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Identifying Mycobacterium tuberculosis membrane proteins involved in binding to and invasion of host cells is important in designing subunit-based anti-tuberculosis vaccines. The Rv2969c gene sequence was identified by PCR in M. tuberculosis complex strains, being transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG. Rabbits immunized with synthetic peptides from highly specific conserved regions of this protein produced antibodies recognizing 27 and 29 kDa bands in M. tuberculosis lysate, which is consistent with the molecular weight of the Rv2969c gene product in M. tuberculosis H37Rv. Immunoelectron microscopy revealed the protein was localized on the bacillus surface. Four and three specific high activity binding peptides (HABPs) to the A549 alveolar epithelial and U937 monocyte cell lines were found, respectively. Two of the HABPs found inhibited M. tuberculosis invasion of A549 cells, suggesting that these peptides might be good candidates to be included in a multiepitopic, subunit-based anti-tuberculosis vaccine. © 2008 Elsevier Inc. All rights reserved.

A rhizosphere isolate Streptomyces sp. CAH29 was found to possess potent antibacterial and antifungal activity against a variety of test organisms. Based on 16S ribosomal ribonucleic acid sequence homology studies, this strain was found to be similar to Streptomyces stramineus (gene sequence similarity 99 %). The major bioactive metabolite produced by Streptomyces sp. CAH29 isolate was extracted, purified andidentified by nuclear magnetic resonance as tetrangomycin. This known anthraquinone-exhibited antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes, methicillin resistant Staphylococcus aureus and Candida albicans with inhibition zones of 14, 10, 12 and 8 mm, respectively. Docking results demonstrate that tetrangomycin has a similar mode of action and a comparable docking score to bind to the dehydrosqualene synthase (CrtM) enzyme of methicillin resistant Staphylococcus aureus compared to the current inhibitor. Hence, this suggests that tetrangomycin has a potential to be used as an anti-methicillin resistant Staphylococcus aureus agent. Tetrangomycin also showed moderate free radical scavenging activity with 1,1-diphenyl-2-picryl-hydrazil. Tetrangomycin apparently decreased all of the studied cytokine (pro-inflammatory: interleukin 1B, interleukin 2, tumor necrosis factor and interleukin L6 and anti-inflammatory: interleukin 10) expression levels at IC50 concentrations in A459 (adenocarcinomic human alveolar basal epithelial) and LNCAP (human prostate adenocarcinoma) cell lines. In addition, it reduced Caspase 8 and 3 mRNA levels in LNCAP and A549 cells. This study describes for the first time novel in vitro immunosuppressive function of tetrangomycin by reducing the transcription of cytokine genes. © 2016, Springer Science+Business Media New York.

A rhizosphere isolate Streptomyces sp. CAH29 was found to possess potent antibacterial and antifungal activity against a variety of test organisms. Based on 16S ribosomal ribonucleic acid sequence homology studies, this strain was found to be similar to Streptomyces stramineus (gene sequence similarity 99 %). The major bioactive metabolite produced by Streptomyces sp. CAH29 isolate was extracted, purified andidentified by nuclear magnetic resonance as tetrangomycin. This known anthraquinone-exhibited antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes, methicillin resistant Staphylococcus aureus and Candida albicans with inhibition zones of 14, 10, 12 and 8 mm, respectively. Docking results demonstrate that tetrangomycin has a similar mode of action and a comparable docking score to bind to the dehydrosqualene synthase (CrtM) enzyme of methicillin resistant Staphylococcus aureus compared to the current inhibitor. Hence, this suggests that tetrangomycin has a potential to be used as an anti-methicillin resistant Staphylococcus aureus agent. Tetrangomycin also showed moderate free radical scavenging activity with 1,1-diphenyl-2-picryl-hydrazil. Tetrangomycin apparently decreased all of the studied cytokine (pro-inflammatory: interleukin 1B, interleukin 2, tumor necrosis factor and interleukin L6 and anti-inflammatory: interleukin 10) expression levels at IC50 concentrations in A459 (adenocarcinomic human alveolar basal epithelial) and LNCAP (human prostate adenocarcinoma) cell lines. In addition, it reduced Caspase 8 and 3 mRNA levels in LNCAP and A549 cells. This study describes for the first time novel in vitro immunosuppressive function of tetrangomycin by reducing the transcription of cytokine genes. © 2016, Springer Science+Business Media New York.

In the present study, in vitro cytotoxic and genotoxic effect of copper-zinc alloy nanoparticles (Cu-Zn ANPs) on human lung epithelial cells (BEAS-2B) were investigated. XTT test and clonogenic assay were used to determine cytotoxic effects. Cell death mode and intracellular reactive oxygen species formations were analyzed using M30, M65 and ROS Elisa assays. Genotoxic effects were evaluated using micronucleus, comet and gamma-H2AX foci assays. Cu-Zn ANPs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential measurements. Characterization of Cu-Zn ANPs showed an average size of 200 nm and zeta potential of -22 mV. TEM analyses further revealed the intracellular localization of Cu-Zn ANPs in cytoplasm within 24 h. Analysis of micronucleus, comet and gamma-H2AX foci counts showed that exposure to Cu-Zn ANPs significantly induced chromosomal damage as well as single and double stranded DNA damage in BEAS-2B cells. Our results further indicated that exposure to Cu-Zn ANPs significantly induced intracellular ROS formation. Evaluation of M30:M65 ratios suggested that cell death was predominantly due to necrosis.

The specific function of putative cut2 protein (or CFP25), encoded by the Rv2301 gene from Mycobacterium tuberculosis H37Rv, has not been identified yet. The aim of this study was to assess some of CFP25 characteristics and its possible biological role in Mycobacterium tuberculosis H37Rv invasion process to target cells. Molecular assays indicated that the gene encoding Rv2301 is present and transcribed in M. tuberculosis complex strains. The presence of Rv2301 protein over the bacilli surface was confirmed by Western blot and immunoelectron microscopy analyses, using goats sera inoculated with synthetic peptides derived from Rv2301 protein. Receptor-ligand binding assays with carcinomic human alveolar basal epithelial cells (A549) and macrophages derived from human histolytic lymphoma monocytes (U937) allowed us to identify five high activity binding peptides (HABPs) in both cell lines, and two additional HABPs only in A549 cells. U937 HABPs binding interactions were characterized by saturation assays, finding dissociation constants (K d) within the nanomolar range and positive cooperativity (n H and gt; 1). Inhibition assays were performed to assess the possible biological role of Rv2301 identified HABPs, finding that some of them were able to inhibit invasion at a 5 ?M concentration, compared with the cytochalasin control. On the other hand, HABPs, and especially HABP 36507 located at the N-terminus of the protein, facilitated the internalization of fluorescent latex beads into A549 cells. These findings are of vital importance for the rational selection of Rv2301 HABPs, to be included as components of an antituberculosis vaccine. © 2011 Springer-Verlag.

Intercellular tight junctions define epithelial apicobasal polarity and form a physical fence which protects underlying tissues from pathogen invasions. PALS1, a tight junction-associated protein, is a member of the CRUMBS3-PALS1-PATJ polarity complex, which is crucial for the establishment and maintenance of epithelial polarity in mammals. Here we report that the carboxy-terminal domain of the SARS-CoV E small envelope protein (E) binds to human PALS1. Using coimmunoprecipitation and pull-down assays, we show that E interacts with PALS1 in mammalian cells and further demonstrate that the last four carboxy-terminal amino acids of E form a novel PDZ-binding motif that binds to PALS1 PDZ domain. PALS1 redistributes to the ERGIC/Golgi region, where E accumulates, in SARS-CoV-infected Vero E6 cells. Ectopic expression of E in MDCKII epithelial cells significantly alters cyst morphogenesis and, furthermore, delays formation of tight junctions, affects polarity, and modifies the subcellular distribution of PALS1, in a PDZ-binding motif-dependent manner. We speculate that hijacking of PALS1 by SARS-CoV E plays a determinant role in the disruption of the lung epithelium in SARS patients. © 2010 K.-T. Teoh et al., link_to_subscribed_fulltext

This study reports the Rv1490 gene presence and transcription in members of the Mycobacterium tuberculosis complex, and characterises the encoded Rv1490 putative membrane protein in M. tuberculosis H37Rv. Rv1490 derived peptides were synthesised and their A549 and U937 cell binding ability was tested, finding five high activity binding peptides (HABPs) for A549 and five for U937. Only two HABPs (11060 and 11073) were shared by both cell lines, both of which affected M. tuberculosis' invading ability to target cells, thus indicating an important role for these sequences in M. tuberculosis entry to A549 alveolar epithelial cells and supporting their inclusion in further studies on the development of a subunit-based multi-epitopic, chemically synthesised anti-tuberculosis vaccine. © 2008 Elsevier Ltd. All rights reserved.

Influenza A viruses continue to represent a severe threat worldwide, causing large epidemics and pandemics responsible for thousands of deaths every year. Excessive inflammation due to overabundant production of proinflammatory cytokines by airway epithelial cells is considered an important factor in disease pathogenesis. Here we report that influenza A virus induced IkappaB kinase (IKK) activity in human airway epithelial A549 cells, resulting in persistent activation of nuclear factor-kappaB (NF-kappaB), a critical regulator of the inflammatory response. Although lung epithelial cells are highly sensitive to stimulation of the IKK/NF-kappaB pathway by influenza virus infection, NF-kappaB was not activated in several non-pulmonary cells permissive to the virus, indicating a cell-specific response. Moreover, NF-kappaB was not essential for virus replication but triggered the expression of proinflammatory cytokines in infected lung cells and was directly responsible for production of high levels of interleukin-8, a chemokine associated with influenza-induced inflammation and airway pathology. We also report that 9-deoxy-delta9,delta12-13,14-dihydro-prostaglandin D2, a cyclopentenone prostanoid with therapeutic efficacy against influenza in preclinical studies, was a powerful inhibitor of influenza virus-induced IKK activity and interleukin-8 production by human pulmonary cells. The results identify IKK as an important factor in triggering influenza virus-induced inflammatory reactions in pulmonary epithelium, suggesting novel therapeutic approaches in the treatment of influenza.