Mitogen-activated protein kinase phosphatase (Mkp)-1 exerts its anti-inflammatory activities during Gram-negative sepsis by deactivating p38 and c-Jun N-terminal kinase (JNK). We have previously shown that Mkp-1+/+ mice, but not Mkp-1−/− mice, exhibit hypertriglyceridemia during severe sepsis. However, the regulation of hepatic lipid stores and the underlying mechanism of lipid dysregulation during sepsis remains an enigma. To understand the molecular mechanism underlying the sepsis-associated metabolic changes and the role of Mkp-1 in the process, we infected Mkp-1+/+ and Mkp-1−/− mice with Escherichia coli i.v., and assessed the effects of Mkp-1 deficiency on tissue lipid contents. We also examined the global gene expression profile in the livers via RNA-seq. We found that in the absence of E. coli infection, Mkp-1 deficiency decreased liver triglyceride levels. Upon E. coli infection, Mkp-1+/+ mice, but not Mkp-1−/− mice, developed hepatocyte ballooning and increased lipid deposition in the livers. E. coli infection caused profound changes in the gene expression profile of a large number of proteins that regulate lipid metabolism in wildtype mice, while these changes were substantially disrupted in Mkp-1−/− mice. Interestingly, in Mkp-1+/+ mice E. coli infection resulted in downregulation of genes that facilitate fatty acid synthesis but upregulation of Cd36 and Dgat2, whose protein products mediate fatty acid uptake and triglyceride synthesis, respectively. Taken together, our studies indicate that sepsis leads to a substantial change in triglyceride metabolic gene expression programs and Mkp-1 plays an important role in this process.

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Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in redox regulation. We have previously shown that Gsr facilitates neutrophil bactericidal activities and is pivotal for host defense against bacterial pathogens. However, it is unclear whether Gsr is required for immune defense against fungal pathogens. It is also unclear whether Gsr plays a role in immunological functions outside of neutrophils during immune defense. In this study, we report that Gsr 2/2 mice exhibited markedly increased susceptibility to Candida albicans challenge. Upon C. albicans infection, Gsr 2/2 mice exhibited dramatically increased fungal burden in the kidneys, cytokine and chemokine storm, striking neutrophil infiltration, histological abnormalities in both the kidneys and heart, and substantially elevated mortality. Large fungal foci surrounded by massive numbers of neutrophils were detected outside of the glomeruli in the kidneys of Gsr 2/2 mice but were not found in wild-type mice. Examination of the neutrophils and macrophages of Gsr 2/2 mice revealed several defects. Gsr 2/2 neutrophils exhibited compromised phagocytosis, attenuated respiratory burst, and impaired fungicidal activity in vitro. Moreover, upon C. albicans stimulation, Gsr 2/2 macrophages produced increased levels of inflammatory cytokines and exhibited elevated p38 and JNK activities, at least in part, because of lower MAPK phosphatase (Mkp)-1 activity and greater Syk activity. Thus, Gsr-mediated redox regulation is crucial for fungal clearance by neutrophils and the proper control of the inflammatory response by macrophages during host defense against fungal challenge.

We have previously shown that Mkp-1deficient mice produce elevated TNF-a, IL-6, and IL-10 following systemic Escherichia coli infection, and they exhibited increased mortality, elevated bacterial burden, and profound metabolic alterations. To understand the function of Mkp-1 during bacterial infection, we performed RNA-sequencing analysis to compare the global gene expression between E. coliinfected wild-type and Mkp-1 2/2 mice. A large number of IFN-stimulated genes were more robustly expressed in E. coliinfected Mkp-1 2/2 mice than in wild-type mice. Multiplex analysis of the serum cytokine levels revealed profound increases in IFN-b, IFN-g, TNF-a, IL-1a and b, IL-6, IL-10, IL-17A, IL-27, and GMSF levels in E. coliinfected Mkp-1 2/2 mice relative to wildtype mice. Administration of a neutralizing Ab against the receptor for type I IFN to Mkp-1 2/2 mice prior to E. coli infection augmented mortality and disease severity. Mkp-1 2/2 bone marrowderived macrophages (BMDM) produced higher levels of IFN-b mRNA and protein than did wild-type BMDM upon treatment with LPS, E. coli, polyinosinic:polycytidylic acid, and herring sperm DNA. Augmented IFN-b induction in Mkp-1 2/2 BMDM was blocked by a p38 inhibitor but not by an JNK inhibitor. Enhanced Mkp-1 expression abolished IFN-b induction by both LPS and E. coli but had little effect on the IFN-b promoter activity in LPSstimulated RAW264.7 cells. Mkp-1 deficiency did not have an overt effect on IRF3/7 phosphorylation or IKK activation but modestly enhanced IFN-b mRNA stability in LPS-stimulated BMDM. Our results suggest that Mkp-1 regulates IFN-b production primarily through a p38-mediated mechanism and that IFN-b plays a beneficial role in E. coliinduced sepsis.

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.