The Mechanism Of M-Like Protein Facilitates Streptococcus Equi SSP.Zooepidemicus Resisting Macrophage Phagocytize

Streptococcus equi ssp. zooepidemicus (SEZ), a member of the Lancifield’s group C, is an pathogen that could infect a wide variety of species, including important domesticated animals such as horses, cows, swine, sheep, and dogs, causing septicemia, arthritis, endocarditis and meningitis.. In China, SEZ is the major cause of diseases in swine. It can infect humans via zoonotic transmission from domesticated animals and cause invasive infections in humans. Pathogenic microorganisms in a host must evade from the immune system before the infection can be established. Our paper was about the mechanism of M-like protein facilitates antiphagocytosis and the relationship among UDP-Glucose Pyrophosphorylase, hyaluronic acid capsule and bacterial virulence.1. Screen the interacting protein of SEZ M-like protein in macrophage cDNA libraryMacrophage plays an important role in the host immunity system. Streptococcus equi ssp. zooepidemicus can resist macrophage phagocytize with the help of its surface M-like protein (SzP). In order to indicate the molecular mechanism of this antiphagocytosis, we constructed a yeast two-hybrid cDNA library from porcine pulmonary alveolar macrophage (PAM). Total RNA was prepared from porcine PAM. First-strand cDNA was synthesized from the purified RNA. Double-stranded cDNA was amplified and ligated to adaptor, digested with Sfil enzyme and removed the small fragments, then cloned into the pPR3-N vector. The recombinant vector was electro-transformed into E. coli DH10B to obtain a primary cDNA library. The primary library was amplified and used to determine the size of cDNA inserts through enzyme digestion. The total RNA was good quality. The primary cDNA library titer was2×106CFU/mL and the amplified library titer was1×109CFU/mL. The size of the inserts varied from750to2500bp, with an average value of about1000bp. A yeast two-hybrid cDNA library has been successfully generated from porcine PAM and can be used for future screening of proteins interacting with M-like protein of SEZ. We constructed the SzP-pDHB1yeast bait strain and confirmed the expression of SzP and the function of this bait strain. After the heterotrophia selection and β-galactosidase activity analysis, we got12candidate proteins which maybe interact with SzP from the cDNA library of PAM with split-ubiquitin yeast two-hybrid system. The co-immunoprecipitation results showed that the thioredoxin of PAM interacted with SzP. Our findings could contribute to the general understanding on how SzP could confer phagocytosis resistance.2. The mechanism of interaction between M-like protein and thioredoxin facilitates antiphagocytosis for SEZM-like protein (SzP) is an important virulence factor of SEZ and contributes to bacterial infection and antiphagocytosis. The interaction between SzP of SEZ and porcine thioredoxin (TRX) was revealed by Yeast Two-hybrid and further confirmed by co-immunoprecipitation. SzP interacted with both reduced and oxidized TRX without inhibiting TRX activity. We found that when TRX was recruited by SzP anchored on the surface of SEZ, it facilitated antiphagocytosis of the bacteria. Further experiments revealed that TRX regulated the alternative complement pathway by inhibiting the activity of C3convertase and had affinity to Factor H (FH). TRX alone inhibited C3cleavage and C3a production, the inhibitory effect was additive when FH was also present. TRX inhibited C3deposition on the bacterial surface when recruited by SzP of SEZ. These new findings indicated SEZ recruited TRX by SzP and regulated alternative complement pathways to evade host immune phagocytosis.3. The effect of M-like protein in SEZ infecting macrophageTo increase our knowledge of the mechanism of SzP in infection, we profiled the response of porcine pulmonary alveolar macrophage (PAM) to infection of SEZ ATCC35246wild strain and SzP-knockout strain using the Roche NimbleGen Porcine Genome Expression Array. We found SzP contributed to differential expression (DE) of446genes, with upregulation of134genes and downregulation of312genes, some of these downregulated genes were related to defence pathogen infection. Gene Ontology category showed that these genes were represented in a variety of functional categories, including genes involved in immune response, regulation of chemokine production, signal transduction and regulation of apoptosis. Three important signal pathways were revealed by the KEGG pathway analysis, including Jak-STAT signal pathway, Cytokine-cytokine receptor interaction pathway and Toll-like receptor signal pathway. STRING anlysis show the relationship among some of these DE genes. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR on12representative genes. The data will contribute to understanding of SzP mediated mechanisms of SEZ pathogenesis.4. Molecular cloning and analysis of the UDP-glucose gene pyrophosphorylase in SEZCapsula of hyaluronic acid (HA) is considered an important virulence factor in other streptococci. UDP-Glucose Pyrophosphorylase (EC2.7.7.9, UGPase) plays an important role in SEZ hyaluronic acid capsula biosynthesis and it is also recognized as a virulence determinant in several bacterial species. In this study, The UGPase gene fragment (789bp) obtained from previous research was amplified using PCR, and located by Genome walking technology (GenBank No.GQ423507). The UGPase was expressed, purified and identified using UGPase antibody. The enzyme kinetic parameters were determined, the temperature and pH of the highest activity for the cloned UGPase were37℃, pH7.5. The Km and Kcat value against UTP and G-1-P was8.5μM,69.05s-1and36.41uM,48.81s-1respectively. The homology-modeling was operated. Overexpression of the UGPase in SEZ, its virulence was affected. Real-Time PCR was carried out to determine the UGPase expression levels of both SEZp and SEZugp in different grow period, the level is high in logarithmic phase and low in Decline phase.