Suilysin remodels the cytoskeletons of human brain microvascular endothelial cells by activating RhoA and Rac1 GTPase
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KeywordsActin Cytoskeleton Bacterial Meningitis Stress Fiber Hemolytic Activity Human Brain Microvascular Endothelial Cell
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 Epidemic S. suis ST7 strains were found to produce more suilysin than other ST strains (unpublished data).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 This ability is also associated with the increased severity of S. suis infection (unpublished data).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 microbial-host interactions has not yet been fully explained.
Because the suilysin-induced changes in the organization of the actin cytoskeleton were found to be cholesterol-dependent, the issue of whether the suilysin-induced activation of RhoA is also cholesterol-dependent was addressed. Suilysin was pretreated with cholesterol at mass ratios of 1:1 and 1:5 for 15 min at 37°C. Then, the hBMEC cells were treated with this suilysin-cholesterol mixture for 10 min. As shown in Fig. 2E, suilysin pretreated with cholesterol at a 1:1 ratio still activated RhoA. When suilysin was pretreated with cholesterol at a 5:1 cholesterol: suilysin ratio, the activation of RhoA induced by suilysin was completely inhibited. Previous studies have shown that hemolysin-positive S. suis strains used adherence and suilysin-induced BMEC injury to move from the circulatory system to the central nervous system (Charland et al., 2000). This indicates that hemolytic activity is the basis for suilysin activity. However, in the present work, sublytic concentrations of suilysin were found to activate RhoA and Rac1, causing remodeling of the actin cytoskeleton. This indicated that suilysin might have functions independent of hemolytic activity. To further analyze the correlation between the GTPase activation induced by suilysin and the hemolytic activity of suilysin, a recombinant suilysin mutant, SLY (P353L), which was proven to lack hemolytic ability, was constructed (Xu et al., 2010). Different concentrations of purified SLY (P353L) protein were used to treat hBMECs for 10 min, and active GTPases were analyzed using pull-down assays. As shown in Fig. 2F, SLY (P353L), cannot activate RhoA, but can activate Rac1 at relatively low concentrations (0.27 μg/mL). These results suggested that the ability of suilysin to activate Rac1 was independent of its hemolytic activity.
Pathogenic microbes subvert normal host cell processes to create a specialized niche, facilitating their survival. Pathogens often target the cytoskeleton of the host cell, which they use for attachment, entry into cells, movement within and between cells, vacuole formation and remodeling, and avoidance of pathocytosis (Barbieri et al., 2002; Rottner et al., 2005). In this work, non-cytotoxic and sublytic concentrations of S. suis suilysin were found to remodel the actin cytoskeleton. These results contribute to understanding of the manner by which S. suis traverses the human blood-brain barrier (BBB). Recent studies also have shown that microbial translocation of the BBB involves host cell actin cytoskeletal rearrangements (Kim, 2006). For example, human BMEC actin cytoskeleton rearrangements were shown to be a prerequisite for human BMEC invasion by E. coli K1, group B streptococcus, and L. monocytogenes (Kim, 2006). In addition to invasion, some pathogens, such as Neisseria meningococcus, induce changes in the host cell cytoskeleton, recruiting endothelial cell adhesion molecules such as VE-cadherin to cortical plaques. This recruitment leads to the formation of ectopic intercellular junctional domains at the site of bacteria-host cell interaction, depletion of junctional proteins at the cell-cell interface, opening of intercellular junctions, and crossing the BBB in a paracellular manner (Coureuil et al., 2009; Coureuil et al., 2010). For this reason, actin rearrangement induced by suilysin might initiate the paracellular traversal of S. suis across the human BBB.
Several signal transduction pathways have been shown to be involved in rearranging the actin cytoskeleton. These include the FAK, PI3K, Src kinase, and Rho GTPase pathways. Among the members of the CDC group, pneumolysin is the only known pore-forming toxin that uses the small GTPases found in intact cells to alter the actin cytoskeleton (Iliev et al., 2007; Hupp et al., 2013). The present results demonstrate another case, showing that actin cytoskeleton rearrangment is a novel function of suilysin. These findings contribute to the current understanding of the mechanism by which S. suis traverses the human BBB, suggesting new potential targets for the treatment of meningitis.
This work was supported by the National Natural Science Foundation of China (Grant Nos. 81171528 and 81371766), the National Basic Research Program (973 Program) (No. 2012CB518804), Chinese State Key Project Specialized for Infectious Diseases (2013ZX10004101-003) and China Postdoctoral Science Foundation (2012M510583).
Qingyu Lv, Huaijie Hao, Lili Bi, Yuling Zheng, Xuyu Zhou, and Yongqiang Jiang declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by the any of the authors.
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