A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri

The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation

A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri

The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation

The Architecture of Communal Living: Lessons from Arcosanti in Arizona

Paolo Soleri's arcology model (architecture + ecology addresses issues of sustainability by advocating living in a balanced relationship between urban morphology and performance within dense, integrated and compact structures. Within these structures material recycling, waste reduction and the use of renewable energy sources are adopted as part of a sustainable strategy aimed at reducing the flow of resources and energy through the urban system. Today, as governments, eager to deliver major environmental improvements, press on with as yet untried and largely untested 'centrist' policies of urban living, there is a need to research relevant models of the 'compact city' approach. Issues involved with the intensification in the use of space, higher residential densities, centralisation, compactness, the integration of land uses and aspects of self-containment need to be examined. Over the last ten years, as the criteria of sustainability have become more widely accepted and understood, the relevance of the Soleri's urban model has become clearer. Arcosanti, begun in 1970, offers a laboratory for testing the validity of Soleri's ideas. This paper examines arcology and Arcosanti within the context of sustainability. Since the energy crisis of the mid 1970s, efforts at Arcosanti have been directed toward the definition and testing of various architectural effects on a community-wide scale that could offer a response to many of today's environmental problems. But progress is painstakingly slow. Lacking the level of funding and resources that would enable it to be convincing, Arcosanti now represents not so much a specific prototypal solution, but an activist-engaged strategy that advocates the possibility of building our dreams and visions. In a world plagued by so many problems and so few alternatives, it nevertheless continues to offer a beacon of hope on the threshold of a new millennium

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

RWU Announces 2017 Class of RWU Athletics Hall of Fame

Four athletes and one team will be inducted into the sixth class during Homecoming & Family Weekend

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

Different usage profiles for listeners of music delivered via interactive television, free-to-air radio, and personal music collections

This study examines the audience profile and usage patterns of interactive cable/satellite audio (ICSA) listeners and compares them with listeners of free-to-air radio (FTA) and personal music collections(PMC). A survey of ICSA users in Australia reveals that heavy users of ICSA are also likely to be heavy users of FTA and PMC. However, ICSA listeners are significantly older than FTA or PMC listeners and prefer music formats different to those reflected in radio audiences and CD sales

IcsA autotransporter passenger promotes increased fusion protein expression on the cell surface

Background: Autotransporters are attractive cell surface display vehicles as they lack complex adaptor proteins necessary for protein export. Recent reports have suggested that the native effector domain (α domain) and translocation domain (β domain) interact with each other to drive translocation of the effector domain to the outer membrane. In this report we compared the expression, surface localisation and folding of TEM-1 β-lactamase (Bla) and maltose binding protein (MalE or MBP) fused to either full length Shigella flexneri IcsA (IcsA) autotransporter or to the β domain alone (IcsAβ) to determine the contribution of the native IcsA α domain in presenting the fusion proteins on the surface of E. coli K-12 UT5600 (ΔompT). Results: Expression of IcsA-Bla was greater than IcsAβ-Bla. High levels of IcsA-MalE were detected but IcsAβ-MalE was not expressed. All fusion proteins other than IcsAβ-MalE were localised to the outer membrane and were detected on the surface of UT5600 via immunofluorescence microscopy. All bacteria expressing IcsA-MalE were labelled with both α-IcsA and α-MBP. UT5600 expressing IcsAβ-MalE was not labelled with α-MBP. A third of UT5600 expressing IcsA-Bla were detectable with α-Bla but only 5% of UT5600 (IcsAβ-Bla) were labelled with α-Bla. The correct folding of the Bla moiety when fused to IcsA and IcsAβ was also retained as UT5600 expressing either fusion protein exhibited a decreased zone of inhibition in the presence of ampicillin. UT5600 expressing IcsA-Bla was more resistant compared to UT5600 expressing IcsAβ-Bla. Conclusions: The export mechanism of autotransporters is not well understood but accumulating evidence suggest a critical role for the native effector or α domain in facilitating its own export via interactions with the translocation or β domain. This is the first report directly comparing expression of heterologous proteins fused to the full length IcsA autotransporter and fusion to the β domain alone. Protein expression and surface presentation of the fusion proteins were dramatically improved when fused to IcsA rather than IcsAβ. Future studies involved in designing autotransporters as cell surface display vehicles would benefit from including the native α domain. This work also provides further evidence for a key interaction between the autotransporter α and β domains.Mabel Lum and Renato Moron