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Ashok Kumar, Pawan Kumar Singh, Manoj K Bhasin; Temporal transcriptome and systems biology analysis to identify key pathways and hub genes in experimental Staphylococcus aureus endophthalmitis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):858.
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© ARVO (1962-2015); The Authors (2016-present)
Bacterial endophthalmitis is a sight threatening complication of ocular trauma and surgery. Despite extensive studies on bacterial pathogenesis, the host response in this disease is not well understood. In this study, we performed a whole genome-wide transcriptome analysis on the Staphylococcus (S.) aureus-infected retinal tissue and used the systems biology approach to identify key pathways and their associated genes.
Endophthalmitis was induced via intravitreal injections of S. aureus (strain RN6390). For the purposes of a temporal analysis, the retinal tissue was surgically removed from enucleated eyes at 3, 6, 12, and 24 h post-infection. In another experiment, prior to S. aureus infection, the eyes were pretreated with the TLR-2 agonist Pam3Cys for 24 h. The transcriptional analysis of differentially expressed genes in S. aureus-infected retinal tissue was performed using the Affymetrix microarray. The expression of selective genes was validated by qRT-PCR, and their respective protein levels were checked by ELISA or western blot.
Analysis of our whole genome-wide microarray data revealed progressive changes in the expression of 1,234 genes. Gene ontology and pathway analyses of differentially expressed genes indicate that S. aureus infection affects many aspects of retinal tissue function, including: inflammatory and immune responses, antimicrobial effects, cell trafficking, apoptosis, lipid biosynthesis, and extracellular matrix reorganization. Interactive network-based analyses identified 13 focus hub genes (IL-6, IL-1β, CXCL2, STAT3, NUPR1, Jun, CSF1, CYR61, CEBPB, IGF-1, and TGM2) within these important pathways. RT-PCR and ELISA data confirmed the time-dependent up-regulation of selected inflammatory mediators (IL-6, IL-1β, and CXCL2) in infected retinal tissue. Moreover, TLR-2 ligand pretreatment modulated the expression of all the hub genes. In addition, our in vivo data revealed the up-regulation of Alox5 and Alox12/15, key enzymes involved in the generation of bioactive lipid mediators. These results were further confirmed by performing lipidomics analysis on infected retinal tissue.
This study provides, for the first time, a comprehensive analysis of the transcriptomic response and identifies novel molecular signatures in staphylococcal endophthalmitis using a systems biology approach.
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