Non-Opsonic Phagocytosis of Legionella pneumophila by Macrophages Is Mediated by Phosphatidylinositol 3-Kinase

Background: Legionella pneumophila, is an intracellular pathogen that causes Legionnaires ’ disease in humans, a potentially lethal pneumonia. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis. Methodology/Principal Findings: Phagocytosis was measured by cell invasion assays. Construction of PI3K mutant by PCR cloning and expression of dominant negative mutant was detected by Western blot. PI3K activity was measured by 32 P labeling and detection of phospholipids products by thin layer chromatography. Infection of macrophages with virulent L. pneumophila stimulated the formation of phosphatidylinositol 3-phosphate (PIP3), a phosphorylated lipid product of PI3K whereas two structurally distinct phosphatidylinositol 3 kinase (PI3K) inhibitors, wortmannin and LY294002, reduced L. pneumophila entry into macrophages in a dose-dependent fashion. Furthermore, PI3K activation led to Akt stimulation, a serine/threonine kinase, which was also inhibited by wortmannin and LY294002. In contrast, PI3K and protein kinase B (PKB/Akt) activities were lower in macrophages infected with an avirulent bacterial strain. Only virulent L. pneumophila increased lipid kinase activity present in immunoprecipitates of the p85a subunit of class I PI3K and tyrosine phosphorylated proteins. In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells. Conclusion/Significance: Entry of L. pneumophila is mediated by PI3K/Akt signaling pathway. These results suggest an important role for PI3K and Akt in the L. pneumophila infection process. They point to possible novel strategies fo

Genome-Wide Analysis Reveals a Major Role in Cell Fate Maintenance and an Unexpected Role in Endoreduplication for the Drosophila FoxA Gene Fork Head

Transcription factors drive organogenesis, from the initiation of cell fate decisions to the maintenance and implementation of these decisions. The Drosophila embryonic salivary gland provides an excellent platform for unraveling the underlying transcriptional networks of organ development because Drosophila is relatively unencumbered by significant genetic redundancy. The highly conserved FoxA family transcription factors are essential for various aspects of organogenesis in all animals that have been studied. Here, we explore the role of the single Drosophila FoxA protein Fork head (Fkh) in salivary gland organogenesis using two genome-wide strategies. A large-scale in situ hybridization analysis reveals a major role for Fkh in maintaining the salivary gland fate decision and controlling salivary gland physiological activity, in addition to its previously known roles in morphogenesis and survival. The majority of salivary gland genes (59%) are affected by fkh loss, mainly at later stages of salivary gland development. We show that global expression of Fkh cannot drive ectopic salivary gland formation. Thus, unlike the worm FoxA protein PHA-4, Fkh does not function to specify cell fate. In addition, Fkh only indirectly regulates many salivary gland genes, which is also distinct from the role of PHA-4 in organogenesis. Our microarray analyses reveal unexpected roles for Fkh in blocking terminal differentiation and in endoreduplication in the salivary gland and in other Fkh-expressing embryonic tissues. Overall, this study demonstrates an important role for Fkh in determining how an organ preserves its identity throughout development and provides an alternative paradigm for how FoxA proteins function in organogenesis

Estrogen-like activity of seafood related to environmental chemical contaminants

BACKGROUND: A wide variety of environmental pollutants occur in surface waters, including estuarine and marine waters. Many of these contaminants are recognised as endocrine disrupting chemicals (EDCs) which can adversely affect the male and female reproductive system by binding the estrogen receptor and exhibiting hormone-like activities. In this study the estrogenic activity of extracts of edible marine organisms for human consumption from the Mediterranean Sea was assayed. METHODS: Marine organisms were collected in two different areas of the Mediterranean Sea. The estrogenic activity of tissues was assessed using an in vitro yeast reporter gene assay (S. cerevisiae RMY 326 ER-ERE). Concentrations of polychlorinated biphenyls (PCBs) (congeners 28, 52, 101, 118, 138, 153, 180) in fish tissue was also evaluated. RESULTS: Thirty-eight percent of extracts showed a hormone-like activity higher than 10% of the activity elicited by 10 nM 17b-estradiol (E2) used as control. Total PCB concentrations ranged from 0.002 up to 1.785 ng/g wet weight. Chemical analyses detected different levels of contamination among the species collected in the two areas, with the ones collected in the Adriatic Sea showing concentrations significantly higher than those collected in the Tyrrhenian Sea (p < 0.01). CONCLUSION: The more frequent combination of chemicals in the samples that showed higher estrogenic activity was PCB 28, PCB 101, PCB 153, PCB 180. The content of PCBs and estrogenic activity did not reveal any significant correlation

Molecular detection of rifampin and isoniazid resistance to guide chronic TB patient management in Burkina Faso

<p>Abstract</p> <p>Background</p> <p>Drug-resistant tuberculosis (DR-TB) is considered a real threat to the achievement of TB control. Testing of mycobacterial culture and testing of drug susceptibility (DST) capacity are limited in resource-poor countries, therefore inadequate treatment may occur, favouring resistance development. We evaluated the molecular assay GenoType^®MTBDR<it>plus </it>(Hain Lifescience, Germany) in order to detect DR-TB directly in clinical specimens as a means of providing a more accurate management of chronic TB patients in Burkina Faso, a country with a high TB-HIV co-infection prevalence.</p> <p>Methods</p> <p>Samples were collected in Burkina Faso where culture and DST are not currently available, and where chronic cases are therefore classified and treated based on clinical evaluation and sputum-smear microscopy results. One hundred and eight chronic TB patients (sputum smear-positive, after completing a re-treatment regimen for pulmonary TB under directly observed therapy) were enrolled in the study from December 2006 to October 2008. Two early morning sputum samples were collected from each patient, immediately frozen, and shipped to Italy in dry ice. Samples were decontaminated, processed for smear microscopy and DNA extraction. Culture was attempted on MGIT960 (Becton Dickinson, Cockeysville, USA) and decontaminated specimens were analyzed for the presence of mutations conferring resistance to rifampin and isoniazid by the molecular assay GenoType^®MTBDR<it>plus</it>.</p> <p>Results</p> <p>We obtained a valid molecular test result in 60/61 smear-positive and 47/47 smear-negative patients.</p> <p>Among 108 chronic TB cases we identified patients who (i) harboured rifampin- and isoniazid-susceptible strains (n 24), (ii) were negative for MTB complex DNA (n 24), and (iii) had non-tuberculous mycobacteria infections (n 13). The most represented mutation conferring rifampin-resistance was the D516V substitution in the hotspot region of the <it>rpoB </it>gene (43.8% of cases). Other mutations recognized were the H526D (15.6%), the H526Y (15.6%), and the S531L (9.4%).</p> <p>All isoniazid-resistant cases (n 36) identified by the molecular assay were carrying a S315T substitution in the <it>katG </it>gene. In 41.7% of cases, a mutation affecting the promoter region of the <it>inhA </it>gene was also detected.</p> <p>Conclusion</p> <p>The GenoType^®MTBDR<it>plus </it>assay performed directly on sputum specimens improves the management of chronic TB cases allowing more appropriate anti-TB regimens.</p

Zelda Binding in the Early Drosophila melanogaster Embryo Marks Regions Subsequently Activated at the Maternal-to-Zygotic Transition

The earliest stages of development in most metazoans are driven by maternally deposited proteins and mRNAs, with widespread transcriptional activation of the zygotic genome occurring hours after fertilization, at a period known as the maternal-to-zygotic transition (MZT). In Drosophila, the MZT is preceded by the transcription of a small number of genes that initiate sex determination, patterning, and other early developmental processes; and the zinc-finger protein Zelda (ZLD) plays a key role in their transcriptional activation. To better understand the mechanisms of ZLD activation and the range of its targets, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to map regions bound by ZLD before (mitotic cycle 8), during (mitotic cycle 13), and after (late mitotic cycle 14) the MZT. Although only a handful of genes are transcribed prior to mitotic cycle 10, we identified thousands of regions bound by ZLD in cycle 8 embryos, most of which remain bound through mitotic cycle 14. As expected, early ZLD-bound regions include the promoters and enhancers of genes transcribed at this early stage. However, we also observed ZLD bound at cycle 8 to the promoters of roughly a thousand genes whose first transcription does not occur until the MZT and to virtually all of the thousands of known and presumed enhancers bound at cycle 14 by transcription factors that regulate patterned gene activation during the MZT. The association between early ZLD binding and MZT activity is so strong that ZLD binding alone can be used to identify active promoters and regulatory sequences with high specificity and selectivity. This strong early association of ZLD with regions not active until the MZT suggests that ZLD is not only required for the earliest wave of transcription but also plays a major role in activating the genome at the MZT

FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer

FOXA1 expression correlates with the breast cancer luminal subtype and patient survival. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with the downregulation of FOXA1 expression. Knockdown of BRCA1 resulted in the downregulation of FOXA1 expression and enhancement of FOXA1 promoter methylation in MCF-7 breast cancer cells, whereas the reconstitution of BRCA1 in Brca1-deficent mouse mammary epithelial cells (MMECs) promoted Foxa1 expression and methylation. These data suggest that BRCA1 suppresses FOXA1 hypermethylation and silencing. Consistently, the treatment of MMECs with the DNA methylation inhibitor 5-aza-2'-deoxycitydine induced Foxa1 mRNA expression. Furthermore, treatment with GSK126, an inhibitor of EZH2 methyltransferase activity, induced FOXA1 expression in BRCA1-deficient but not in BRCA1-reconstituted MMECs. Likewise, the depletion of EZH2 by small interfering RNA enhanced FOXA1 mRNA expression. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNA methyltransferases (DNMT)1/3a/3b and H3K27me3 are recruited to the endogenous FOXA1 promoter, further supporting the hypothesis that these proteins interact to modulate FOXA1 methylation and repression. Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity. We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter. These associations were validated in a familial breast cancer patient cohort. Integrated analysis of the global gene methylation and expression profiles of a set of 33 familial breast tumours revealed that FOXA1 promoter methylation is inversely correlated with the transcriptional expression of FOXA1 and that BRCA1 mutation breast cancer is significantly associated with FOXA1 methylation and downregulation of FOXA1 expression, providing physiological evidence to our findings that FOXA1 expression is regulated by methylation and chromatin silencing and that BRCA1 maintains FOXA1 expression through suppressing FOXA1 gene methylation in breast cancer.Oncogene advance online publication, 22 December 2014; doi:10.1038/onc.2014.421.published_or_final_versio

The Coxiella burnetii Dot/Icm System Delivers a Unique Repertoire of Type IV Effectors into Host Cells and Is Required for Intracellular Replication

Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication

A Histone-Like Protein of Mycobacteria Possesses Ferritin Superfamily Protein-Like Activity and Protects against DNA Damage by Fenton Reaction

Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe2+ into Fe3+ and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1), a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The Km values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c), Mycobacterium tuberculosis (Rv2986c), and Mycobacterium leprae (ML1683; ML-LBP) were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage

Priming with a Recombinant Pantothenate Auxotroph of Mycobacterium bovis BCG and Boosting with MVA Elicits HIV-1 Gag Specific CD8+ T Cells

A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔpanCD) that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag) and BCGΔpanCD[pHS400] (BCGpan-Gag) were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag). After the boost high frequencies of predominantly Gag-specific CD8+ T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4+ T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge