Fig 1.
BNI52 specifically detects TG rickettsiae.
(A) L929 cells were infected with the indicated TG and SFG rickettsiae or Orientia tsutsugamushi and stained with isotype antibody as a negative control (upper panel), BNI52 (middle panel) or serum from patients suffering from the respective infection (lower panel). Bacteria are shown in green. Nuclei were stained with DAPI (blue). (B) L929 cells were infected with TG rickettsiae (R. typhi, R. prowazekii), SFG rickettsiae (R. conorii) or transitional rickettsiae (R. felis), stained with BNI52 and gold-labeled secondary antibody and analyzed by electron microscopy. The antibody binds to the TG rickettsiae R. typhi and R. prowazekii but not to the SFG rickettsiae R. conorii, R. rickettsii and R. africae or to R. felis.
Fig 2.
BNI52 detects immunodominant peptides of 25–35 kDa.
(A-C) Proteins in the lysate of R. typhi were seperated by SDS PAGE and blotted on a nitrocellulose membrane. (A) The membrane was incubated with the monoclonal BNI52 antibody and HRP-labeled anti-mouse Ig secondary antibody. (B) Western Blots from R. typhi lysate were detected with serum from a BALB/c mouse immunized with inactivated R. typhi (#1), serum from mice that were infected with viable R. typhi (#2: BALB/c; #3: C57BL/6) or serum from a naive BALB/c mouse (Control) and HRP-labeled anti-mouse Ig secondary antibody. (C) Western Blots from R. typhi lysate were detected with serum from patients who acquired the infection with TG rickettsiae, most likely R. typhi, in different countries (#1: Greece, #2: Laos/Cambodia; #3: Thailand) and HRP-labeled anti-human Ig antibody. Patient serum was taken at different points in time (a: early, b: late). Serum from two healthy indivuals was used as a control (Control #1 and #2). BNI52 as well as antibodies in the sera from patients and immunized or infected mice predominantly recognize three to five antigen bands ranging from 25 to 35 kDa.
Fig 3.
The antigen recognized by BNI52 is a protein.
(A, B) Proteins in the lysate of R. typhi were digested with Proteinase K for indicated periods. Control bacterial lysate was left undigested. (A) A Western Blot and (B) a Dot blot was performed. The membranes were incubated with the BNI52 antibody followed by the incubation with a HRP-labeled secondary antibody. The antigen was no longer detectable after 24h proteinase K digestion.
Fig 4.
The antigen recognized by BNI52 is surface-exposed.
(A) R. typhi surface proteins were biotinylated prior to generating lysate. Biotinylated surface antigens from R. typhi were then enriched by streptavidine-coupled agarose. Lysates, flow through, wash fraction and eluate were applied to SDS PAGE and Western Blots. Western blots were first incubated with BNI52 (left) and after stripping with streptavidin-HRP (right). The antibody recognizes mainly the protein of 30 kDa and a slightly larger and a slightly smaller protein of about 27 kDa and 32 kDa. (B) BNI52 and gold-labeled secondary antibody were used for the staining of R. typhi-infected L929 cells and electron microscopy. The antigen is located on the outer cell membrane of R. typhi and in the periplasmic space between outer and the inner membrane.
Fig 5.
BNI52 binds the GroEL protein of R. typhi.
(A) Proteins in the lysate of R. typhi were precipitated with the BNI52 antibody by a protein A/G column. A Western Blot was performed from total lysate (R. typhi), flow through, eluates and wash fraction. BNI52 antibody was loaded as a control. The membrane was incubated with the BNI52 antibody. The antibody predominantly precipitates a 30 kDa protein. (B) For the identification of the antigen lysate of R. typhi was separated by two-dimensional SDS PAGE. Gels were silver stained (left) and applied to Western blotting. The membrane was incubated with the BNI52 antibody (right). The indicated protein spots (1–4, 5–13 and 14–18) of the silver stained gels that were recognized by the antibody in Western blots were analyzed by mass spectrometry. These analyses revealed the presence of GroEL in all spots recognized by BNI52. In addition, OmpB peptides were found in the lower molecular weight spots 1–4 and 14–18 but not in the 60 kDa spots. (C) His-tagged GroEL protein of R. typhi was expressed in E. coli, purified and subjected to Western blotting. Total lysate from R. typhi and BSA protein were used as a control. The membranes were incubated with a polyhistidine antibody (left) or the antibody BNI52 (right). The BNI52 antibody recognizes the 60 kDa recombinant GroEL protein but only weakly binds to the 60 kDa protein in R. typhi lysate.
Fig 6.
BNI52 enhances the uptake of R. typhi into phagocytic cells.
(A) Bone marrow-derived cells dendritic cells (bmDC) and macrophages (bmMΦ) were incubated with R. typhi (10 particles per cell) in the presence of of either IgG3 isotype antibody or BNI52 (8 μg/mL each) for 24h. Afterwards R. typhi was stained with patient serum (green) and nuclei were stained with DAPI (blue). An example staining is shown (above). Cells and bacteria were counted from five different views of the slides and the percentage of infected cells (y-axis; below, left) and the amount of R. typhi particles per cell (y-axis; below, right) were determined and statistically analyzed with Mann Whitney U test (**p<0.01, ***p<0.001). (B) Z232 macrophages were incubated with R. typhi (5 particles per cell) in the presence of increasing amounts of IgG3 isotype antibody or BNI52 for 3h. PBS was used as a control. Cells were then fixed and bacteria were stained with BNI52 (green). Nuclei were stained with DAPI (blue). An example staining is shown (above). From that experiment the percentage of infected cells (y-axis) in five different microscopic views was determined and statistically analyzed with Mann Whitney U test (**p<0.01). The x-axis shows the antibody concentration used (below).
Fig 7.
BNI52 does not detect R. typhi anymore during passage through BALB/c CB17 SCID mice.
(A) Lung sections were prepared on day 3 post infection from a R. typhi-infected BALB/c CB17 SCID mouse (left) and a BALB/c wildtype mouse (right) and stained with BNI52. BNI52 detects the bacteria in both sections, although the staining in BALB/c CB17 SCID already appears weaker compared to the staining in the BALB/c wildtype mouse. (B) Lung sections from a R. typhi-infected BALB/c CB17 SCID mouse prepared on day 14 post infection were stained with either BNI52 or patient serum. BNI52 does not detect the bacteria in BALB/c CB17 SCID tissue sections anymore although bacterial loads were high.
Fig 8.
GroEL is downregulated by R. typhi after passage through BALB/c CB17 SCID mice.
(A) R. typhi bacteria were reisolated from the spleens of BALB/c CB17 SCID mice and cultured in L929 cells in vitro. Bacteria reisolated from two different mice (R. typhiSCID1, R. typhiSCID2) were purified from L929 cell cultures and lysates were prepared for Western blotting in comparison to lysates from R. typhi prior to the passage through BALB/c CB17 SCID mice. 6 μg protein of each lysate was loaded. The membrane was incubated with BNI52. The antibody detects the same 25–35 kDa protein bands in R. typhiSCID lysates as in R. typhi prior to the infection of the mice but with remarkably reduced signal intensity. (B) An ELISA with the indicated amounts of proteins in the lysate of R. typhi prior to the infection of BALB/c CB17 SCID mice (R. typhi) and re-isolated bacteria from these animals (R. typhiSCID) (x-axis) was performed. Plate-coated proteins were detected with the BNI52 antibody. The OD450 is depicted as a measure for the binding of BNI52 (y-axis). The binding of BNI52 to plate-coated R. typhi antigen from R. typhiSCID was reduced compared to antigen from R. typhi prior to the infection of CB17 SCID mice. (C) Finally, the protein content in the lysates from R. typhi and R. typhiSCID bacteria (reisolate from one mouse) was analyzed three times each and quantified by mass spectrometry. With this method differentially regulated proteins in R. typhi and R. typhiSCID were identified (cut-off: ≥2fold change in expression). The expression of 32 proteins was reduced in R. typhiSCID bacteria compared to R. typhi prior to the infection of CB17 SCID mice and the expression of 8 proteins was enhanced. The GroEL protein that is recognized by BNI52 was found among the proteins that are downregulated in R. typhiSCID bacteria.