Hyejin Jeon

Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

BackgroundPlasminogen activator inhibitor type 1 (PAI-1) is the primary inhibitor of urokinase type plasminogen activators (uPA) and tissue type plasminogen activators (tPA), which mediate fibrinolysis. PAI-1 is also involved in the innate immunity by regulating cell migration and phagocytosis. However, little is known about the role of PAI-1 in the central nervous system.MethodsIn this study, we identified PAI-1 in the culture medium of mouse mixed glial cells by liquid chromatography and tandem mass spectrometry. Secretion of PAI-1 from glial cultures was detected by ELISA and western blotting analysis. Cell migration was evaluated by in vitro scratch-wound healing assay or Boyden chamber assay and an in vivo stab wound injury model. Phagocytic activity was measured by uptake of zymosan particles.ResultsThe levels of PAI-1 mRNA and protein expression were increased by lipopolysaccharide and interferon-γ stimulation in both microglia and astrocytes. PAI-1 promoted the migration of microglial cells in culture via the low-density lipoprotein receptor-related protein (LRP) 1/Janus kinase (JAK)/signal transducer and activator of transcription (STAT)1 axis. PAI-1 also increased microglial migration in vivo when injected into mouse brain. PAI-1-mediated microglial migration was independent of protease inhibition, because an R346A mutant of PAI-1 with impaired PA inhibitory activity also promoted microglial migration. Moreover, PAI-1 was able to modulate microglial phagocytic activity. PAI-1 inhibited microglial engulfment of zymosan particles in a vitronectin- and Toll-like receptor 2/6-dependent manner.ConclusionOur results indicate that glia-derived PAI-1 may regulate microglial migration and phagocytosis in an autocrine or paracrine manner. This may have important implications in the regulation of brain microglial activities in health and disease.

Analysis of glial secretome: The long pentraxin PTX3 modulates phagocytic activity of microglia

Microglia, as the phagocytes of the central nervous system, play an important role in the recognition, engulfment, and clearance of apoptotic cells and invading microbes. Proteins secreted from activated glial cells may affect microglial phagocytic activity. Secreted proteins of mixed glial cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) for 24h were identified for the first time by liquid chromatography and tandem mass spectrometric analysis. Several proteins were newly identified as a glia-secreted protein. Among the proteins identified by the mixed glia secretome analysis, pentraxin 3 (PTX3) secretion was most highly induced by LPS/IFN-γ stimulation. Expression of PTX3 mRNA was detected in primary microglia and astrocyte cultures as well as glial cell lines. Glial secretion of PTX3 and its inflammatory induction was confirmed by Western blot analysis of conditioned media of mixed glial cultures. PTX3 did not influence LPS-induced nitric oxide production or neurotoxicity of BV-2 microglial cells. Most importantly, PTX3 selectively modulated microglial phagocytosis activity; it promoted engulfment of zymosan particles, while it inhibited uptake of apoptotic cells. Our results indicate that glia-derived PTX3 may modulate phagocytic functions of microglia, and this may have important implications in the regulation of microglial activity in health and disease.

Role of soluble CD14 in cerebrospinal fluid as a regulator of glial functions.

Proteomic analysis of cerebrospinal fluid (CSF) samples derived from patients with Alzheimers disease (AD) or Parkinsons disease (PD) was performed. On the basis of liquid chromatography–tandem mass spectrometry, two‐dimensional gel electrophoresis analysis, and Western blot validation, it was found that the level of soluble form of monocyte differentiation antigen CD14 precursor was elevated in CSF from AD or PD patients compared with normal subjects. The soluble CD14 protein and mRNA expression was detected in microglia cells, indicating that microglia may be a cellular source of soluble CD14 in CSF. Next, the role of soluble CD14 in the regulation of glial functions was investigated. Soluble CD14 inhibited lipopolysaccharide (LPS)‐ or LPS/interferon‐gamma‐induced nitric oxide production and cell death of microglia and astrocytes. Soluble CD14 suppressed glial neurotoxicity in a coculture of glia/neuroblastoma. In addition, soluble CD14 moderately enhanced phagocytic activity of microglia. These results suggest that microglia‐derived soluble CD14 is a candidate CSF biomarker for AD and PD, and the soluble CD14 may inhibit glial activation by interfering with LPS effects.

Genetic basis of antimicrobial resistance and clonal dynamics of carbapenem-resistant Acinetobacter baumannii sequence type 191 in a Korean hospital.

This study investigated the genetic basis of antimicrobial resistance and the epidemiological characteristics of 125 carbapenem-resistant Acinetobacter baumannii (CRAB) isolates collected from 2011 to 2012 in a Korean hospital. All CRAB isolates showed an extensively drug-resistant phenotype, but were susceptible to tigecycline. The blaOXA-23 and armA genes were mainly responsible for resistance to carbapenems and aminoglycosides, respectively. Four colistin-resistant CRAB isolates with different pulsotypes were identified. All four colistin-resistant isolates had a deletion at nucleotide 776 in lpxA, while one also had an insertion at nucleotide 732 in lpxA. All CRAB isolates belonged to three sequence types (STs): ST191 (n=118), ST208 (n=6), and ST436 (n=1), but were classified into 33 arbitrary pulsotypes. Of the CRAB ST191 isolates, two main arbitrary pulsotypes 5 (n=20) and 18 (n=17) emerged sequentially, but were not clonally related to CRAB isolates collected from 2009 to 2010 in the same hospital. Furthermore, of the two main pulsotypes identified among CRAB ST191 isolates from 2009 to 2010, one was clonally related to sporadic CRAB ST191 isolates from 2011 to 2012, but the other was not related to any CRAB isolate from 2011 to 2012. In conclusion, this study shows the clonal dynamics of CRAB ST191 isolates in a Korean hospital during the last four years.

Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells.

Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs.

Comparative analysis of the role of small G proteins in cell migration and cell death: cytoprotective and promigratory effects of RalA.

Small G protein superfamily consists of more than 150 members, and is classified into six families: the Ras, Rho, Rab, Arf, Ran, and RGK families. They regulate a wide variety of cell functions such as cell proliferation/differentiation, cytoskeletal reorganization, vesicle trafficking, nucleocytoplasmic transport and microtubule organization. The small G proteins have also been shown to regulate cell death/survival and cell shape. In this study, we compared the role of representative members of the six families of small G proteins in cell migration and cell death/survival, two cellular phenotypes that are associated with inflammation, tumorigenesis, and metastasis. Our results show that small G proteins of the six families differentially regulate cell death and cell cycle distribution. In particular, our results indicate that Rho family of small G proteins is antiapoptotic. Ras, Rho, and Ran families promoted cell migration. There was no significant correlation between the cell death- and cell migration-regulating activities of the small G proteins. Nevertheless, RalA was not only cytoprotective against multiple chemotherapeutic drugs, but also promigratory inducing stress fiber formation, which was accompanied by the activation of Akt and Erk pathways. Our study provides a framework for further systematic investigation of small G proteins in the perspectives of cell death/survival and motility in inflammation and cancer.

Interaction between optineurin and Rab1a regulates autophagosome formation in neuroblastoma cells

Optineurin (OPTN) is an autophagy receptor protein that has been implicated in glaucoma and amyotrophic lateral sclerosis. OPTN‐mediated autophagy is a complex process involving many autophagy‐regulating proteins. Autophagy plays a critical role in removing damaged organelles, intracellular pathogens, and protein aggregates to maintain cellular homeostasis. We identified Ypt1 as a novel interaction partner of OPTN by performing a large‐scale yeast‐human two‐hybrid assay. Coimmunoprecipitation assay showed that OPTN interacted with Rab1, the mammalian homolog of yeast Ypt1, in N2a mouse neuroblastoma cell line. We confirmed this interaction by confocal microscopy showing intracellular colocalization of the two proteins. We observed that a zinc finger domain of OPTN is important for Rab1a binding. Rab1a activity is also required for the binding with OPTN. The role of the OPTN‐Rab1a complex in neuronal autophagy was determined by measuring the translocation of microtubule‐associated protein light chain 3–EGFP to autophagosomes. In N2a cells, OPTN‐induced autophagosome formation was inhibited by Rab1a knockdown, indicating the important role of OPTN‐Rab1a interaction in neuronal autophagy processes. Similarly, in N2a cells overexpressing Rab1a, serum starvation–induced formation of autophagosome was enhanced, while OPTN knockdown reduced the Rab1a‐induced autophagy. These results show that the OPTN‐Rab1a complex modulates autophagosome formation in neuroblastoma cells.

Mutational inactivation of OprD in carbapenem-resistant Pseudomonas aeruginosa isolates from Korean hospitals.

This study investigated the mechanisms underlying the carbapenem resistance of bloodstream isolates of Pseudomonas aeruginosa obtained from two Korean hospitals. Of the 79 P. aeruginosa isolates, 22 and 21 were resistant to imipenem and meropenem, respectively. The 22 imipenem-resistant P. aeruginosa isolates were classified into 7 sequence types (STs) and 13 pulsotypes. Twelve imipenem-resistant isolates from one hospital were found to belong to the international clone ST111. Two imipenem-resistant P. aeruginosa ST235 isolates carried the blaIMP-6 gene, but the remaining 20 isolates did not produce carbapenemases. Mutations in the oprD gene and a related decrease in gene expression were found in 21 and 5 isolates, respectively. However, all imipenemresistant P. aeruginosa isolates showed no significant expression of OprD in the outer membrane as compared with that of carbapenem-susceptible PAO1 strain. Overexpression of genes associated with efflux pumps, including mexB, mexD, mexF, and mexY, was not found in any imipenem-resistant isolate. One imipenem-resistant P. aeruginosa isolate overexpressed the ampC gene. Our results show that the low permeability of drugs due to the mutational inactivation of OprD is primarily responsible for carbapenem resistance in bloodstream isolates of P. aeruginosa from Korean hospitals.

Outer membrane Protein A plays a role in pathogenesis of Acinetobacter nosocomialis

ABSTRACT Acinetobacter nosocomialis is an important nosocomial pathogen that causes a variety of human infections. However, the specific virulence factors of this microorganism have not yet been determined. We investigated the role of outer membrane protein A (OmpA) in the pathogenesis of A. nosocomialis. A ΔompA mutant of the A. nosocomialis ATCC 17903T strain was constructed using markerless gene deletion. The ΔompA mutant displayed reduced biofilm formation in polystyrene tubes and reduced adherence to A549 cells in comparison to the wild-type strain. These virulence traits of the ΔompA mutant strain were restored when the ompA gene was complemented. Cytotoxicity was not significantly different between the wild-type strain and the ΔompA mutant when A549 cells were infected with bacteria or treated with outer membrane vesicles (OMVs). However, OMVs from the wild-type strain induced cytotoxicity in HEp-2 cells, whereas OMVs from the ΔompA mutant did not induce cytotoxicity. Proteomic analysis of OMVs revealed that OmpA influenced the distribution of envelope and periplasmic proteins. Overall, this study is the first report that links OmpA to A. nosocomialis pathogenesis, and highlights OmpA as a putative target to develop anti-virulence agents or vaccines against A. nosocomialis infection.

Differences in Biofilm Mass, Expression of Biofilm-Associated Genes, and Resistance to Desiccation between Epidemic and Sporadic Clones of Carbapenem-Resistant Acinetobacter baumannii Sequence Type 191

Understanding the biology behind the epidemicity and persistence of Acinetobacter baumannii in the hospital environment is critical to control outbreaks of infection. This study investigated the contributing factors to the epidemicity of carbapenem-resistant A. baumannii (CRAB) sequence type (ST) 191 by comparing the differences in biofilm formation, expression of biofilm-associated genes, and resistance to desiccation between major epidemic (n = 16), minor epidemic (n = 12), and sporadic (n = 12) clones. Biofilm mass was significantly greater in the major epidemic than the minor epidemic and sporadic clones. Major and minor epidemic clones expressed biofilm-associated genes, abaI, bap, pgaABCD, and csuA/BABCDE, higher than the sporadic clones in sessile conditions. The csuC, csuD, and csuE genes were more highly expressed in the major epidemic than minor epidemic clones. Interestingly, minor epidemic clones expressed more biofilm-associated genes than the major epidemic clone under planktonic conditions. Major epidemic clones were more resistant to desiccation than minor epidemic and sporadic clones on day 21. In conclusion, the epidemic CRAB ST191 clones exhibit a higher capacity to form biofilms, express the biofilm-associated genes under sessile conditions, and resist desiccation than sporadic clones. These phenotypic and genotypic characteristics of CRAB ST191 may account for the epidemicity of specific CRAB ST191 clones in the hospital.