Binding Of Isaac Exploits

[Manric Hock]

Thecritical step for meningitis development is the transmigration of blood-borne GBS into the CNS. The brain is normally protected by physiological barriers that separate the blood from the brain parenchyma or the cerebrospinal fluid. Brain microvascular endothelial cells and choroid plexus epithelial cells that compose the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB), respectively, are characterized by the presence of tight junctions that are crucial for the maintenance of the barrier function. To penetrate those barriers, blood-borne bacteria can either use a transcellular mechanism that requires pathogen internalization and/or a paracellular mechanism that requires tight-junction disruption. Alternatively, bacteria can use phagocytes as Trojan horses (12). GBS has been described to be internalized in cerebral endothelial cells, but also to inhibit tight-junction protein expression, suggesting that GBS could use transcellular and/or paracellular mechanisms to penetrate the brain (13, 14).

Here, we demonstrated that Srr2 directly and specifically bound to integrins α5β1 and αvβ3, thereby contributing to the adhesion and invasion of brain endothelial cells and to the crossing of the brain barrier, leading to meningitis development. Most importantly, we showed that these receptors were overexpressed during the postnatal period in brain vessels. This likely accounts for the CC17-GBS increased invasiveness of the CNS in neonates.

We therefore compared the interaction of BRSrr2 and BRSrr1 with α5β1 and αvβ3 integrins. We first measured by immunoblotting the direct binding of BRSrr2 and BRSrr1 to immobilized recombinant soluble human integrins α5β1 and αvβ3. Intense signals were obtained with BRSrr2 where α5β1 and αvβ3 integrins were spotted but not where ICAM1, another transmembrane protein, was spotted, indicating that BRSrr2 bound to recombinant human α5β1 and αvβ3 integrins (Figure 1B). In contrast, weak signals were observed when BRSrr1 was assayed in the same conditions with α5β1 and αvβ3 integrins (Figure 1B).

αvβ3-BRSrr2 interaction depends on the RGD and SDV motifs. In order to test the role of the RGD and SDV motifs present on BRSrr2 in their interaction with integrins, mutated forms of BRSrr2 were produced in which the RGD and/or SDV motifs were replaced by 3 alanines (Supplemental Figure 1B and C). The interaction between BRSrr2 mutated forms with integrins αvβ3 or α5β1 was assessed by ELISA. RGD and/or SDV substitutions strongly affected αvβ3 integrin binding (Figure 2A) but not α5β1 integrin binding (Figure 2B). No additive inhibition was observed when both motifs (RGD and SDV) were mutated (Figure 2A).

The contribution of these motifs to integrin binding was further supported by competitive ELISA assays using synthetic peptides containing an RGD sequence (RGDS and RGDfV peptides) or an SDV sequence (P11 peptide) widely used to inhibit integrin recognition (20, 21). RGD and SDV peptides significantly inhibited αvβ3-BRSrr2 interaction in a dose-dependent manner, whereas they had no significant effect on α5β1-BRSrr2 interaction (Figure 2, C and D). Altogether, these results demonstrated that the interaction of BRSrr2 with integrin αvβ3 required both RGD and SDV motifs, whereas the interaction with integrin α5β1 was independent from these motifs.

BRSrr2 interaction with integrin α5β1 involves 2 other motifs. To identify BRSrr2 residues involved in α5β1 integrin recognition, we used the RaptorX web server that enables, by combining coevolution and deep learning, the prediction of residue-residue interactions (28). Using this approach, 2 putative motifs of interaction between BRSrr2 and α5β1 were identified (Supplemental Figure 4A and Figure 2E). The first was an FSVKI motif located in the N-terminal subdomain of BRSrr2 at position 362 and the second was an ETYVI motif located in the C-terminal subdomain of BRSrr2 at position 496 (Figure 2, E and F). These 2 motifs are absent from the BRSrr1 sequence. To address the role of these 2 motifs in the interaction with integrin α5β1, we first addressed the binding capacities of BRSrr2 subdomains containing these motifs. The N-terminal and C-terminal subdomains of BRSrr2 containing either motif were produced, and an equimolar amount of full-length BRSrr2, N-terminal, and C-terminal domains was used to perform ELISA binding assays. The α5β1 integrin displayed a similar capacity to bind to all 3 forms of BRSrr2 (Figure 2G), indicating the presence of at least one binding motif on each BRSrr2 subdomain.

We next generated mutated forms of BRSrr2 in which the FSVKI or the ETYVI sequences were replaced by FAAAI and AAYAI, respectively (Supplemental Figure 1, D and E), and the interaction with integrin α51 was assessed by ELISA. Although the mutation of the ETYVI motif affected the interaction with α5β1 integrin only at the highest concentration, the mutation of the FSVKI motif significantly reduced the binding to α5β1 integrin at most concentrations (Figure 2H).

Altogether, we identified the FSVKI motif of BRSrr2 as required for α5β1 integrin recognition. In addition, we demonstrated that BRSrr2 contains at least one other integrin α5β1 recognition motif, which is located in its C-terminal region but remains to be identified. Interestingly, when integrin-binding motifs were highlighted on BRSrr2, we noticed that both αvβ3 binding motifs (RGD and SDV) were localized on the same side of BRSrr2, whereas the α5β1 binding motif, FSVKI, was located on the opposite side (Supplemental Figure 4B).

Numerous CC17-GBS bacteria were observed adhering to CHO-α5β1 cells (Figure 3A arrows) as compared with nontransfected CHO or CHO-ICAM1 cells that were nonpermissive for CC17 adhesion where most streptococci were found unbound to cells (Figure 3 A, arrowheads). Immunostaining with anti-α5 antibody showed that CC17-GBS bacteria were almost exclusively associated with cells expressing integrin α5β1, whereas few bacteria were associated with cells that had lost its expression (Figure 3B). In addition, few CC17-GBS bacteria adhered to CHO-αvβ3 (Figure 3A). However, only the adhesion to α5β1-expressing cells was statistically significant (Figure 3C). We next analyzed the specificity of CC17-GBS interaction with α5β1 integrin by testing adhesion of a non-CC17 GBS strain (CC23) expressing the Srr1 surface protein to CHO transfected cells. The CC23-GBS strain did not show proficient adhesion to any CHO cell lines tested (Figure 3C). Altogether, these results indicate that integrin α5β1 is an effective and specific host receptor for CC17-GBS.

Finally, using differential staining to distinguish extracellular from intracellular streptococci, we were able to detect internalized CC17-GFP bacteria in CHO-α5β1 cells (Figure 3E). Comparing invasion rates obtained with CHO-α5β1 and with untransfected cells, CFU counting indicated that α5β1 integrin also promoted CC17-GBS internalization in an Srr2-dependent manner (Figure 3F). In conclusion, our data demonstrated that Srr2 expression allowed α5β1 integrin recognition, promoting CC17-GBS adhesion and invasion in a simplified cellular model.

α5β1 and αvβ3 integrins are overexpressed during the postnatal period. CC17-GBS is overrepresented among GBS neonatal meningitis, accounting for more than 80% of the cases (4, 9, 11). In contrast, epidemiological data show that GBS is an uncommon cause of meningitis in adults and CC17 is only found in 21% of the cases (37, 38). Meningeal pathogens can enter the brain via the BBB and/or the BCSFB located in the choroid plexuses of the ventricular area (12). We therefore hypothesized that neonatal susceptibility to CC17-GBS meningitis might be correlated with the expression of α5β1 and αvβ3 integrins at the BBB/BCSFB level. To test this hypothesis, cerebral blood vessels from neonatal and adult rats were purified and the expression of α5β1 and αvβ3 was assessed by immunofluorescence staining and Western blot analysis. A striking, intense α5 integrin staining was observed on brain vessels from pups, whereas adult brain vessels displayed a much less intense staining (Figure 5A). Overexpression of α5 integrin in neonatal brain vessels was confirmed by Western blot analysis (Figure 5B). Although no β3 expression was detected on adult brain vessels, either by immunofluorescence or by Western blot, a faint but easily detectable β3 expression was observed in brain vessels from rat pups (Figure 5, B and C). These results indicate that α5β1 and αvβ3 integrins are overexpressed in rat brain vessels during the neonatal period.

Choroid plexuses are composed of blood vessels that are different from those of the BBB, and of stroma and choroid epithelial cells that form the BCSFB (39). Therefore, choroid plexus tissues from the fourth ventricle (CP4V) or the lateral ventricles (CPLV) were collected and analyzed for α5 and β3 expression. We found that α5 integrin was overexpressed in rat pups compared with adults, and β3 integrin expression could only be detected in the choroid plexuses of rat pups (Figure 5D).

Importantly, similar results were obtained for human brain samples by immunohistochemistry analyses of the brain of a newborn (9 days old) and an adult (52 years old), for whom the causes of death were unrelated to meningitis. In the cortex, α5 integrin staining mainly revealed cerebral blood vessels in the neonate and adult. However, the staining intensity of the cerebral blood vessels was strikingly more intense in the neonatal cortex compared with the adult. In the neonate, β3 expression was very faint and observed in slightly bigger blood vessels than capillaries, whereas it was totally absent in the adult cortex (Figure 5E).

When brain sections from the ventricular area were specifically analyzed for α5 expression, a massive staining of all choroid plexus blood vessels was observed in the human neonatal specimen (Figure 5F, arrows). In contrast to and except for a few infiltrating cells, α5 expression was not detected in the adult specimen (Figure 5F, arrowheads). Similar to what was observed in the cortex, β3 expression was restricted to the biggest blood vessels in the choroid plexuses (Figure 5F, arrows) and absent from smaller blood vessels (Figure 5F, arrowheads), whereas it was totally absent in the adult choroid plexuses (Figure 5F). Importantly, neither α5 nor β3 integrins were detected in choroid plexus epithelial cells of either the neonate or the adult (Figure 5F). Altogether, these data indicate that α5β1 and αvβ3 integrins are overexpressed in human blood vessels of the BBB and the BCSFB during the neonatal period.

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