12,868 research outputs found
The Multifaceted Activity of the VirF Regulatory Protein in the Shigella Lifestyle
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
Investigating the DNA-Binding Site for VirB, a Key Transcriptional Regulator of Shigella Virulence Genes, Using an In Vivo Binding Tool
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 Shigella flexneri OmpA amino acid residues 188EVQ190 are essential for the interaction with the virulence factor PhoN2
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
Identification of critical residues of the serotype modifying O-acetyltransferase of Shigella flexneri
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
Shigella flexneri infection in a newly acquired rhesus macaque (Macaca mulatta)
A 3.4 year-old rhesus macaque weighing 4.5 kg, was suffering from anorexia, acute mucous and bloody diarrhea. On physical examination, the monkey showed a loss of activity, hunched posture, abdominal pain, dehydration, mild gingivitis and unclean anus with discharge. Whole blood was collected for the examination of electrolytes, hematology and serum chemistry; fresh stool was also collected for bacterial culture. Blood profiles showed leukocytosis (14.5 K/µL) and neutrophilia (11.0 K/µL) on complete blood cell count and imbalanced electrolytes associated with diarrhea. As a result of bacterial culture, Shigella flexneri was identified through Mac/SS, IMVIC test, TCBS and VITEK II. Based on these results, this monkey was diagnosed as having acute enteritis caused by Shigella flexneri. Treatment was performed with enrofloxacin prior to the isolation of Shigella flexneri to prevent the transmission of disease. Fortunately, mucus and bloody diarrhea did not persist and general conditions fully recovered. Our results show that the use of enrofloxacin is effective in controlling Shigella flexneri infection in newly acquired rhesus monkeys
Open-source genomic analysis of Shiga-toxin–producing E. coli O104:H4
An outbreak caused by Shiga-toxin–producing Escherichia coli O104:H4 occurred in Germany in May and June of 2011, with more than 3000 persons infected. Here, we report a cluster of cases associated with a single family and describe an open-source genomic analysis of an isolate from one member of the family. This analysis involved the use of rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses. In less than a week, these studies revealed that the outbreak strain belonged to an enteroaggregative E. coli lineage that had acquired genes for Shiga toxin 2 and for antibiotic resistance
Uji Aktivitas Antibakteri Infusa Daun Mangga Bacang (Mangifera Foetida L.) Terhadap Pertumbuhan Shigella Flexneri
Latar Belakang: Shigellosis merupakan penyakit endemik di banyak negara berkembang termasuk Indonesia dan menyebabkan morbiditas serta mortalitas. Shigella flexneri merupakan bakteri dengan isolat terbanyak yang ditemukan pada kasus-kasus shigellosis. Studi literatur menunjukkan bahwa infusa daun mangga bacang (Mangifera foetida L.) mengandung senyawa-senyawa metabolit yang memiliki efek sebagai antibakteri. Tujuan: Penelitian ini bertujuan untuk mengetahui aktivitas antibakteri infusa daun mangga bacang (Mangifera foetida L.) terhadap pertumbuhan Shigella flexneri, mengetahui kandungan senyawa metabolit sekunder yang terdapat dalam infusa daun mangga bacang (Mangifera foetida L.), dan menentukan konsentrasi hambat minimum infusa daun mangga bacang (Mangifera foetida L.) terhadap pertumbuhan Shigella flexneri. Metodologi: Penelitian ini merupakan penelitian eksperimental murni. Daun mangga bacang (Mangifera foetida L.) diekstraksi dengan metode infundasi menggunakan pelarut akuades, kemudian dilakukan uji fitokimia dan uji aktivitas antibakteri dengan metode tuang dan sumuran pada konsentrasi 100%, 80%, 40%, 20%, dan 10%. Kontrol positif yang digunakan adalah siprofloksasin 5 ÎĽg/sumuran dan kontrol negatif yang digunakan adalah akuades. Hasil: Hasil uji fitokimia infusa daun mangga bacang (Mangifera foetida L.) mengandung alkaloid, fenol, flavonoid, terpenoid, saponin, dan tanin. Infusa daun mangga bacang (Mangifera foetida L.) pada konsentrasi 100%, 80%, 40%, 20%, dan 10% tidak menunjukkan adanya zona hambat. Kesimpulan: Infusa daun mangga bacang (Mangifera foetida L.) tidak memiliki aktivitas antibakteri terhadap pertumbuhan Shigella flexneri
12 TEXTILE DYES DECOLORIZATION BY COPPER-RESISTANT-BACTERIA KLEBSIELLA GRIMONTII, SHIGELLA FLEXNERI, ENTEROBACTER CLOACAE ISOLATED FROM CISADANE RIVER TANGERANG
Dyes and copper are dangerous contaminants because they are toxic. Bioremediation using indigenous bacteria is the best solution to overcome water pollution. Copper resistant bacteria usually have resistance to dyes thereby helping the bioremediation of dye and copper wastes. This study aims to examine the ability of indigenous bacteria isolated from the Cisadane River, namely Klebsiella grimontii IrCis3, Shigella flexneri IrCis5, Enterobacter cloacae IrCis6, and Enterobacter cloacae IrCis9 in terms of resistance and ability to decolorize 12 textile dyes namely methylene blue, malachite green, congo red, mordant orange, reactive black, direct yellow, basic fuchsin, reactive orange, dispersed orange, remasol red, wantex yellow and wantex red. The results showed that Shigella flexneri IrCis5, Enterobacter cloacae IrCis6, and Enterobacter cloacae IrCis9 were resistant to all dye concentrations of 200 and 500 ppm except Klebsiella grimontii IrCis3 did not grow on malachite green and basic fuchsin at concentrations of 200 ppm and methylene blue, malachite green and basic fuchsin concentration of 500 ppm. Only Shigella flexneri IrCis5 has the ability to decolorize 200 ppm basic fuchsin up to 87.23% after 3 days of incubation. The addition of 3 mM CuSO4 reduced the ability to decolorize Shigella flexneri IrCis5 to 0.57%
Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from \u3cem\u3eShigella flexneri\u3c/em\u3e
Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents
Escherichia coli EHEC Germany outbreak preliminary functional annotation using BG7 system
We have annotated the European outbreak E. coli EHEC genome sequenced by BGI (6-2-2011) and assembled with MIRA by Nick Loman (6-2-2011 ). Our system BG7, Bacterial Genome annotation of Era7 Bioinformatics, predicts ORFs and annotates them based on fragments of similarity with Uniprot proteins. We have predicted 6327 genes, 6156 encoding proteins y 171 corresponding to ribosomal and tRNA. Based on the preliminary results of our semi-automated method of annotation we have selected some predicted proteins with potential implications in pathogenicity and virulence.
There are 33 predicted genes annotated as toxins and we have found three putative hemolysins: Hemolysin E, a putative hemolysin expression modulating protein and a channel protein, hemolysin III family. We have found 31 predicted genes that could be related to specific antibiotic resistance: beta-lactamic, aminoglycoside, macrolide, polymyxin, tetracycline, fosfomycin and deoxycholate, novobiocin, chloramphenicol, bicyclomycin, norfloxacin and enoxacin and 6-mercaptopurine. This strain is rich in adhesion, secretion systems, pathogenicity and virulence related proteins. It seems to have a restriction-modification system, many proteins involved in Fe transport and utilization (siderophores as aerobactin and enterobactin), lysozyme, one inhibitor of pancreatic serine proteases, proteins involved in anaerobic respiration, antimicrobial peptides, and proteins involved in quorum sensing and biofilm formation that could confer competitive advantage to this strain
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