The serine/threonine protein kinase of Streptococcus suis serotype 2 affects the ability of the pathogen to penetrate the blood-brain barrier.

Streptococcus suis serotype 2 (SS2) is a zoonotic agent that causes meningitis in humans and pigs. However, the mechanism whereby SS2 crosses the microvasculature endothelium of the brain is not understood. In this study, transposon (TnYLB-1) mutagenesis was used to identify virulence factors potentially associated with invasive ability in pathogenic SS2. A poorly invasive mutant was identified and was found to contain a TnYLB-1 insertion in the serine/threonine kinase (stk) gene. Transwell chambers containing hBMECs were used to model the blood-brain barrier (BBB). We observed that the SS2 wild-type ZY05719 strain crossed the BBB model more readily than the mutant strain. Hence, we speculated that STK is associated with the ability of crossing blood-brain barrier in SS2. In vitro, compared with ZY05719, the ability of the stk-deficient strain (Δstk) to adhere to and invade both hBMECs and bEnd.3 cells, as well as to cross the BBB, was significantly attenuated. Immunocytochemistry using antibodies against claudin-5 in bEnd.3 cells showed that infection by ZY05719 disrupted BBB tight junction proteins to a greater extent than in infection by Δstk. The studies revealed that SS2 initially binds at or near intercellular junctions and crosses the BBB via paracellular traversal. Claudin-5 mRNA levels were indistinguishable in ZY05719- and Δstk-infected cells. This result indicated that the decrease of claudin-5 was maybe induced by protein degradation. Cells infected by ZY05719 exhibited higher ubiquitination levels than cells infected by Δstk. This result indicated that ubiquitination was involved in the degradation of claudin-5. Differential proteomic analysis showed that E3 ubiquitin protein ligase HECTD1 decreased by 1.5-fold in Δstk-infected bEnd.3 cells relative to ZY05719-infected cells. Together, the results suggested that STK may affect the expression of E3 ubiquitin ligase HECTD1 and subsequently increase the degradation of claudin-5, thus enabling SS2 to traverse the BBB.

Virulent and Vaccine Strains of Streptococcus equi ssp. zooepidemicus Have Different Influences on Phagocytosis and Cytokine Secretion of Macrophages.

Swine streptococcosis is a significant threat to the Chinese pig industry, and Streptococcus equi ssp. zooepidemicus (SEZ) is one of the major pathogens. SEZ ATCC35246 is a classical virulent strain, while SEZ ST171 is a Chinese attenuated vaccine strain. In this study, we employed stable isotope labeling by amino acids in cell culture and liquid chromatography-mass spectrometry (LC-MS) to determine the differential response of macrophages to infection by these two strains. Eighty-seven upregulated proteins and 135 downregulated proteins were identified. The proteomic results were verified by real-time polymerase chain reaction for 10 chosen genes and Western blotting for three proteins. All differentially abundant proteins were analyzed for their Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations. Certain downregulated proteins were associated with immunity functions, and the upregulated proteins were related to cytomembrane and cytoskeleton regulation. The phagocytosis rate and cytokine genes transcription in Raw264.7 cells during SEZ ATCC35246 and ST171 infection were detected to confirm the bioinformatics results. These results showed that different effects on macrophage phagocytosis and cytokine expression might explain the different phenotypes of SEZ ATCC35246 and ST171 infection. This research provided clues to the mechanisms of host immunity responses to SEZ ST171and SEZ ATCC35246, which could identify potential therapy and vaccine development targets.

SILAC and LC-MS/MS identification of Streptococcus equi ssp. zooepidemicus proteins that contribute to mouse brain microvascular endothelial cell infection.

Streptococcus equi ssp. zooepidemicus (SEZ) causes meningitis in both humans and animals. Some dissociative proteins of SEZ are cytotoxic to mouse brain microvascular endothelial cells (mBMECs) and may contribute to the penetration of SEZ across the blood-brain barrier (BBB). In this study, the ability of SEZ to penetrate across an in vitro BBB model was confirmed. We used stable isotope labeling with amino acids in cell culture (SILAC) to label SEZ proteins with heavy or light isotope-tagged amino acids, along with LC-MS/MS to determine which SEZ proteins were involved in interactions with mBMECs. The efficiency of SEZ protein isotope labeling was 94.7 %, which was sufficient for further analysis. Forty-nine labeled peptides were identified as binding to mBMECs, which matched to 25 SEZ proteins. Bioinformatic analysis indicated that most of these proteins were cytoplasmic. These proteins may have functions in breaching the host BBB, and some of them are known virulence factors in other bacteria. Indirect immunofluorescence results indicated that SEZ enolase had binding activity toward mBMECs. Protective test results showed that enolase was a protective antigen against SEZ infection. This research is the first application of SILAC combined with LC-MS/MS to identify SEZ proteins that may contribute to the infection of mBMECs and potentially show functions related to breaching the BBB. The outcomes provide many future avenues for research into the mechanism of SEZ-induced meningitis.