Streptococcus Suis Human Brain Microvascular Endothelial Cell Interaction Studies

Streptococcus suis (S.suis) is an important emerging pathogen that can cause severe systemic infection in humans with high mortality and morbility. Two large outbreaks of S.suis infection occurred in Jiangsu Province in 1998 and Sichuan Province in 2005. More than 200 cases were infected and 50 cases died during these two outbreaks. Meningitis is the most important clinical feature of S.suis infection in humans, however, the pathogenesis mechanism of S.suis meningitis is still not well elucidated. In general, S.suis could develop meningitis through several steps of bacteria-host interactions. These steps include that how bacteria present at low levels on mucosal surfaces are able to traverse the first mucosal barriers into the bloodstream, how bacteria survive/multiply in blood and result in bacteraemia, how bacteria traverse the blood brain barrier (BBB) into centre nervous system.The interactions of S.suis with brain microvascular endothelial cells (BMEC) are an important process for S.suis passage across the BBB. At present, the interactions of S.suis with different originated BMEC have been charactered, for example, S.suis could adhere to porcine and human BMEC, but could only invade pBMEC, S.suis could stimulate BMEC to secrete cytokines. But the detailed cellular and molecular mechanisms of the interactions between S.suis and BMEC are still unclear.The interactions of S.suis with hBMEC were studied in three aspects: the self-regulation of S.suis to respond the hBMEC cell-contact; the gene expression profile of hBMEC during the S.suis infection; the remodeling of cytoskeleton by suilysin.Part I: The gene expression profile of S.suis upon hBMEC cell-contact.DNA microarray was used to study the gene expression profile of S.suis upon hBMEC cell-contact. After cell-contact with hBMEC 1 h and 3 h, S.suis changed the expression of 219 genes (131 up-regulated genes and 88 down-regulated genes) and 175 genes (123 up-regulated genes and 52 down-regulated genes), respectively. Differential regulated genes were grouped by Clusters of Orthologous Groups of proteins (COGs) according to S.suis 05ZYH33 genome, and among the regulated genes which respond for translation, amino acid transport, energy production, cell cycle control, cell wall biogenesis as well as lipid transport, the number of up-regulated genes was more than that of down-regulated genes. Among the regulated genes which respond for nucleotide transport and carbohydrate transport, the number of down-regulated genes were more than that of up-regulated genes.The expression of capsule polysaccharide synthesis related genes was increased after S.suis contacted with hBMEC and capsule was confirmed to be thickened by transmission electron microscopy. Besides CPS, some known virulence genes such as epf, mrp, ofs were also up-regulated, but ADS and sly genes were down-regulated. After cell-contact with hBMEC, S.suis up-regulated the expression of those genes encoding for cell wall biogenesis, pili (sfp1, sfp2), cell wall proteins (such as sao, SSU05₀272) as well as dltB gene which is required to catalyze the incorporation of D-alanine residues into the lipoteichoic acids, suggesting that upon cell-contact, S.suis undergoes substantial surface remodeling. Besides, after cell-contact with hBMEC, S.suis up-regulated the expression of genes involved in lipid synthesis, gene replication, transcript and translation as well as the expression of the gene cluster encoding the ATP synthase F1 and F0 subunits (atpC, atpD, atpA, atpH, atpF) and cell division related genes. These regulated genes might contribute to increase metabolism and cell cycle of S.suis.Based on the data of expression profile of S.suis upon cell-contact, we studied the molecular mechanism of the capsule thickening. Experiment results showed that the capsule thickening of S.suis upon cell-contact was related to two component signal transduction 2148/2149 and Rgg, but not to covR. The detailed mechanism is that, Rgg could sense the cell-contact signal by unknown mechanism and transduct the signal to 2148/2149 which could upregulate the expression of CPS related genes by unknown mechanism. CovR could negatively regulate the expression of CPS genes, but after cell-contact, there are some unknown mechanisms which could hinder the negative regulation of covR to CPS genes.After the analysis of the gene expression profile of S.suis upon hBMEC cell-contact, we presume that the regulated genes might contribute to S.suis passage across the BBB. Firstly, Thickened capsule and D-alanylation of LTAs may allow S.suis to escape the clearance from phagocytes and the killing from cationic antimicrobial peptides. The capsule of S.suis serotype 2 itself could also induce the MCP-1 release through a TLR2- and MyD88- independent pathway. And MCP-1 has been confirmed to down-regulate the expression and/or distribution of the tight junction-associated proteins such as ZO-1 and occludin in BMEC to alter BBB interity. Secondly, the up-regulated genes which encode pili, cell wall proteins and biogenesis might contribute to the recognization of S.suis with hBMEC receptors, the adhesion of S.suis upon hBMEC and the activation of hBMEC to secrete cytokines. Thirdly, the up-regulated genes which encode cell cycle, metabolism and protein translation might contribute to the extracellular multiplication of S.suis.Part II: The expression profile of hBMEC infected by S.suis.Wild-type S.suis 261 strain and its 2148/2149 knock-out mutant were used to infect hCMEC/D3 with the multiplicity of infection (MOI) 1:1, respectively, and the untreated hCMEC/D3 cells were used as negative control. After the infection, the total RNA of hCMEC/D3 cells were isolated, reversed transcript and labeled fluorescent dyes, then hybridized with Agilent SurePrint G3 human GE 8×60K.To analyze the chip data, differential expression genes were screened according to the criteria that q-value(%) of gene is not more than 5 and Fold Change of gene is more than 1.5. Compared to untreated groups, 2780 genes (446 genes up-regulated, 2334 genes down-regulated) showed differential expression in S.suis 261-infected groups at 4 h post infection (p.i.), and 2926 genes (611 genes up-regulated, 2315 genes down-regulated) showed differential expression in S.suis 2148/2149 knock-out mutant groups.Further analysis showed that hCMEC/D3 up-regulated the expression of several chemokines such as IL-8, MCP-1, CXCL-1, CXCL-2 and cytokines such as IL-1?, IL-6, IL-11, GM-CSF as well as cell adhesion molecules such as selectin E, ICAM-4, and down-regulated the expression of tight junction-related genes such as ZO-1, claudin-5, after infected 4 h by S.suis 261 strain or ?2148/2149 strain. Besides, hCMEC/D3 also changed the expression of genes encoding cell surface antigens such as CD34, CD59, CD93, CD83 and CD274.Based on the expression profile of hCMEC/D3 infected by S.suis and the progress of study on the interactions of S.suis and hBMEC, we presumed that S.suis might traverse the BMEC through paracellular pathway. First, S.suis could not invade hBMEC and S.suis has low adhesion ratio to BMEC. Second, S.suis could up-regulate the expression of genes encoding cytokines and chemokines as well as cell adhesion molecules, and these regulated genes might contribute to recruit the neutrophils and monocytes and increase the adhesion of leukocytes to endothelial cells. Once neutrophils adhere to endothelial cells, they could activate the signal pathway of endothelial cells, regulate the distribution of tight junction proteins and increase the endothelial permeability. Third, S.suis could down-regulate the expression of genes such as ZO-1, Claudin 5 and par-6 which are very important to maintain the function of tight junction.Part III: The remodeling of cytoskeleton in hBMEC by suilysin.Fluorescence microscope observation showed that S.suis culture supernatant and SLY protein could induce hBMEC to form stress fibers, filipodium and lamellipodia at sublytic and non-cytotoxic concentration. These effects were cholesterol-dependent, because after pretreatment of SLY with cholesterol at different ratios, the SLY-induced actin changes were partly or completely inhibited, and similarly inhibited effects were also observed after cellular cholesterol depletion by M?CD.To elucidate the molecular mechanism of the cytoskeleton remodeling induced by SLY protein, the GTP-RhoA proteins in the cell lysates treated by S.suis supernatant or SLY protein were pulled down using anti-Rhotekin, the GTP-Rac1 and GTP-Cdc42 proteins in the cell lysates treated by S.suis supernatant or SLY protein were pulled down using anti-p21 activated kinase antibody, and the activation of GTPase was identified by Western blot analysis. Experimental results showed that S.suis supernatant and SLY protein could time-dependently activate RhoA and Rac1, and the activation effects reach to highest value at 10 min. But S.suis supernatant and SLY protein could not activate Cdc42 in our study.In conclusion, the interactions of S.suis with hBMEC were studied in two aspects, the responses of S.suis upon hBMEC cell-contact, the responses of hBMEC by S.suis infection. For the pathogen, S.suis up-regulated the expression of CPS genes through Rgg and 2148/2149 two-component systems, and thickened capsule and D-alanylation of LTA might help S.suis to escape killing from phagocytes and cationic antimicrobial peptides. Besides, after cell-contact, S.suis up-regulated the expression of those genes involved in pili, cell wall biogenesis and cell wall proteins. The remodeling of S.suis surface might contribute to the contact of S.suis with hBMEC and the activation of hBMEC signal pathway to secrete cytokines. On other hands, S.suis also up-regulated the expression of genes encoding cell division to increase the cell cycle. All these regulated genes might help S.suis to survive/multiply upon hBMEC surface. For the host cells, after S.suis infection, hBMEC up-regulated the expression of chemokines and cytokines as well as cell adhesion molecules, down-regulated the expression of those proteins involved in tight junction formation and altered the expression of cell surface antigens. All these changes might increase the BMEC permeability. Besides, S.suis supernatant and suilysin could remodel the cytoskeleton of hBMEC to form stress fibers, filipodium and lamellipodia by activating RhoA and Rac1 of hBMEC. Since actin could bind all tight junction proteins, the re-distribution of actin might re-distribute the tight junction proteins and increase the space between two BMEC cells. We presume all these changes of S.suis and brain microvascular endothelial cells might contribute to the passage of S.suis across blood brain barrier.