NLRP3 selectively drives IL-1ß secretion by Pseudomonas aeruginosa infected neutrophils and regulates corneal disease severity.

Macrophages infected with Gram-negative bacteria expressing Type III secretion system (T3SS) activate the NLRC4 inflammasome, resulting in Gasdermin D (GSDMD)-dependent, but GSDME independent IL-1ß secretion and pyroptosis. Here we examine inflammasome signaling in neutrophils infected with Pseudomonas aeruginosa strain PAO1 that expresses the T3SS effectors ExoS and ExoT. IL-1ß secretion by neutrophils requires the T3SS needle and translocon proteins and GSDMD. In macrophages, PAO1 and mutants lacking ExoS and ExoT (ΔexoST) require NLRC4 for IL-1ß secretion. While IL-1ß release from ΔexoST infected neutrophils is also NLRC4-dependent, infection with PAO1 is instead NLRP3-dependent and driven by the ADP ribosyl transferase activity of ExoS. Genetic and pharmacologic approaches using MCC950 reveal that NLRP3 is also essential for bacterial killing and disease severity in a murine model of P. aeruginosa corneal infection (keratitis). Overall, these findings reveal a function for ExoS ADPRT in regulating inflammasome subtype usage in neutrophils versus macrophages and an unexpected role for NLRP3 in P. aeruginosa keratitis.

Transcriptomic and Metabolomic Analysis Revealed Roles of Yck2 in Carbon Metabolism and Morphogenesis of Candida albicans.

Candida albicans is a part of the normal microbiome of human mucosa and is able to thrive in a wide range of host environments. As an opportunistic pathogen, the virulence of C. albicans is tied to its ability to switch between yeast and hyphal morphologies in response to various environmental cues, one of which includes nutrient availability. Thus, metabolic flexibility plays an important role in the virulence of the pathogen. Our previous study has shown that C. albicans Yeast Casein Kinase 2 (CaYck2) regulates the yeast-to-hyphal switch, but its regulatory mechanisms remain unknown. This study further elucidated the role of Yck2 in governing morphology and carbon metabolism by analyzing the transcriptome and metabolome of the C. albicans YCK2 deletion mutant strain (yck2Δ strain) in comparison to the wild type strain. Our study revealed that loss of CaYck2 perturbs carbon metabolism, leading to a transcriptional response that resembles a transcriptional response to glucose starvation with coinciding intracellular accumulation of glucose and depletion of TCA cycle metabolites. This shift in the metabolome is likely mediated by derepression of glucose-repressed genes in the Mig1/2-mediated glucose sensing pathway and by downregulation of glycolytic genes, possibly through the Rgt1-mediated SRR pathway. In addition, genes involved in beta-oxidation, glyoxylate cycle, oxidative stress response, and arginine biosynthesis were upregulated in the yck2Δ strain, which is highly reminiscent of C. albicans engulfment by macrophages. This coincides with an increase in arginine degradation intermediates in the yck2Δ strain, suggesting arginine catabolism as a potential mechanism of CaYck2-mediated filamentation as seen during C. albicans escape from macrophages. Transcriptome analysis also shows differential expression of hyphal transcriptional regulators Nrg1 and Ume6. This suggests dysregulation of hyphal initiation and elongation in the yck2Δ strain which may lead to the constitutive pseudohyphal phenotype of this strain. Metabolome analysis also detected a high abundance of methyl citrate cycle intermediates in the yck2Δ strain, suggesting the importance of CaYck2 in this pathway. Taken together, we discovered that CaYck2 is an integral piece of carbon metabolism and morphogenesis of C. albicans.

Yeast casein kinase 2 governs morphology, biofilm formation, cell wall integrity, and host cell damage of Candida albicans.

The regulatory networks governing morphogenesis of a pleomorphic fungus, Candida albicans are extremely complex and remain to be completely elucidated. This study investigated the function of C. albicans yeast casein kinase 2 (CaYck2p). The yck2Δ/yck2Δ strain displayed constitutive pseudohyphae in both yeast and hyphal growth conditions, and formed enhanced biofilm under non-biofilm inducing condition. This finding was further supported by gene expression analysis of the yck2Δ/yck2Δ strain which showed significant upregulation of UME6, a key transcriptional regulator of hyphal transition and biofilm formation, and cell wall protein genes ALS3, HWP1, and SUN41, all of which are associated with morphogenesis and biofilm architecture. The yck2Δ/yck2Δ strain was hypersensitive to cell wall damaging agents and had increased compensatory chitin deposition in the cell wall accompanied by an upregulation of the expression of the chitin synthase genes, CHS2, CHS3, and CHS8. Absence of CaYck2p also affected fungal-host interaction; the yck2Δ/yck2Δ strain had significantly reduced ability to damage host cells. However, the yck2Δ/yck2Δ strain had wild-type susceptibility to cyclosporine and FK506, suggesting that CaYck2p functions independently from the Ca+/calcineurin pathway. Thus, in C. albicans, Yck2p is a multifunctional kinase that governs morphogenesis, biofilm formation, cell wall integrity, and host cell interactions.