Genetic deletion of Sphk2 confers protection against Pseudomonas aeruginosa mediated differential expression of genes related to virulent infection and inflammation in mouse lung

Abstract

BACKGROUND Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative bacteria that causes serious life threatening and nosocomial infections including pneumonia. PA has the ability to alter host genome to facilitate its invasion thus increasing the virulence of the organism. Sphingosine-1 phosphate (S1P), a bioactive lipid is known to play a key role in facilitating infection. Sphingosine kinases (SPHK)1&2 phosphorylate sphingosine to generate S1P in mammalian cells. We reported earlier that Sphk2-/- mice offered significant protection against lung inflammation, compared to wild type (WT) animals. Therefore, we profiled the differential expression of genes between the protected group of Sphk2-/- and the wild type controls to better understand the underlying protective mechanisms Sphk2 deletion in lung inflammatory injury. Whole transcriptome shotgun sequencing (RNA seq) was performed on mouse lung tissue using NextSeq 500 sequencing system. RESULTS: Two-way ANOVA analysis was performed and differentially expressed genes following PA infection were identified using whole transcriptome of Sphk2-/- mice and their wild WT counterparts. Pathway (PW) enrichment analyses of the RNA seq data identified several signaling pathways that are likely to play a crucial role in pneumonia caused by PA such as those involved in: 1. Immune response to PA infection and NF-κB signal transduction; 2. PKC signal transduction; 3. Impact on epigenetic regulation; 4. Epithelial sodium channel pathway; 5. Mucin expression; and 6. Bacterial infection related pathways. Our data clearly suggests a role for SPHK2 in PA-induced pneumonia through elevated expression of inflammatory genes in lung tissue. Further, validation by RT-PCR on 10 differentially expressed genes showed 100% concordance in terms of vectoral changes, and 70% of genes showed concordance for significant fold change. CONCLUSION: Using Sphk2-/- mice and differential gene expression analysis, we have shown here that S1P/SPHK2 signaling plays a key role in promoting PA pneumonia. The identified genes promote inflammation, and suppress others that naturally inhibit inflammation and host defense. We therefore propose a key role for SPHK2/S1P signaling in PA-induced lung inflammation, and could serve as a potential therapeutic target to combat PA-induced pneumonia.

NOX4 Mediates Pseudomonas aeruginosa-Induced Nuclear Reactive Oxygen Species Generation and Chromatin Remodeling in Lung Epithelium

“…Deletion of Sphk2 , but not Sphk1 , modulated P. aeruginosa- stimulated NOX4 expression in mouse lung ( Figure 3 D), suggesting that S1P generated in the nucleus and not in the cytoplasm might be involved in regulating NOX4 expression. However, deletion of Sphk2 in the mouse had no significant effect on Nox4 mRNA level [ 8 ], suggesting the degradation of NOX4 possibly via the ubiquitination pathway, which needs further investigation.…”

“…P. aeruginosa infection is associated with significant morbidity and mortality, prevalent with immuno-compromised patients and those with severe burns, diabetes, cancer, organ transplant, cystic fibrosis, chronic obstructive pulmonary disease, and on mechanical ventilation support [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ]. P. aeruginosa infection of the lung alters the host genome to facilitate its replication and virulence and also initiates cascade of events in the host, including innate immune responses, reactive oxygen species (ROS) generation, cytokine production, inflammation, dysregulated lipid metabolism, and modulation of epigenetic factors [ 8 , 9 , 10 , 11 ]. Recent studies strongly suggest the regulation of sphingolipid metabolism and signaling pathways by P. aeruginosa infection of the mouse lung.…”

“…Several inflammatory response genes including monocyte chemotactic protein (MCP-1), C-C motif chemokine ligand 2 (CCL2), tumor necrosis factor (TNF) -α-induced protein A20 (TNFAIP3), nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB), and E74-Like ETS Transcription factor (Elf) 3, were upregulated in response to P. aeruginosa infection of the mouse lung [ 8 , 10 , 11 ]. Interestingly, genetic deletion of Sphk2 attenuated P. aeruginosa -mediated activation of many of the aforementioned pro-inflammatory genes belonging to the NF-κB pathway of inflammation in the mouse lung [ 14 ].…”

Lipid larceny: channelizing host lipids for establishing successful pathogenesis by bacteria

“…Despite their bactericidal functions, a wide array of pro-bacterial roles of sphingolipids have also been extensively studied. The mice's enhanced resistance against the infection caused by Pseudomonas aeruginosa upon deleting sphingosine kinase 2, the enzyme required to synthesize sphingosine-1-phosphate, clearly indicates the contribution of sphingosine to the establishment of bacterial pathogenesis [200].…”