Study On The Microarray Gene Expression Profile Of Host After Streptococcus Suis Infection

Streptococcus suis (S. suis) is an important swine gram positive bacterial pathogen, and 33 S. suis serotypes have been identified based on the differences in their capsular antigens. Among the known 33 serotypes, Streptococcus suis serotype 2 (SS2) is the most frequently isolated serotype worldwide considered a major swine pathogen and an important zoonotic disease causing a variety of life- threatening infections in humans. SS2 can cause septicemia, meningitis, pneumonia, arthritis, endocarditis, and even sudden death in pigs. In addition to causing disease in pigs, it is also an important zoonotic agent for humans in contact with diseased pigs or their products which can cause streptococcal toxic shock syndrome (STSS), streptococcal meningitis syndrome (SMS) leading to meningitis, septicemia, pneumonia, endocarditis, permanent hearing loss, sudden death and high mortality. In 1998 and 2005, SS2 outbreaks in humans were reported in China, and hundreds of people were infected, leading to several fatalities.SS2 current research focuses on the virulence factors discovery and functional identification. Many virulence associated factors are well characterized include capsular polysaccharide (CPS), muramidase released protein (MRP), extracellular protein factor (EF), hemolysin, fibrinogen binding protein (FBPS), glutamate dehydrogenase protein (GDH), secreted nuclease SsnA and so on. In addition, The Chinese isolated SS2 strains 98HAH12 and 05ZYH33 genome were sequenced. These works made an important foundation for SS2 research. However, the pathogenic mechanism of SS2 infection is still not very clear. In this work, the microarray was used to study the genes different expression (DE) of human monocytic cell line THP-1 cells and the pigs tissues brain, lungs, and PBMC after the SS2 infection.1. Human monocytic cell line THP-1 cells gene DE response to SS2 infectionThree hours after SS2 infection, the control and experimental groups THP-1 cells were collected. The cells RNA were extracted, reverse transcripted into cDNA, and then in vitro (IVT) synthetic cRNA. The cRNA were hybridized with Human Affymetrix GenChip Genome U133 plus 2.0. After the hybridization, the DE genes compared to the infection group and control group were scanned. By using the statistical analysis, gene p-value less than or equal to 0.05 and Fold Change (FC) greater than or equal to 2 is up regulated, gene p-value less than or equal to 0.05 and FC less than or equal to 0.5 is down regulated. In total 38,500 probes, there were 2,021 transcripts p-value is less than or equal to 0.05 and selected 386 DE transcripts. By DAVID analysis of online software, according to Gene Ontology (GO) annotation,328 genes are known, including 230 up regulated genes and 98 down regulated genes. We use real-time quantitative PCR technique to verify our microarray results. Six genes include homeobox A4, (HOXA4), CC chemokine receptor type 8 (CCR8), carboxypeptidase enzyme O (CPO), RNA polymeraseâ…¡subunit 2 (POLR2B), pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1), Janus kinase 1 (JAK1) were selected. The results were almost the same with the microarray.By Gene Ontology biological processes (Biological process) classification, the DE genes major involved in biological processes include translation, chemokine, defense/ immune response, ubiquitin - proteasome system, cell division, apoptosis, signal transduction, metabolism, signal transduction, transcription and so on. In addition, we use online software STRING to analysis the functional relationships between DE genes. We selected the INSR, BCL2, CTNNB, UBE2D1 and TIF2 associated pathway, the main focus in the NF-kappa-B signaling pathway and immune response associated pathway.2. Genes DE response of the brain, lungs, and PBMC of the pig after SS2 infection.Twenty four hours after SS2 infection, the experiment and control group piglets were slaughtered and the tissues brain, lung and blood were collected. Then PBMC was isolated from blood. The total RNA was extracted and reverse transcripted into cDNA, and then in vitro (IVT) synthetic cRNA. The cRNA fragments were hybridized with the Porcine Genome gene chip of Affymetrix GenChip. After the hybridization and the washing, the DE genes compared to the infection group and control group were detected. In this experiment, gene p-value less than or equal to 0.05 and FC greater than or equal to 2 is up regulated and gene p-value less than or equal to 0.05 and less than or equal to 0.5 is down regulated. In total 20,201 probes,1608 DE genes were detected in brain including 344 up regulated and 1264 down regulated,617 DE genes were detected in lung including 293 up regulated and 324 down regulated,685 DE genes were detected in PMBC including 403 up regulated and 282 down regulated. There were 31 significant genes DE in all three tissues including 26 genes up regulated and 5 down regulated genes. We use real-time quantitative PCR technique to verify our microarray results. Five genes include ICAM-1, TLR2, CD 163, TTR, VEGFA were selected. The results were almost the same with the microarray.By using the online analysis software Visualization and Integrated Discoveiy (DAVID), in brain 417 known DE genes were detected, in lung 210 known DE genes were detected, in PMBC 213 known DE genes were detected. These detected DE genes were classificated mainly in the following categories, including defense response, immune response, inflammatory response, natural immunity, cell adhesion, cell death, cell differentiation, cell surface receptor linked signal transduction, cytokine activity, cytokine binding, response to external stimuli, response to stimulus, response to stress, response to wounding, signal transducer activity, gene expression and growth factor activity.