The Study On The Interaction Between Streptococcus Suis2and Pig Neutrophils

Streptococcus suis serotype2（S. suis2, SS2） is one of the most important swine pathogens, and an emerging, life-threatening zoonotic agent in both pigs and humans. Since high economic losses as well as threat to human life, the study of SS2pathogenesis and the development of new vaccines is crucial for prevention and control of this disease.Development of the disease requires temporal and coordinated expression of a series of genes that allow the prospective pathogen to shift to its pathogenic state and adapt to a hostile environment in the host. Two-component regulatory systems （TCS） control the expression of virulence factors in a wide range of bacterial species in response to external stimuli. For bacterial pathogens, they have evolved virulence factors and regulation systems that allow adaptation to different environments within a host ensuring their survival.SS2current research focuses on the virulence factors discovery and functional identification. The Chinese isolated SS2strains98HAH12and05ZYH33genome were sequenced. The results predict that there are15TCS in both the strains together with single, unpaired response regulator. These works made an important foundation for SS2research. However, the pathogenic mechanism of SS2infection is still not very clear. In this work, the microarray was used to study the genes different expression （DE） of S. suis2incubated with Neutrophils. Furthermore, the mutant strains△1660HK/RR,△1910HK/RR, and△1094HK/RR were attenuated in vivo in CD1mice with reduced mortality and inflammatory factors.1. Cell extraction and culture of Pig neutrophilsIn this study, the neutrophils were achieved by using the Ficoll-Hypaque density gradient centrifugation. Detected by Giemsa staining, the purity of neutrophils was98%, and the cell viability was96%when detected by trypan blue （0.01%concentration） exclusion assay. So the neutrophils met the requirements of the experiment.2. Different expression genes （DE） of SS2after infection in PMNsHalf hour, one hour and three hours after SS2infection, the experiment and control group RNA were collected for the gene chip. In this experiment, gene p-value less than or equal to0.05and FC greater than or equal to2is up regulated and gene p-value less than or equal to0.05and less than or equal to0.5is down regulated. At half hour,342DE genes were detected including154up regulated and188down regulated. At one hour,463DE genes were detected including213up regulated and223down regulated. At three hours,1196DE genes were detected including531up regulated and665down regulated. There were156significant genes DE in all three times including75genes up regulated and81down regulated genes.Further analysis of the different expression genes of two-component regulatory system （TCS） shows that:There were seven TCS genes including SSU05₀427, SSU05,0906, SSU05,0944, SSU05₁358, SSU05₁595and SSU05₁660up regulated all the time; and there were two genes SSU05₀883and SSU05₂090down regulated all the time. SSU05₀430、SSU05₁910and SSU05₂148three genes were down regulated at first and up regulated in later. On the contrary, SSU05₁094gene was down regulated at first and up regulated in later.3. The research of TCS mutants about pathogenic and inflammatory responsesTwo-component regulatory system genes play different roles in the interaction of SS2and pig neutrophils in different time. Respectively,1094HK/RR,1660HK/RR and1910HK/RR play the role in the immune response in early, middle and late time.CD1mice as a good model, when infection with TCS mutants△1910HK/RR,△1094HK/RR and△1660HK/RR, the expression of proinflammatory cytokine is reduced. And the highest expression is after12hours of infection. The resistion to neutrophil phagocytosis and the pathogenicity significantly reduced.