Fig 1.
SssP1 is critical for S. suis to cause meningitis.
Since the inoperability of constructing the complementary strain by reintroducing a 13,944 bp sssP1 gene, a mutant (SssP1T182A) with a stop codon at position 182 bp was constructed by artificially changing T to A, which was used as an alternative method to confirm that ΔsssP1 is a non-polar deletion mutant. (A) The red blood and white blood cell counts of cerebrospinal fluid (CFS) from the mice infected with the indicated strains. Data are represented as mean ± SEM of three independent repeats (**** P < 0.0001, *** P < 0.001). (B) Transcriptional level of IL-8 encoding gene in the brains of the mice infected with the indicated strains. Data are represented as mean ± SEM of three independent repeats (**** P < 0.0001). (C) Histopathological analysis of the brains from the mice challenged with CZ130302, ΔsssP1 or SssP1T182A. Scale, 50 μm.
Fig 2.
SssP1 is required for S. suis to cross the BBB in vivo.
(A) The bacterial load of the wild-type and ΔsssP1 strains in the brain of the mice infected with the indicated strains. Data are represented as mean ± SEM of triplicate samples (**** P < 0.0001). (B) The curves showed the bacterial loads in CFS of the mice challenged with different S. suis strains until 20 h post-infection. Mean values and SEMs of triplicate samples are indicated. (C, D) Evans Blue (EB) permeability in brains of the mice infected with the indicated strains. EB concentration of the infected brains was measured. Data are represented as mean ± SEM of triplicate samples (** P < 0.01).
Fig 3.
SssP1 promotes S. suis to cross the BBB in vitro.
All data are represented as mean ± SEM of triplicate samples (**** P < 0.0001, ** P < 0.01, *P < 0.05). (A) The curves showed the TEER values of three types of models within 4 days. The HBMECs and astrocyte cell HA1800 were respectively or jointly inoculated into a transwell chamber to establish three types of BBB models in vitro (S1 Fig). (B) The curves showed the TEER values of the HBMEC&HA18000 joint BBB model infected with the indicated S. suis strains. (C) The penetration rates of three types of models infected with the indicated strains were measured, and the data showed the calculated value of the bacterial CFU in the bottom chamber/ upper + bottom chambers. (D) The fluorescence values of the bottom chambers at OD428 were measured to show the permeability of Fluorescein (FLU) in three type of BBB models infected with the indicated bacterial strains. The boiled CZ130302 was used as control.
Fig 4.
The sssP1 plays significant roles in adherence and invasion.
All data are represented as mean ± SEM of triplicate samples (**** P < 0.0001, *** P < 0.001, ** P < 0.01). (A, B) The adhesion and invasion levels of the indicated S. suis strains to HBMECs were detected at the MOI 100:1. (C) The transcriptional level of IL-8 encoding gene in the HBMECs interacted with different S. suis strains were detected using an RT-qPCR assay at the indicated times post-infection. (D) The releasing levels of IL-8 in the cultural supernatant of the HBMECs infected with different S. suis strains were measured at 4 h post-infection. (E) Cytoskeletal changes of HBMECs in response to infection by S. suis CZ130302 and ΔsssP1. Cells were stained with phalloidin to show actin filaments and were imaged using confocal microscopy. Control HBMECs were incubated without bacteria. Otherwise, HBMECs were incubated with S. suis for 0.5, 1.0, and 1.5 h before phalloidin staining. (F) Levels of the three major components of TJs, ZO-1, occludin and claudin-5, were detected using a Western blot assay to the total protein samples from the HBMECs infected with the indicated strains. β-actin was used as a loading control.
Fig 5.
Vimentin of HBMECs was screened as a vital receptor of SssP1.
(A) Schematic diagram of the domain architecture of SssP1 and the location of NR1-1298 and NR1225-2214. (B) The analysis of the screened host proteins by pull-down assays of SssP1-NR1-1298 and SssP1-NR1225-2214 is based on the data shown in Tables 1 and S2. The binding score represents the ability to interact with SssP1 based on the protein loads measured in the pull-down assays. (C) The Far-Western blot was performed to identify the interaction between vimentin and the indicated NR fragments of SssP1. (D) Visual observation of the localisation of vimentin and S. suis co-incubated with the HBMECs. The vimentin skeleton was stained by indirect immunofluorescence (IIF) in red, the nuclei were counterstained with DAPI in blue, and the S. suis cells with green fluorescent plasmid pKSM410-rss29 were observed in green.
Table 1.
The potential receptor proteins of HBMEC were screened jointly by SssP1-NR1-1298 and SssP1-NR1225-2214 binding.
Fig 6.
Identification of the smaller fragments required for SssP1-NR2 attachment to vimentin.
(A) Schematic diagram of the NR domain divided into 5 fragments according to the protein structure. (B) The Far-Western blot was performed to identify the interaction between vimentin and the indicated fragments of NR domain. (C) Contributions of the NR216-781 and NR1711-2214 fragments in bacterial adhesion to HBMECs. Several indicated cell samples were pretreated for 0.5 h at 37°C using the sialidase (Sigma). The images show the DAPI-stained nuclei in blue, and the adhered proteins detected by goat anti-mouse IgG-FITC in green. The NR1225-1781 was used as control. (D) The Far-Western blot analyses of NR domains using the sialidase (Sigma) pretreated vimentin (for 0.5 h at 37°C). The hyaluronidase (Sigma) pretreated vimentin was used as control.
Fig 7.
The adhesion of S. suis to HBMECs was attenuated by the blocking of vimentin, NR216-781 or NR1711-2214.
All data are represented as mean ± SEM of triplicate samples (*** P < 0.001, ** P < 0.01, *P < 0.05). (A) The adhesion levels of S. suis strain CZ130302 were detected using the HBMECs pretreated with the NR216-781, NR1711-2214 and NR1225-1781 fragments. (B) The adhesion levels of S. suis strain CZ130302 to HBMECs were detected when anti-NR216-781 or anti-NR1711-2214 serums were added during co-incubation at MOI 100:1. The values were normalised against cells incubated with the pre-immune serum. (C) The adhesion levels of S. suis strain CZ130302 were detected using the HBMECs pretreated with the vimentin or Recombinant-keratin-2 (KRT2). KRT2 protein was use as the negative control. The cells without any treatments were used as the blank control. (D) The adhesion levels of S. suis strain CZ130302 to HBMECs were detected when the anti-vimentin or negative serums were added during the co-incubation at the MOI 100:1. (E) Confocal microscopy observation of vimentin skeleton and bacterial tracer. The images showed that the nuclei were stained with DAPI in blue, the vimentin was stained by IIF in red, and the S. suis cells with green fluorescent plasmid pKSM410-rss29 were observed in green.
Fig 8.
Summary of the role of the SssP1-vimentin interaction in promoting meningitis.
SssP1 interacts with vimentin on the surface of HBMECs to promote S. suis attachment and induce robust inflammatory responses, which contributes to destroying the BBB to cause meningitis indirectly. The transmission electron microscope (TEM) image of S. suis strain CZ130302 showing the fimbria-like construction of SssP1 proteins was observed by this study, which was is consistent with a previous study of our lab [16].