11,098 research outputs found

    IDENTIFIKASI DAN UJI AKTIVITAS ANTIBAKTERI FRAKSI TERAKTIF DAUN MIMBA (Azadirachta indica A. Juss)

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    Penelitian ini bertujuan untuk mengetahui aktivitas antibakteri fraksi daun mimba (Azadirachta indica A. Juss) terhadap beberapa bakteri patogen dan mengidentifikasi fraksi teraktifnya. Serbuk daun mimba dimaserasi dengan etanol dan difraksinasi dengan kromatografi vakum cair berturut-turut menggunakan eluen heksana, etil asetat, dan etanol. Aktivitas antibakteri dilakukan dengan metode difusi agar, kemudian fraksi teraktif antibakteri ditentukan berdasarkan Diameter Daerah Hambat (DDH). Fraksi teraktif antibakteri ditentukan Konsentrasi Hambat Minimum (KHM) dan nilai bandingnya terhadap amoksisilin dan kloramfenikol. Selanjutnya fraksi ini diidentifikasi menggunakan skrining fitokimia dan Kromatografi Gas-Spektrofotometer Massa (GC-MS). Fraksi daun mimba hasil pemisahan KVC mempunyai aktivitas antibakteri terhadap Staphylococcus epidermidis, Bacillus cereus, dan Shigella flexneri. Fraksi etil asetat menunjukkan fraksi teraktif antibakteri terhadap semua bakteri uji. Fraksi etil asetat memiliki KHM 0,075% terhadap bakteri Staphylococcus epidermidis, 0,05% terhadap bakteri Bacillus cereus dan Shigella flexneri. Aktivitas antibakteri fraksi etil asetat dibandingkan dengan amoksisilin adalah 0,01% untuk bakteri S. epidermidis, 0,02% untuk bakteri B. cereus, dan 0,02% untuk bakteri S. flexneri, sedangkan dibandingkan dengan kloramfenikol adalah 0,04% untuk bakteri S. epidermidis, 0,02% untuk bakteri B. cereus, dan 0,06% untuk bakteri S. flexneri. Identifikasi dengan skrining fitokimia menunjukkan bahwa fraksi etil asetat mengandung senyawa alkaloid, terpenoid, steroid, tanin (polifenolik), antrakuinon, dan asam lemak. Hasil GC-MS menunjukkan bahwa fraksi etil asetat mengandung senyawa asam palmitat, etil linoleolat, asam stearat, trans-fitol, DOF (di oktil ftalat), dan dimungkinkan 1 senyawa golongan asam lemak serta 5 senyawa golongan triterpenoid. The purpose of this research was to evaluate antibacterial activity of Neem (Azadirachta indica A. Juss) leaves fraction against some pathogenic bacterial and to identificate the most active fraction. Leaves powder of Neem was macerated with ethanol and factionated by vacuum liquid chromatography using hexane, ethyl acetate and ethanol as eluent, respectively. The antibacterial activity of the fraction was evaluated by diffusion method, then the most active fraction of antibacterial was evaluated by zone of inhibition. The most active fraction of antibacterial was evaluated for Minimum Inhibitory Concentration (MIC) and equivalent value, compared with amoxicillin and chloramphenicol, then identified using phytochemical screening and Gass Chromatography-Mass Spectroscopy (GC-MS). The fraction of Neem leaves fractionated by vacuum liquid chromatography had antibacterial activity against Staphylococcus epidermidis, Bacillus cereus, and Shigella flexneri. The ethyl acetate fraction showed the most active fraction of antibacterial against all bacterial tested. The ethyl acetate fraction had MIC 0.075% against Staphylococcus epidermidis 0.05% against Bacillus cereus and Shigella flexneri. The antibacterial activity of ethyl acetat fraction compared to amoxicillin was 0.01% for S. epidermidis, 0.02% for B. cereus, and 0.02% for S. flexneri. Then compared to chloramphenicol was 0.04% for S. epidermidis, 0.02% for B. cereus, and 0.06% for S. flexneri. Identification using phytochemical screening showed that the ethyl acetate fraction contained alkaloids, terpenoids, steroids, tannins (polyphenolics), anthraquinones, and fatty acid. The result of GC-MS showed that the ethyl acetate fraction contained palmitic acid, ethyl linoleolate, stearic acid, trans-phytol, DOP (di octyl phthalate), and suggested 1 class of compound fatty acid and 5 classes of compound triterpenoids

    The Shigella flexneri OmpA amino acid residues 188EVQ190 are essential for the interaction with the virulence factor PhoN2

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    Shigella flexneri is an intracellular pathogen that deploys an arsenal of virulence factors promoting host cell invasion, intracellular multiplication and intra- and inter-cellular dissemination. We have previously reported that the interaction between apyrase (PhoN2), a periplasmic ATP-diphosphohydrolase, and the C-terminal domain of the outer membrane (OM) protein OmpA is likely required for proper IcsA exposition at the old bacterial pole and thus for full virulence expression of Shigella flexneri (Scribano et al., 2014). OmpA, that is the major OM protein of Gram-negative bacteria, is a multifaceted protein that plays many different roles both in the OM structural integrity and in the virulence of several pathogens. Here, by using yeast two-hybrid technology and by constructing an in silico 3D model of OmpA from S. flexneri 5a strain M90T, we observed that the OmpA residues 188EVQ190 are likely essential for PhoN2-OmpA interaction. The 188EVQ190 amino acids are located within a flexible region of the OmpA protein that could represent a scaffold for protein-protein interaction

    Cell Death Dis.

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    The Gram-negative bacterium Shigella flexneri invades the colonic epithelium and causes bacillary dysentery. S. flexneri requires the virulence factor invasion plasmid antigen B (IpaB) to invade host cells, escape from the phagosome and induce macrophage cell death. The mechanism by which IpaB functions remains unclear. Here, we show that purified IpaB spontaneously oligomerizes and inserts into the plasma membrane of target cells forming cation selective ion channels. After internalization, IpaB channels permit potassium influx within endolysosomal compartments inducing vacuolar destabilization. Endolysosomal leakage is followed by an ICE protease-activating factor-dependent activation of Caspase-1 in macrophages and cell death. Our results provide a mechanism for how the effector protein IpaB with its ion channel activity causes phagosomal destabilization and induces macrophage death. These data may explain how S. flexneri uses secreted IpaB to escape phagosome and kill the host cells during infection and, may be extended to homologs from other medically important enteropathogenic bacteria

    Identification of critical residues of the serotype modifying O-acetyltransferase of Shigella flexneri

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    BACKGROUND Thirteen serotypes of Shigella flexneri (S. flexneri) have been recognised, all of which are capable of causing bacillary dysentery or shigellosis. With the emergence of the newer S. flexneri serotypes, the development of an effective vaccine has only become more challenging. One of the factors responsible for the generation of serotype diversity is an LPS O-antigen modifying, integral membrane protein known as O-acetyltransferase or Oac. Oac functions by adding an acetyl group to a specific O-antigen sugar, thus changing the antigenic signature of the parent S. flexneri strain. Oac is a membrane protein, consisting of hydrophobic and hydrophilic components. Oac bears homology to several known and predicted acetyltransferases with most homology existing in the N-terminal transmembrane (TM) regions. RESULTS In this study, the conserved motifs in the TM regions and in hydrophilic loops of S. flexneri Oac were targeted for mutagenesis with the aim of identifying the amino acid residues essential for the function of Oac. We previously identified three critical arginines-R73, R75 and R76 in the cytoplasmic loop 3 of Oac. Re-establishing that these arginines are critical, in this study we suggest a catalytic role for R73 and a structural role for R75 and R76 in O-acetylation. Serine-glycine motifs (SG 52-53, GS 138-139 and SYG 274-276), phenylalanine-proline motifs (FP 78-79 and FPV 282-84) and a tryptophan-threonine motif (WT141-142) found in TM segments and residues RK 110-111, GR 269-270 and D333 found in hydrophilic loops were also found to be critical to Oac function. CONCLUSIONS By studying the effect of the mutations on Oac's function and assembly, an insight into the possible roles played by the chosen amino acids in Oac was gained. The transmembrane serine-glycine motifs and hydrophilic residues (RK 110-111, GR 269-270 and D333) were shown to have an affect on Oac assembly which suggests a structural role for these motifs. The phenylalanine-proline and the tryptophan-threonine motifs affect Oac function which could suggest a catalytic role for these amino acids.This work was supported by a grant from the National Health and Medical Research Council of Australia

    Investigating the DNA-Binding Site for VirB, a Key Transcriptional Regulator of Shigella Virulence Genes, Using an In Vivo Binding Tool

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    The transcriptional anti-silencing and DNA-binding protein, VirB, is essential for the virulence of Shigella species and, yet, sequences required for VirB-DNA binding are poorly understood. While a 7-8 bp VirB-binding site has been proposed, it was derived from studies at a single VirB-dependent promoter, icsB. Our previous in vivo studies at a different VirB-dependent promoter, icsP, found that the proposed VirB-binding site was insufficient for regulation. Instead, the required site was found to be organized as a near-perfect inverted repeat separated by a single nucleotide spacer. Thus, the proposed 7-8 bp VirB-binding site needed to be re-evaluated. Here, we engineer and validate a molecular tool to capture protein-DNA binding interactions in vivo. Our data show that a sequence organized as a near-perfect inverted repeat is required for VirB-DNA binding interactions in vivo at both the icsB and icsP promoters. Furthermore, the previously proposed VirB-binding site and multiple sites found as a result of its description (i.e., sites located at the virB, virF, spa15, and virA promoters) are not sufficient for VirB to bind in vivo using this tool. The implications of these findings are discussed

    The Multifaceted Activity of the VirF Regulatory Protein in the Shigella Lifestyle

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    Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV). The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella. In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non-invasive to the invasive phenotype of Shigella. We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis
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