Moo-Seung Lee

Small heterodimer partner interacts with NLRP3 and negatively regulates activation of the NLRP3 inflammasome

Excessive activation of the NLRP3 inflammasome results in damaging inflammation, yet the regulators of this process remain poorly defined. Herein, we show that the orphan nuclear receptor small heterodimer partner (SHP) is a negative regulator of NLRP3 inflammasome activation. NLRP3 inflammasome activation leads to an interaction between SHP and NLRP3, proteins that are both recruited to mitochondria. Overexpression of SHP competitively inhibits binding of NLRP3 to apoptosis-associated speck-like protein containing a CARD (ASC). SHP deficiency results in increased secretion of proinflammatory cytokines IL-1β and IL-18, and excessive pathologic responses typically observed in mouse models of kidney tubular necrosis and peritoneal gout. Notably, the loss of SHP results in accumulation of damaged mitochondria and a sustained interaction between NLRP3 and ASC in the endoplasmic reticulum. These data are suggestive of a role for SHP in controlling NLRP3 inflammasome activation through a mechanism involving interaction with NLRP3 and maintenance of mitochondrial homeostasis.

Evaluation of anti-influenza effects of camostat in mice infected with non-adapted human influenza viruses

SummaryThe anti-influenza effects of camostat, a serine protease inhibitor, on in vivo influenza infections were evaluated. Mice which received non-adapted human influenza viruses intranasally, developed a reproducible infection with very low mortality. The infection was readily detected by the recovery of the virus from an oropharyngeal swab. Five-week-old ICR mice received intraperitoneal injections of saline (control), amantadine (known positive drug), or camostat, after infection with influenza A/Taiwan/1/86 virus. Virus detection was performed on day 1, 2, 3, 5, and 7 of postinfection. Both camostat and amantadine were effective in ameliorating mouse influenza. On day 5, mice injected with camostat (45%) or amantadine (50%) showed a lower virus secreting rate than those receiving saline (90%). Additionally, camostat showed strong anti-influenza effects on an amantadine-resistant type A virus and a type B virus infection in vitro. The results show that blocking the hemagglutinin cleavage is an effective target for development of an anti-influenza agent. They also demonstrate that virus detection from the oropharynx of mice, infected with non-adapted virus, is a useful in vivo influenza model.

Shiga toxins expressed by human pathogenic bacteria induce immune responses in host cells

Shiga toxins are a family of genetically and structurally related toxins that are the primary virulence factors produced by the bacterial pathogens Shigella dysenteriae serotype 1 and certain Escherichia coli strains. The toxins are multifunctional proteins inducing protein biosynthesis inhibition, ribotoxic and ER stress responses, apoptosis, autophagy, and inflammatory cytokine and chemokine production. The regulated induction of inflammatory responses is key to minimizing damage upon injury or pathogen-mediated infections, requiring the concerted activation of multiple signaling pathways to control cytokine/chemokine expression. Activation of host cell signaling cascades is essential for Shiga toxin-mediated proinflammatory responses and the contribution of the toxins to virulence. Many studies have been reported defining the inflammatory response to Shiga toxins in vivo and in vitro, including production and secretion of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), macrophage inflammatory protein-1α/β (MIP-1α/β), macrophage chemoattractant monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), interleukin 6 (IL-6), and Groβ. These cytokines and chemokines may contribute to damage in the colon and development of life threatening conditions such as acute renal failure (hemolytic uremic syndrome) and neurological abnormalities. In this review, we summarize recent findings in Shiga toxin-mediated inflammatory responses by different types of cells in vitro and in animal models. Signaling pathways involved in the inflammatory responses are briefly reviewed.

Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1β and Apoptotic Cell Death

ABSTRACT Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1β (IL-1β), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1β secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1β. Processing and release of both caspase-1 and IL-1β were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1β as well as to promote apoptotic cell death.

Short-term differential adaptation to anaerobic stress via genomic mutations by Escherichia coli strains K-12 and B lacking alcohol dehydrogenase

Microbial adaptations often occur via genomic mutations under adverse environmental conditions. This study used Escherichia coli ΔadhE cells as a model system to investigate adaptation to anaerobic conditions, which we then compared with the adaptive mechanisms of two closely related E. coli strains, K-12 and B. In contrast to K-12 ΔadhE cells, the E. coli B ΔadhE cells exhibited significantly delayed adaptive growth under anaerobic conditions. Adaptation by the K-12 and B strains mainly employed anaerobic lactate fermentation to restore cellular growth. Several mutations were identified in the pta or pflB genes of adapted K-12 cells, but mostly in the pta gene of the B strains. However, the types of mutation in the adapted K-12 and B strains were similar. Cellular viability was affected directly by severe redox imbalance in B ΔadhE cells, which also impaired their ability to adapt to anaerobic conditions. This study demonstrates that closely related microorganisms may undergo different adaptations under the same set of adverse conditions, which might be associated with the specific metabolic characteristics of each strain. This study provides new insights into short-term microbial adaptation to stressful conditions, which may reflect dynamic microbial population changes in nature.

Production of recombinant human procollagen type I C-terminal propeptide and establishment of a sandwich ELISA for quantification

Procollagen type I carboxy-terminal propeptide (PICP), derived from type I procollagen, has been identified as an indicator of type I collagen synthesis in bone matrix formation and skin recovery. PICP is a heterotrimeric glycoprotein consisting of two α1 chains (PICPα1) and one α2 chain (PICPα2). Here, we report the recombinant expression of human PICP using a mammalian expression system. Co-expression of PICPα1 and PICPα2 in HEK293F cells resulted in the production of functional PICP in the correctly assembled heterotrimeric form. Using the recombinant PICP as an antigen, we isolated PICP-specific human monoclonal antibodies from phage-displayed antibody libraries and raised rabbit polyclonal antibodies. Using those antibodies, we then developed a sandwich ELISA for PICP with a limit of detection of 1u2009ng/mL and a measurable range of 1–640u2009ng/mL. Both intra- and inter-assay imprecision values were <10%. For measuring PICP levels in human fibroblast cellular extracts and culture supernatants and a human serum, the developed ELISA kit displayed comparable performance to that of a commercialized kit. Our results provide an efficient production strategy for recombinant PICP, facilitating the generation of PICP-specific antibodies and development of PICP sandwich ELISA, with potential use in clinical diagnosis of serum samples and testing of cosmeceutical ingredients in fibroblast cell cultures.

Simultaneous detection of severe acute respiratory syndrome, Middle East respiratory syndrome, and related bat coronaviruses by real-time reverse transcription PCR

Since severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) share similar characteristics with respect to clinical signs, etiology, and transmission, methods for a rapid and accurate differential diagnosis are important. Therefore, the aim of this study was to develop a duplex real-time reverse transcription (RT)-PCR method for the simultaneous detection of these viruses. Primers and probes that target the conserved spike S2 region of human SARS-CoV, MERS-CoV, and their related bat CoVs were designed. The results of real-time RT-PCR showed specific reactions for each virus with adequate detection limits of 50–100xa0copies/mL and 5–100xa0copies/mL using pUC57-SARS-pS2 (a template for SARS-CoV) and pGEM-MERS-S2 (a template for MERS-CoV), respectively. In addition, this real-time RT-PCR system was able to detect the target viruses SARS-like bat CoV and MERS-CoV in bat fecal samples and sputum of MERS patients, respectively. Therefore, this newly developed real-time RT-PCR method is expected to detect not only SARS-CoV and MERS-CoV in humans but also several bat CoVs that are closely related to these viruses in bats.

Complete mitochondrial genome of the invasive semi-aquatic mammal, nutria Myocastor coypus (Rodentia; Myocastoridae)

The nutria or coypu (Myocastor coypus, Molina, 1782) is one of the world’s worst invasive alien species. Nutria have significant harmful effects on natural ecosystems and agricultural industries on a global scale, including in South Korea. Herein, we report the complete mitochondrial genome of M. coypus. The 16,874xa0bp sequenced genome exhibited a typical rodential mitochondrial gene arrangement, and consisted of the typical set of 37 genes, one replication origin, and a D-loop. The mitogenome of nutria displayed the highest similarity with that of Cuvier’s spiny rat, Proechimys cuvieri, and was distinct from that of the house mouse, Mus musculus. Multigene phylogenetic analysis also revealed that M. coypus was well clustered with other species in Myocastorini, and the overall tree topology accorded well with recent molecular phylogenetic analysis of South American spiny rats. The results will provide information fundamental for the scientific management of the nutria populations in South Korea.

Complete genome sequence of multidrug-resistant Staphylococcus sciuri strain SNUDS-18 isolated from a farmed duck in South Korea

OBJECTIVESnThis study aimed to determine the complete genome sequence of multidrug-resistant Staphylococcus sciuri strain SNUDS-18 isolated from a farmed duck in South Korea.nnnMETHODSnGenomic DNA was sequenced using a PacBio RS II system. The obtained genome was annotated and antimicrobial resistance and virulence genes were identified.nnnRESULTSnThe sequenced genome possessed a mecA homologue (mecA1) that was almost identical to that of other oxacillin-susceptible S. sciuri strains, whereas the staphylococcal cassette chromosome mec (SCCmec) was not detected. Moreover, various antimicrobial resistance genes conferring resistance to β-lactams, aminoglycosides, phenicols, tetracycline and macrolide-lincosamide-streptogramin B (MLSB) antimicrobials were identified.nnnCONCLUSIONSnThe SNUDS-18 genome and its associated genomic data will provide important insights into the biodiversity of the S. sciuri group as well as valuable information for the control of this potential pathogen.

Complete genome sequence of multidrug-resistant Staphylococcus cohnii ssp. urealyticus strain SNUDS-2 isolated from farmed duck, Republic of Korea

OBJECTIVESnStaphylococcus cohnii has become increasingly recognized as a potential pathogen of clinically significant nosocomial and farm animal infections. This study was designed to determine the genome of a multidrug-resistant S. cohnii subsp. urealyticus strain SNUDS-2 isolated from a farmed duck in Korea.nnnMETHODSnGenomic DNA was sequenced using the PacBio RS II system. The complete genome was annotated and the presence of antimicrobial resistance and virulence genes were identified.nnnRESULTSnThe annotated 2,625,703 bp genome contained various antimicrobial resistance genes conferring resistance to β-lactam, aminoglycosides, fluoroquinolones, phenicols and trimethoprim. The virulence-associated three synergistic hemolysins have been identified in the strain.nnnCONCLUSIONSnTo the best of our knowledge, this is the first complete genome of S. cohnii, and will provide important insights into the biodiversity of CoNS and valuable information for the control of this emerging pathogen.