Suilysin remodels the cytoskeletons of human brain microvascular endothelial cells by activating RhoA and Rac1 GTPase

Streptococcus suis (S. suis) is a Gram-positive, facultative anaerobic coccus and an important emerging pathogen. It is associated with bacterial meningitis in adults, especially in southeastern Asia (Wertheim et al., 2009). A striking feature of S. suis infection is the resulting complications, which may include deafness and vestibular dysfunction. Complications of some kind affect 50% of S. suis patients in Europe and 73% of those in Asia (Gottschalk et al., 2010). Some S. suis surface-associated factors, such as the capsule and Fhb, may affect the pathogenesis of meningitis. Of these secreted factors, suilysin was found to be the most important. It has been found to be toxic to various types of cells (Lalonde et al., 2000). It is also involved in modulation of the interactions of S. suis with different host cells (Charland et al., 2000; Segura et al., 2006). Epidemic S. suis ST7 strains were found to produce more suilysin than other ST strains (unpublished data). This has been shown to contribute to their ability to travel across the epithelial barrier, which they do in a TLR4-dependent manner. This ability is also associated with the increased severity of S. suis infection (unpublished data). Recently, subcytolytic suilysin was shown to promote S. suis association with epithelial cells without causing the formation of functional (cytolytic) pores. This indicated that sublytic concentrations of suilysin also contributed to pathogenesis by modification of host-pathogen interactions (Seitz et al., 2013). However, the mechanism underlying suilysin-mediated modulation of microbialhost interactions has not yet been fully explained. In the present study, human brain microvascular endothelial cells (hBMECs) were treated with non-cytotoxic and sublytic concentrations of the culture supernatant of S. suis serotype 2 strain 05ZYH33 and stained with FITC-labeled phalloidin at different time points after challenging the culture with sublytic supernatant. Fluorescence microscopy showed stress fibers, lamellipodia, and filopodia to be visible 5– 40 min after treatment (Fig. S1). These results demonstrated that sublytic concentrations of S. suis culture supernatant rearranged the cytoskeletons of the hBMECs. Suilysin has been shown to be the most important virulence factor in the secreted supernatant of S. suis. Suilysin is presumed to be the effective component of S. suis culture supernatant, and the factor primarily responsible for the remodeling of hBMEC cytoskeletons. To confirm this, hBMEC cells were treated with sublytic concentrations of suilysin protein (0.3 μg/mL) purified from S. suis cell culture supernatant over different periods of time. As shown in Fig. 1, suilysin led to the rapid formation of filopodia, stress fibers, and lamellipodia in hBMECs. Lipids and cholesterol have been identified as the receptors of suilysin, as well as some cholesterol-dependent cytolysins (CDCs). To confirm the cholesterol dependence of the effects of suilysin, suilysin was exposed to cholesterol at mass ratios of 1:1 and 1:5 for 15 min at 37°C immediately before it was applied to the hBMEC cells for 10 min. As shown in Fig. 1, pretreatment of suilysin with cholesterol at a 1:1 ratio reduced the formation of lamellipodia and filopodia, although actin stress fibers still formed. When suilysin was pretreated with cholesterol at a 5:1 cholesterol:suilysin ratio, the suilysininduced changes in actin were completely absent. The changes in actin were also absent when cellular cholesterol was removed using 2 mmol/L methyl-β-cyclodextrin (MβCD) to inhibit suilysin binding. Taken together, these results indicate that the changes in actin cytoskeleton organization induced by suilysin were cholesterol-dependent. Generally, Rho-subclass GTPases produce stress fibers, Rac produces filopodia and lamellipodia, and Cdc42 produces filopodia (Tapon and Hall, 1997). To confirm the effects of suilysin on these pathways, RhoA-associated kinase (ROCK) inhibitor Y27632 was used to pretreat hBMECs for 1 h before suilysin challenge. The inhibitor Y27632 prevented the formation of stress fibers after 10 min of suilysin treatment (Fig. 1). However, RhoA, which is upstream of ROCK in the signaling pathway, was still activated (Fig. 2E). These results indicated that the changes in actin cytoskeleton organization induced by suilysin were GTPase-dependent. Next, the molecular basis of the changes in actin induced by S. suis culture supernatant was assessed. A rhotekin-based pull-down assay showed approximately 3-fold activation of RhoA 5 min after treatment by sublytic concentration of S. suis culture supernatant, which peaked at 10 min and was found to have returned to


Bacteria, cells, and culture conditions
Streptococcus suis serotype 2 strain 05ZYH33 was originally isolated from human patients in Sichuan, China. The strain was positive for muramidase-released protein (MRP), extracellular factor (EF), and suilysin (SLY). Streptococcus suis serotype 2 strain 1330 was originally isolated from healthy piglets in Canada. The strain was negative for MRP, EF, and SLY. Bacteria were grown overnight on sheep blood agar plates at 37 °C and isolated colonies were used as inocula to culture in Todd-Hewitt broth (THB) at 37 °C in 5% CO2 atmosphere.
Cells of a human brain microvascular endothelial cell line, hBMECs, were immortalized by transfection with the SV40 large T antigen and retain the morphologic and functional characteristics of primary brain endothelial cells. They were successfully used in other studies to model human BBB interactions with host cells and infectious microorganisms. Here, the cells were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 10% Nu-Serum (BD BioSciences, Palo Alto, CA, U.S.), 2 mM glutamine, 1 mM pyruvate, penicillin (100 U/ml), streptomycin (100 μg/ml), essential amino acids, and vitamins. They were incubated at 37 °C in a humidified 5% CO2 atmosphere.

Preparation of S. suis culture supernatant
Single colonies of S. suis strain was inoculated into THB medium and cultured overnight at 37 °C in 5% CO2. The next day, bacteria were inoculated at 1% into new THB medium and cultured further for 5 h. Bacteria were collected and centrifuged at 10,000 × g at 4 °C for 10 min, the supernatant was collected and passed through a 0.2 μm sterile filter.

Preparation of suilysin protein
Natural suilysin protein was isolated and purified from the culture supernatant of S. suis strain 05ZYH33 by ammonium sulfate precipitation, followed by anion-exchange, and then hydrophobic chromatography. Escherichia coli BL21 (DE3) were induced to express histidine-tagged recombinant suilysin (wild and P353L mutant) proteins with 1 mM IPTG. They were expressed as inclusion bodies. The pellet was purified and refolded using a simple and efficient purification process and on-column refolding according to the recombinant protein purification handbook (GE Healthcare).

Hemolytic activity and cytotoxicity activity assays
The hemolytic activity of S. suis culture supernatant and purified suilysin proteins was determined by titration as described previously (Jacobs et al., 1994). The reciprocal of the highest dilution of a sample that exhibited at least 50% of red blood cell lysis was

Actin staining
hBMEC cells were seeded in complete culture medium on glass coverslips in a 12-well culture plate. Once the cells were 30-40% confluent, the medium was replaced with culture medium containing 1% FBS and cultured for another day. After serum starvation, cells were incubated in serum-free medium and treated with suilysin.
After fixation in 4% paraformaldehyde in PBS, cells were permeabilized with 0.2% Triton-X 100 in PBS and incubated with FITC-phalloidin for 30 min at room temperature in the dark. Actin filaments were viewed on a fluorescence microscopy (BX51, Olympus) system by using a 100X oil immersion objective.

GTPase activity assays
GTPase activity was evaluated using Rho, Rac1, and Cdc42 activity kits (Cytoskeleton, Inc., USA) according to the manufacturer's instructions. Briefly, cells were grown under appropriate culture conditions. After treatment as described in the manual, hBMEC cells were washed once with ice-cold PBS (pH 7.2) and lysed on ice with lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, 2.5 mM Na3VO4) supplemented with phosphatase cocktail, and complete mini protease inhibitor (Roche). Cell lysates were harvested with a cell scraper and centrifuged at 10,000 × g, 4 °C for 2 min. Cell lysates were incubated with rhotekin-RBD beads (for evaluation of Rho activity) or PAK-PBD beads (for evaluated of Rac1 and Cdc42 activity) for 1 h at 4 °C under gentle rotation and then washed. Bound proteins were analyzed using western blotting. The protein signals were developed with SuperSignal West Dura Extended Duration substrate (Pierce) and imaged using an NE-1000 charge-coupled device (CCD, BGI-GBI Biotech Co., China).