSP1a positive control; (c-f) MSP4; (g-k) HSP70; (l) GroEL; (m) Thioredoxin negative control. Microtubules are shown by arrows in (d). Scale bars, 1 μm (a, c, e, g, i, k-m), 500 nm (b, d, f, h, j).</p

Proposed mechanisms of how stress response and surface proteins facilitate <i>A</i>. <i>phagocytophilum</i> infection in high percentage infected tick cells and tick salivary glands.

<p>The levels of certain bacterial stress response and surface proteins are higher in high percentage <i>A</i>. <i>phagocytophilum</i>-infected tick cells and tick salivary glands. MSP4, GroEL and HSP70 interact and bind to tick cells, thus facilitating rickettsia-tick interactions and infection. In high percentage infected tick cells and tick salivary glands, bacteria reduce multiplication once they infect the cells but infection is required to complete the life cycle and get ready for transmission. The activation of stress response proteins in <i>A</i>. <i>phagocytophilum</i> may represent a mechanism by which rickettsiae increase infection by facilitating interaction with tick cells and protecting bacteria against stress. The T4SS may be associated with the secretion of HSP70 and other stress response proteins. Abbreviations: T4SS, Type IV Secretion System; Question mark indicates that secretion of HSP70 in a T4SS-dependent manner remains to be proved.</p

<i>A</i>. <i>phagocytophilum</i> protein ontology in infected tick cells and adult female guts and salivary glands.

<p>(A) Rickettsia cell protein ontology for biological process of differentially represented proteins in high percentage infected cells when compared to low percentage infected cells. (B) Rickettsia protein ontology for biological process of proteins identified in infected adult female guts. (C) Rickettsia protein ontology for biological process of proteins identified in infected adult female salivary glands. Only proteins identified with a FDR < 0.05 and at least 2 peptides per protein were included in the analysis. After discarding tick proteins, proteins with the same description in the Anaplasmataceae were grouped and the total number of PSM for each protein were normalized against the total number of PSM on each infected tick cell or tissue and compared between low and high percentage infected cells or between salivary glands and guts by Chi2-test (P = 0.05; N = 3 for tick cells and N = 2 for ticks). Biological processes with over-represented proteins in high percentage infected cells and in infected tick guts or salivary glands are indicated with red arrows (P<0.05).</p