Fig 1.
Expression of ScaA by O. tsutsugamushi.
(A) Immunoblot analysis of whole proteins from L929 cells infected with O. tsutsugamushi proteins by using anti-ScaA serum (right panel). Anti-ScaA serum detected a protein with a molecular mass of approximately 150 kDa. Immunoblotting using anti-TAS56 was performed as a control (left panel). (B) Immunofluorescence confocal microscopy using preimmune serum or anti-ScaA serum (α-ScaA) showed ScaA in the O. tsutsugamushi-infected L929 cells. The left panels show bacteria stained with the pooled sera of scrub typhus patients (α-OT). Magnified images are shown in the lower panels (inset boxes). Scale bars, 5 μm.
Fig 2.
(A) HeLa cells were incubated with fluorescent microbeads coated with GST or GST-ScaA (ScaA) for 1 h, washed extensively, and fixed. Cell-bound microbeads (green) were visualized by fluorescence microscopy after staining of cell nuclei (blue). Scale bars, 10 μm. (B) Relative binding of the microbeads coated with GST (dotted line) or GST-ScaA (thick line) to HeLa cells was quantified directly using fluorescence-activated cell sorter (FACS) analysis. The gray histogram represents unbound cells (cells not incubated with microbeads). (C) Immunofluorescence microscopy using an anti-ScaA antibody revealed the presence of ScaA on the surface of the recombinant E. coli (lower panels). Preimmune serum did not detect the recombinant protein (upper panels). Scale bars, 5 μm. (D) E. coli transformed with the pET28a vector or with pScaA was induced with IPTG and incubated with HeLa cells. After being washed to remove adherent bacteria, the cells were fixed, permeabilized, and stained with an anti-E. coli antibody (green) and ToPro-3 for nuclear staining (blue). Scale bars, 10 μm. (E) CFU-based quantification of adherent E. coli transformed with the vector or pScaA was performed. The results are presented as percentages of adherent bacteria relative to the total bacterial input. Data are representative of three independent assays for each of the host cells. **, p < 0.01. (F) Inclusion of anti-ScaA serum in the medium (α-ScaA) significantly inhibited adhesion of E. coli expressing ScaA into host cells. After addition of anti-ScaA or preimmune serum into infection media, CFU-based quantification of adherent E. coli transformed with the vector or pScaA was performed. **, p < 0.01.
Fig 3.
Protective role of anti-ScaA immunity.
(A) Anti-ScaA antibody inhibited O. tsutsugamushi infection into host cells. HeLa cells were infected with the pathogen in the presence of the indicated anti-Sca antibodies or nonimmune serum. At 4 h after infection, bacterial infection was examined using confocal microscopy after differential immunoflourescent staining (see materials and methods). (B) The O. tsutsugamushi per host cell ratio was determined from three independent experiments in (A). **, p < 0.01. (C) Survival curves of immunized mice following lethal challenge with O. tsutsugamushi. Mice (n = 5/group) were immunized with the indicated antigen from the Boryong strain and challenged intraperitoneally with 100 x LD50 of O. tsutsugamushi Boryong strain. Their survival was monitored until all the surviving mice recovered from the disease. This graph is a representative survival curve of two experiments. **, p < 0.01 when compared with non-immunized group (PBS).
Fig 4.
Protective role of ScaA or combined immunization against heterologous strain infection.
Mice (n = 5/group) were immunized with the indicated antigens and challenged intraperitoneally with 10 x LD50 (A) or 100 x LD50 (B) of O. tsutsugamushi. Mice were immunized with antigens from the Boryong strain and challenged with the indicated strains (BR: Boryong, KP: Karp, KT: Kato). p value and median survival are summarized in S3 Fig. *, p < 0.05; **, p < 0.01.
Fig 5.
Similarity plots of a set of tsa56 and scaA sequences from the indicated strains compared to sequences from the Boryong strain.
Each plotted point is the percent identity within a sliding window of 100 bp or 100 amino acids wide centered on the position plotted, with a step size between points of 10 bp or amino acids. Diagrams above the graphs show the relative sizes of TSA56 and ScaA proteins and their sequence motiffs. Yellow box: signal peptide, gray box: antigenic domain, green box: variable domain, blue box: transmembrane domain, pink box: repeated sequences, brown box: autotransporter domain.