Fig 1.
Growth of Rickettsia helvetica AS819 in Vero E6 (A) and L929 (B) cell cultures. Genome equivalents measured by quantitative gltA PCR of infected cell cultures are given at several time points during infection. The values from triplicate infected cultures were averaged; standard deviation error bars are given. Growth studies using Vero E6 (A) and L929 (B) were performed simultaneously. ● indicate gltA-copies/μl template-DNA purified from cell layers, and ■ represent gltA-copies/μl template-DNA obtained from culture supernatants.
Fig 2.
Laser scanning confocal microscopy of Rickettsia helvetica AS819-infected L929 cells five days (A) and ten days (B) after infection. F-actin was stained with Alexa Fluor 568 Phalloidin (red), α-tubulin appears green (Alexa Fluor 488), nuclei were stained with DAPI (blue), and rickettsiae are coloured yellow (magnification 40x). The bar indicates 50 μm.
Fig 3.
Laser scanning confocal microscopy of Rickettsia helvetica AS819-infected Vero E6 cells five days (A) and 20 days (B) after infection. F-actin was stained with Alexa Fluor 568 Phalloidin (red), nuclei were stained with DAPI (blue), and rickettsiae are coloured yellow (magnification 40x). The bar indicates 100 μm (a) and 50 μm (b).
Fig 4.
Quantification of rickettsiae per nucleus area over time.
The infection progress over time is shown as the increase of rickettsiae per cell nucleus area. For both cell lines a continuous increase of rickettsiae can be observed until day 15 p.i. After this point in time, L929 cells detached and could not be further investigated while a slight increase was still observable in Vero E6 cells from day 15 to day 20 p.i. Images with at least 450 host cells were analysed for each time point and cell line.
Fig 5.
Laser scanning confocal microscopy of Rickettsia conorii VR141-infected Vero E6 cells four days after infection.
Actin tails at one pole of R. conorii VR141 can be seen. F-actin was stained with Alexa Fluor 568 Phalloidin (red), nuclei were stained with DAPI (blue), and R. conorii VR141 is presented in yellow (magnification 40x). The bar indicates 50 μm.
Fig 6.
Rickettsia helvetica AS819 plaque-forming assay.
Vero E6 and L929 monolayers were infected for 21 days with serial dilutions of R. helvetica AS819. After crystal violet staining, plaques were absent at 7 d p.i. in Vero E6 (A) and L929 (B) cells as well as after 21 d p.i. (C, Vero E6; D, L929). Detachment of L929 cells (D) increased with incubation time in agreement with earlier observations.
Fig 7.
Plaque formation by Rickettsia honei in different cell lines.
Vero E6 and L929 monolayers were infected for 7 days with serial dilutions of R. honei. After crystal violet staining plaques were noticed in Vero E6 (A) and L929 (B) cells.
Fig 8.
Amino acid sequence alignments of the indicated vinculin-binding-sites.
Sca4 of various Rickettsia shares significant similarity with vinculin-binding-site (VBS) sequences in mammalian talin-1 or Shigella invasion protein IpaA. Gray: aa residues identical to IpaA2 or talin-1; blue: aa residues similar to IpaA2 or talin-1. Abbreviations: Rcon—R. conorii, Rafr—R. africae, Rpar—R. parkeri, Rric—R. rickettsii, Rasi—R. asiatica, Rhel—R. helvetica, Shs—Shigella sonnei, Hs—Homo sapiens.