Search

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.

Tuberculosis (TB) remains one of the most worrying infectious diseases affecting public health around the world; 8.7 million new TB cases were reported in 2011. The search for an Mycobacterium tuberculosis H37Rv protein sequence which is functionally important in host-pathogen interaction has been proposed for developing a new vaccine which will allow efficient and safe control of the spread of this disease. The present study thus reports the results obtained for the Rv1268c protein described in the M. tuberculosis H37Rv genome as a hypothetical unknown, probably secreted, protein based on a highly robust, specific, sensitive and functional approach to the search for potential epitopes to be included in an anti-tuberculosis vaccine. Rv1268c presence was determined by immunoblotting after obtaining polyclonal sera against mycobacterial total sonicate or subcellular fractions. Such sera were used in electron immunomicroscopy (EIM) for confirming protein localisation on the M. tuberculosis envelop by recognising colloidal gold-labelled immunoglobulin. Screening assays revealed the presence of two sequences having high binding activity: one binding A549 alveolar epithelial cells ( 141TGMAALEQYLGSGHAVIVSI160) and other binding U937 monocyte-derived macrophages (21AVALGLASPADAAAGTMYGD40). Such sequences' ability to inhibit mycobacterial entry during in vitro assays was analysed. The structure of synthetic peptides binding to target cells was also determined, bearing in mind the structure-function relationship. These results, together with those obtained for other proteins, have been involved in selecting peptides which might be included in a subunit-based anti-tuberculosis vaccine. © 2013 Elsevier Ltd. All rights reserved.

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.

Given the urgent need for designing a new antituberculosis vaccine conferring total protection on patients of all ages, following the line of research adopted by our institute, this work has identified Mycobacterium tuberculosis (Mtb) Rv3166c protein high-activity binding peptides (HABPs) which are able to inhibit bacterial invasion of U937 (monocyte-derived macrophages) and A549 (type II alveolar epithelial cells) cell lines. The presence and transcription of the rv3166c gene in the Mtb species complex was confirmed by polymerase chain reaction (PCR) and reverse transcriptase-PCR; Rv3166c expression was evaluated by western blot and cellular localisation confirmed by immunoelectron microscopy. Its presence was mainly determined on cell surface. Sixteen peptides covering its entire length were chemically synthesised and tested for their ability to bind to U937 and A549 cells. Two U937 HABPs were identified and three for A549, one of them being shared by both cell lines. The four HABPs found inhibited Mtb entry by 15.0794.06. These results led us to including Rv3166c HABPs as candidates for further studies contributing towards the search for a multiepitope, chemically synthesised, subunit-based antituberculosis vaccine. © 2012 The Author.

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.

Wild-type strains of mice do not express CD46, a high-affinity receptor for human group B adenoviruses including type 35. Therefore, studies performed to date in mice using replication-incompetent Ad35 (rAd35) vaccine carriers may underestimate potency or result in altered vector distribution. Here, it is reported that CD46 transgenic mice (MYII-strain) express CD46 in all major organs and that it functions as a receptor for rAd35 vectors. Similar to monkeys and humans, MYII mice highly express CD46 in their lungs and kidneys and demonstrate low expression in muscle. Upon intravenous administration, rAd35 vector genomes as well as expression are detected in lungs of MYII mice, in contrast to wild-type littermates. Expression was predominantly detected in lung epithelial cells. Upon intramuscular administration, the initial level of luciferase expression is higher in MYII mice as compared with wild-type littermates, in spite of the fact that CD46 expression is low in muscle of MYII mice. The higher level of expression in muscle of MYII mice results in prolonged gene expression as assessed by CCD camera imaging for luciferase activity. Finally, a significant dose-sparing effect in MYII mice as compared with wild-type littermates on anti-SIVgag CD8+ T-cell induction following intramuscular vaccination with an rA35.SIVgag vaccine was observed. This dose-sparing effect was also observed when reinfusing dendritic cells derived from MYII mice after exposure to rAd35.SIVgag vaccine as compared with rAd35.SIVgag exposed dendritic cells from wild-type littermates. It was concluded that MYII mice represent an interesting preclinical model to evaluate potency and safety of rAd35 vectors. © 2006 SGM. Chemicals / CAS: Antigens, CD46

Wild-type strains of mice do not express CD46, a high-affinity receptor for human group B adenoviruses including type 35. Therefore, studies performed to date in mice using replication-incompetent Ad35 (rAd35) vaccine carriers may underestimate potency or result in altered vector distribution. Here, it is reported that CD46 transgenic mice (MYII-strain) express CD46 in all major organs and that it functions as a receptor for rAd35 vectors. Similar to monkeys and humans, MYII mice highly express CD46 in their lungs and kidneys and demonstrate low expression in muscle. Upon intravenous administration, rAd35 vector genomes as well as expression are detected in lungs of MYII mice, in contrast to wild-type littermates. Expression was predominantly detected in lung epithelial cells. Upon intramuscular administration, the initial level of luciferase expression is higher in MYII mice as compared with wild-type littermates, in spite of the fact that CD46 expression is low in muscle of MYII mice. The higher level of expression in muscle of MYII mice results in prolonged gene expression as assessed by CCD camera imaging for luciferase activity. Finally, a significant dose-sparing effect in MYII mice as compared with wild-type littermates on anti-SIVgag CD8+ T-cell induction following intramuscular vaccination with an rA35.SIVgag vaccine was observed. This dose-sparing effect was also observed when reinfusing dendritic cells derived from MYII mice after exposure to rAd35.SIVgag vaccine as compared with rAd35.SIVgag exposed dendritic cells from wild-type littermates. It was concluded that MYII mice represent an interesting preclinical model to evaluate potency and safety of rAd35 vectors. © 2006 SGM. Chemicals / CAS: Antigens, CD46

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.