N-3-Hydroxy Dodecanoyl-DL-homoserine Lactone (OH-dDHL) Triggers Apoptosis of Bone Marrow-Derived Macrophages through the ER- and Mitochondria-Mediated Pathways.

Quorum sensing of Acinetobacter nosocomialis for cell-to-cell communication produces N-3-hydroxy dodecanoyl-DL-homoserine lactone (OH-dDHL) by an AnoR/I two-component system. However, OH-dDHL-driven apoptotic mechanisms in hosts have not been clearly defined. Here, we investigated the induction of apoptosis signaling pathways in bone marrow-derived macrophages treated with synthetic OH-dDHL. Moreover, the quorum-sensing system for virulence regulation was evaluated in vivo using wild-type and anoI-deletion mutant strains. OH-dDHL decreased the viability of macrophage and epithelial cells in dose- and time-dependent manners. OH-dDHL induced Ca2+ efflux and caspase-12 activation by ER stress transmembrane protein (IRE1 and ATF6a p50) aggregation and induced mitochondrial dysfunction through reactive oxygen species (ROS) production, which caused cytochrome c to leak. Pretreatment with a pan-caspase inhibitor reduced caspase-3, -8, and -9, which were activated by OH-dDHL. Pro-inflammatory cytokine and paraoxonase-2 (PON2) gene expression were increased by OH-dDHL. We showed that the anoI-deletion mutant strains have less intracellular invasion compared to the wild-type strain, and their virulence, such as colonization and dissemination, was decreased in vivo. Consequently, these findings revealed that OH-dDHL, as a virulence factor, contributes to bacterial infection and survival as well as the modification of host responses in the early stages of infection.

470 nm LED Irradiation Inhibits the Invasiveness of CD133-positive Human Colorectal Cancer Stem Cells by Suppressing the Cyclooxygenase-2/prostaglandin E2 Pathway.

BACKGROUND/AIM: Recurrence and metastasis of cancer caused by cancer stem cells (CSCs) is a challenge to overcome. Low level laser therapy is a new treatment strategy to suppress their invasiveness. We have assessed the inhibitory effects of 470 nm blue LED on the invasiveness of them to determine the molecular mechanisms of anti-invasiveness. MATERIALS AND METHODS: The effects of blue LEDs on their viability, proliferation and invasion were analyzed using MTT and transwell methods. In addition, the anti-invasiveness effect of blue LED on them was evaluated by zymography, semi-quantitative RT-PCR and western blot analysis. RESULTS: Irradiation with blue LED at 3 J/cm2 resulted in inhibition of their viability, proliferation and invasiveness. Their matrix metalloproteinase 2 (MMP-2) and MMP-9 activities were reduced by blue LED irradiation. Semi-quantitative RT-PCR also showed similar results. In addition, western blotting analyses showed that cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) synthesis were significantly inhibited by LED irradiation in CD133+ colorectal CSCs. CONCLUSION: Down-regulation of the COX-2/PGE2 signaling pathway by blue LED irradiation led to reduce expression of MMP-2 and MMP-9, inhibiting the invasiveness of CD133+ colorectal CSC.

Molecular subtypes of triple-negative breast cancer: understanding of subtype categories and clinical implication.

BACKGROUND: Triple-negative breast cancer (TNBC) is a heterogeneous entity that encompasses several subtypes with distinct molecular characteristics. The patients with TNBCs show unpredictable response to the chemotherapy, and further there is the lack of effective agents. Thus, many studies have been underway to discover targeted therapy suitable for patients with specific genetic alterations in each molecular subtypes. TNBCs are classified as four major molecular subtypes according to the gene expression patterns. These are luminal androgen receptor (LAR), mesenchymal-like, immunomodulatory (IM), and basal-like types. CONCLUSION: Here, we discuss the unique molecular features of each subtype as well as promising targets for anti-cancer therapy.

UmTco1, a Hybrid Histidine Kinase Gene, Is Essential for the Sexual Development and Virulence of Ustilago maydis.

Hybrid histidine kinase is part of a two-component system that is required for various stress responses and pathogenesis of pathogenic fungi. The Tco1 gene in human pathogen Cryptococcus neoformans encodes a hybrid histidine kinase and is important for pathogenesis. In this study, we identified a Tco1 homolog, UmTco1, in the maize pathogen Ustilago maydis by bioinformatics analysis. To explore the role of UmTco1 in the survival of U. maydis under environmental stresses and its pathogenesis, Δumtco1 mutants were constructed by allelic exchange. The growth of Δumtco1 mutants was significantly impaired when they were cultured under hyperosmotic stress. The Δumtco1 mutants exhibited increased resistance to antifungal agent fludioxonil. In particular, the Δumtco1 mutants were unable to produce cytokinesis or conjugation tubes, and to develop fuzzy filaments, resulting in impaired mating between compatible strains. The expression levels of Prf1, Pra1, and Mfa1, which are involved in the pheromone pathway, were significantly decreased in the Δumtco1 mutants. In inoculation tests to the host plant, the Δumtco1 mutants showed significantly reduced ability in the production of anthocyanin pigments and tumor development on maize leaves. Overall, the combined results indicated that UmTco1 plays important roles in the survival under hyperosmotic stress, and contributes to cytokinesis, sexual development, and virulence of U. maydis by regulating the expression of the genes involved in the pheromone pathway.

Acinetobacter nosocomialis secretes outer membrane vesicles that induce epithelial cell death and host inflammatory responses.

Acinetobacter nosocomialis is an important nosocomial pathogen that causes a variety of opportunistic infections; however, pathogenesis of this microorganism has not yet been characterized. The aim of this study was to investigate the secretion of outer membrane vesicles (OMVs) from A. nosocomialis and to determine their cytotoxic effects and their ability to induce inflammatory responses both in vitro and in vivo by using human epithelial HEp-2 cells and a mouse model, respectively. A. nosocomialis ATCC 17903(T) secreted spherical OMVs when cultured in vitro. Proteomic analysis revealed that 147 different proteins were associated with A. nosocomialis OMVs and virulence-associated proteins, such as outer membrane protein A (OmpA), CsuA, CsuC, CsuD, PilW, hemolysin, and serine protease, were identified. A. nosocomialis OMVs were cytotoxic to HEp-2 cells. These vesicles also induced the expression of pro-inflammatory cytokine genes in the HEp-2 cells. Early inflammatory responses, such as congestion and focal neutrophilic infiltration, were observed in the lungs of mice injected with A. nosocomialis OMVs. In conclusion, A. nosocomialis OMVs are important secretory nanocomplexes that induce cytotoxicity of epithelial cells and host inflammatory responses, which may contribute to the pathogenesis of A. nosocomialis.

Distinct characteristics of OxyR2, a new OxyR-type regulator, ensuring expression of Peroxiredoxin 2 detoxifying low levels of hydrogen peroxide in Vibrio vulnificus.

Two peroxiredoxins, Prx1 and Prx2, were previously identified in Vibrio vulnificus. Besides OxyR1, a homologue of Escherichia coli OxyR (EcOxyR), OxyR2 that shares low homology with EcOxyR was first identified in V. vulnificus. OxyR2 activated prx2 during aerobic growth, while OxyR1 activated prx1 only when exposed to exogenous H2O2. OxyR2 was oxidized to form a reversible C206 to C215 disulphide bond by sensing low levels of H2O2, which were insufficient to oxidize OxyR1, and only the oxidized OxyR2 activated prx2. OxyR25CA, in which all cysteine residues except for C206 and C215 were replaced with alanines, and its mutants, OxyR25CA-C206S and OxyR25CA-C215S, were constructed. OxyR25CA and OxyR25CA-C215S directly bound to a specific binding sequence centred at -56.5 from the prx2 transcription start site, albeit with different binding affinities. The binding sequence consisted of four ATCGnt elements spaced by a helical turn and aligned in the twofold dyad symmetry, suggesting that OxyR2 binds DNA as a tetramer. OxyR25CA-C206S also directly bound to DNA comprising more extended sequences, indicating that oxidized and reduced OxyR2 adopt different conformational states, leading to altered DNA contacts. The oxyR2 mutation reduced cytotoxicity and growth during infection, indicating that OxyR2 is essential for the pathogenesis of V. vulnificus.

Distinct characteristics of two 2-Cys peroxiredoxins of Vibrio vulnificus suggesting differential roles in detoxifying oxidative stress.

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes reducing toxic peroxides. Two distinct 2-Cys Prxs, Prx1 and Prx2, were identified in Vibrio vulnificus, a facultative aerobic pathogen. Both Prxs have two conserved catalytic cysteines, C(P) and C(R), but Prx2 is more homologous in amino acid sequences to eukaryotic Prx than to Prx1. Prx2 utilized thioredoxin A as a reductant, whereas Prx1 required AhpF. Prx2 contained GGIG and FL motifs similar to the motifs conserved in sensitive Prxs and exhibited sensitivity to overoxidation. MS analysis and C(P)-SO(3)H specific immunoblotting demonstrated overoxidation of C(P) to C(P)-SO(2)H (or C(P)-SO(3)H) in vitro and in vivo, respectively. In contrast, Prx1 was robust and C(P) was not overoxidized. Discrete expression of the Prxs implied that Prx2 is induced by trace amounts of H(2)O(2) and thereby residential in cells grown aerobically. In contrast, Prx1 was occasionally expressed only in cells exposed to high levels of H(2)O(2). A mutagenesis study indicated that lack of Prx2 accumulated sufficient H(2)O(2) to induce Prx1. Kinetic properties indicated that Prx2 effectively scavenges low levels of peroxides because of its high affinity to H(2)O(2), whereas Prx1 quickly degrades higher levels of peroxides because of its high turnover rate and more efficient reactivation. This study revealed that the two Prxs are differentially optimized for detoxifying distinct ranges of H(2)O(2), and proposed that Prx2 is a residential scavenger of peroxides endogenously generated, whereas Prx1 is an occasional scavenger of peroxides exogenously encountered. Furthermore, genome sequence database search predicted widespread coexistence of the two Prxs among bacteria.

Identification of the Vibrio vulnificus ahpCl gene and its influence on survival under oxidative stress and virulence.

Pathogens have evolved sophisticated mechanisms to survive oxidative stresses imposed by host defense systems, and the mechanisms are closely linked to their virulence. In the present study, ahpCl, a homologue of Escherichia coli ahpC encoding a peroxiredoxin, was identified among the Vibrio vulnificus genes specifically induced by exposure to H(2)O(2). In order to analyze the role of AhpCl in the pathogenesis of V. vulnificus, a mutant, in which the ahpCl gene was disrupted, was constructed by allelic exchanges. The ahpCl mutant was hypersusceptable to killing by reactive oxygen species (ROS) such as H(2)O(2) and t-BOOH, which is one of the most commonly used hydroperoxides in vitro. The purified AhpCl reduced H(2)O(2) in the presence of AhpF and NADH as a hydrogen donor, indicating that V. vulnificus AhpCl is a NADH-dependent peroxiredoxin and constitutes a peroxide reductase system with AhpF. Compared to wild type, the ahpCl mutant exhibited less cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. In addition, the ahpCl mutant was significantly diminished in growth with INT-407 epithelial cells, reflecting that the ability of the mutant to grow, survive, and persist during infection is also impaired. Consequently, the combined results suggest that AhpCl and the capability of resistance to oxidative stresses contribute to the virulence of V. vulnificus by assuring growth and survival during infection.

The capability of catabolic utilization of N-acetylneuraminic acid, a sialic acid, is essential for Vibrio vulnificus pathogenesis.

N-acetylneuraminic acid (Neu5Ac, sialic acid) could provide a good substrate for enteropathogenic bacteria in the intestine, when the bacteria invade and colonize in human gut. In order to analyze the role of Neu5Ac catabolism in Vibrio vulnificus pathogenesis, a mutant with disruption of the nanA gene encoding Neu5Ac lyase was constructed by allelic exchanges. The nanA mutant was not able to utilize Neu5Ac as a sole carbon source and revealed an altered colony morphotype with reduced opacity in the presence of Neu5Ac. Compared to the wild type, the nanA mutant exhibited a low level of cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. The disruption of nanA also resulted in a substantial decrease in histopathological damage in jejunum and colon tissues from the mouse intestine. These results indicated that NanA plays an important role in V. vulnificus pathogenesis. In addition, the nanA mutant was significantly diminished in growth with and adherence to INT-407 epithelial cells in vitro, and was defective for intestinal colonization, reflecting the impaired ability of the mutant to grow and survive with, persist in, and adhere to the intestine in vivo. Consequently, the combined results suggest that NanA and the capability of catabolic utilization of Neu5Ac contribute to V. vulnificus virulence by ensuring growth, adhesion, and survival during infection.

Proteomic identification and characterization of Vibrio vulnificus proteins induced upon exposure to INT-407 intestinal epithelial cells.

Proteomic analysis led to identification of the proteins of Vibrio vulnificus that were induced upon exposure to INT-407 cells, and 7 of which belong to the functional categories such as amino acid transport/metabolism, nucleotide transport/metabolism, posttranslational modification/protein turnover/chaperones, and translation. Among the genes encoding the host-induced proteins, disruption of purH, trpD, tsaA, and groEL2 resulted in reduced cytotoxicity. The purH, trpD, and tsaA mutants showed impaired growth in the INT-407 lysate; however, the growth rate of the groEL2 mutant was not significantly changed, indicating that the possible roles of the host-induced proteins in the virulence of V. vulnificus are rather versatile.

Vibrio vulnificus rtxE is important for virulence, and its expression is induced by exposure to host cells.

Numerous secreted virulence factors have been proposed to account for the fulminating and destructive nature of Vibrio vulnificus infections. A mutant of V. vulnificus that exhibited less cytotoxicity to INT-407 human intestinal epithelial cells was screened from a library of mutants constructed by random transposon mutagenesis. A transposon-tagging method was used to identify and clone an open reading frame encoding an RTX toxin secretion ATP binding protein, RtxE, from V. vulnificus. The deduced amino acid sequence of RtxE from V. vulnificus was 91% identical to that reported from Vibrio cholerae. Functions of the rtxE gene in virulence were assessed by constructing an isogenic mutant whose rtxE gene was inactivated by allelic exchanges and by evaluating the differences between its virulence phenotype and that of the wild type in vitro and in mice. The disruption of rtxE blocked secretion of RtxA to the cell exterior and resulted in a significant reduction in cytotoxic activity against epithelial cells in vitro. Also, the intraperitoneal 50% lethal dose of the rtxE mutant was 10(4) to 10(5) times higher than that of the parental wild type, indicating that RtxE is essential for the virulence of V. vulnificus. Furthermore, the present study demonstrated that the rtxBDE genes are transcribed as one transcriptional unit under the control of a single promoter, P(rtxBDE). The activity of V. vulnificus P(rtxBDE) is induced by exposure to INT-407 cells, and the induction requires direct contact of the bacteria with the host cells.