Evolutionary Dynamics of Clustered Irregularly Interspaced Short Palindromic Repeat Systems in the Ocean Metagenome

Clustered regularly interspaced short palindromic repeats (CRISPRs) form a recently characterized type of prokaryotic antiphage defense system. The phage-host interactions involving CRISPRs have been studied in experiments with selected bacterial or archaeal species and, computationally, in completely sequenced genomes. However, these studies do not allow one to take prokaryotic population diversity and phage-host interaction dynamics into account. This gap can be filled by using metagenomic data: in particular, the largest existing data set, generated from the Sorcerer II Global Ocean Sampling expedition. The application of three publicly available CRISPR recognition programs to the Global Ocean metagenome produced a large proportion of false-positive results. To address this problem, a filtering procedure was designed. It resulted in about 200 reliable CRISPR cassettes, which were then studied in detail. The repeat consensuses were clustered into several stable classes that differed from the existing classification. Short fragments of DNA similar to the cassette spacers were more frequently present in the same geographical location than in other locations (P, <0.0001). We developed a catalogue of elementary CRISPR-forming events and reconstructed the likely evolutionary history of cassettes that had common spacers. Metagenomic collections allow for relatively unbiased analysis of phage-host interactions and CRISPR evolution. The results of this study demonstrate that CRISPR cassettes retain the memory of the local virus population at a particular ocean location. CRISPR evolution may be described using a limited vocabulary of elementary events that have a natural biological interpretation.Prokaryotes are highly diverse (33). One of the explanations of this diversity is the high extinction rate, due to genetic aggression, which leads to the clearance of ecological niches and, as a result, may allow new prokaryotic species to emerge. In the absence of host defense, viral infection of prokaryotic colonies results in colony extinction or the fixation of a fraction of the invader''s genetic material in the host genome, profoundly affecting the life cycle of the host (32). Thus, bacteria and archaea have developed various kinds of defense mechanisms to resist this pressure; the best studied of these mechanisms is restriction-modification systems (4).Along with well-known prokaryotic defense mechanisms, such as rapid evolution of cell receptors or the use of restriction-modification or toxin-antitoxin systems (see, e.g., references 6, 21, and 25), newly discovered clustered regularly interspaced palindromic repeat (CRISPR) systems seem to play an important role in protecting the cell from archaeal virus or bacteriophage assaults (reviewed in reference 36). A typical CRISPR system is a genetic locus comprising CRISPR-associated (cas) genes coding for proteins of several distinct functional classes (8, 19, 29) and a CRISPR cassette. A CRISPR cassette is formed by almost identical direct repeats with an average length of 32 nucleotides (nt), which are separated by similarly sized, unique spacers. A considerable proportion of spacers is similar to known phage or virus sequences, suggesting that the system is involved in antivirus defense (8, 29, 31). This involvement was experimentally demonstrated when a CRISPR system was shown to be essential for cell survival after invasion by foreign DNA (5). The mechanism is thought to be analogous to eukaryotic RNA interference (29), but it has not been characterized in detail yet.CRISPR cassettes retain information that could be used to reveal the evolutionary history of individual systems. First, it has been shown that CRISPR-associated genes could be divided into eight subtypes according to operon organization and gene phylogeny (19). Second, the repeats of different CRISPR cassettes may be similar, which might indicate a common origin of such cassettes. The first attempt to cluster CRISPR cassettes by the similarity of repeat sequences resulted in 12 clusters (27). In that study, the cassettes were obtained by the application of PILER-CR to completely sequenced genomes. Third, pairwise comparison of spacers could also reveal the specific evolutionary history of individual CRISPR cassettes.So far, most large-scale studies of CRISPR systems have been restricted to well-studied organisms with completely sequenced genomes (5, 9, 20, 28, 30). However, the dynamic interaction between viruses or phages and microorganisms in natural environments is of particular interest (2, 10, 15, 23, 35, 38, 40-42). It may be studied using CRISPRs in a metagenome, that is, sequenced DNA fragments collected in one geographical location and therefore representing one ecological niche with all its inhabitants. This approach is interesting for two reasons. First, metagenomic samples provide a common census of coexisting organisms, i.e., in many cases, both the infecting viruses and phages and their victims. Second, most bacteria and archaea from metagenomic samples cannot be cultivated, and hence little is known about their CRISPR systems.To date, three studies have considered host-virus interactions in metagenomes. One study used two thermophilic Synechococcus isolates from microbial mats in hot springs at Yellowstone National Park to demonstrate fast coevolution of the host and phage genomes (22). Two studies described archaeal and bacterial interactions with viruses and phages, respectively, in acidophilic biofilms (2, 39). All environmental communities analyzed so far are extreme and are dominated by few species. Natural samples containing many diverse coexisting organisms may arguably be more interesting.The largest available metagenome, produced by the Sorcerer II Global Ocean Sampling (GOS) expedition, comprises samples of genetic material collected from more than 50 geographical locations of the Pacific and Atlantic oceans (34). This variety provides an opportunity to study the evolution of phage-host interactions reflected in CRISPRs.Three algorithms, PILER-CR (14), the CRISPR recognition tool (CRT) (7), and CRISPRFinder (18), have been developed as tools for the discovery of new CRISPR cassettes. All these algorithms define candidate CRISPR cassette sequences as short direct repeats separated by short unique spacers; they then use a variety of standard repeat-finding techniques. However, the implementation of specific details is different.PILER-CR constructs local alignments of the input sequence to itself; each hit between two close regions is a candidate for an alignment of a repeat with its neighbor copy. In terms of dynamic programming, taking into account the repeat structure of a CRISPR cassette implies looking for hits only within a relatively narrow band around the main diagonal of the dot plot. This process is followed by several refinement steps.CRT does not use alignments to identify candidate repeats; rather, it derives them directly from the analysis of an input sequence. It is based on finding series of short repeats of a specified length (searching for exact k-mer matches) and then extending these repeats (increasing k-mer length) while allowing for a certain level of mismatches.Finally, CRISPRFinder is based on a suffix-tree-based algorithm for repeat discovery, again with additional refinement.All three algorithms were used for the CRISPR cassette search in this study.     

Parasite-Derived Plasma Microparticles Contribute Significantly to Malaria Infection-Induced Inflammation through Potent Macrophage Stimulation

There is considerable debate as to the nature of the primary parasite-derived moieties that activate innate pro-inflammatory responses during malaria infection. Microparticles (MPs), which are produced by numerous cell types following vesiculation of the cellular membrane as a consequence of cell death or immune-activation, exert strong pro-inflammatory activity in other disease states. Here we demonstrate that MPs, derived from the plasma of malaria infected mice, but not naive mice, induce potent activation of macrophages in vitro as measured by CD40 up-regulation and TNF production. In vitro, these MPs induced significantly higher levels of macrophage activation than intact infected red blood cells. Immunofluorescence staining revealed that MPs contained significant amounts of parasite material indicating that they are derived primarily from infected red blood cells rather than platelets or endothelial cells. MP driven macrophage activation was completely abolished in the absence of MyD88 and TLR-4 signalling. Similar levels of immunogenic MPs were produced in WT and in TNF^−/−, IFN-γ^−/−, IL-12^−/− and RAG-1^−/− malaria-infected mice, but were not produced in mice injected with LPS, showing that inflammation is not required for the production of MPs during malaria infection. This study therefore establishes parasitized red blood cell-derived MPs as a major inducer of systemic inflammation during malaria infection, raising important questions about their role in severe disease and in the generation of adaptive immune responses.     

Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium

In this article, a phage-based magnetoelastic sensor for the detection of Salmonella typhimurium is reported. Filamentous bacteriophage specific to S. typhimurium was used as a biorecognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. Phage was immobilized onto the surface of the sensors by physical adsorption. The phage immobilized magnetoelastic sensors were exposed to S. typhimurium cultures with different concentrations ranging from 5x10(1) to 5x10(8) cfu/ml, and the corresponding changes in resonance frequency response of the sensor were studied. It was experimentally established that the sensitivity of the magnetoelastic sensors was higher for sensors with smaller physical dimensions. An increase in sensitivity from 159 Hz/decade for a 2 mm sensor to 770 Hz/decade for a 1 mm sensor was observed. Scanning electron microscopy (SEM) analysis of previously assayed biosensors provided visual verification of frequency changes that were caused by S. typhimurium binding to phage immobilized on the sensor surface. The detection limit on the order of 10(3) cfu/ml was obtained for a sensor with dimensions 1x0.2x0.015 mm.     

Bioluminescent assay of total bacterial contamination of drinking water.

A bioluminescent assay of total bacterial contamination (TBC) of drinking water (DW) with a detection limit of approximately 1 CFU/mL and duration of less than 7 h has been developed. The protocol of the TBC assay comprises: incubation of water sample in nutrition broth supplemented with salts mixture, up to 6 h; filtration of bacterial suspension obtained through membrane filter (pore size 0.45 microm); release of bacterial ATP by dimethyl sulphoxide; determination of bacterial ATP concentration using highly sensitive ATP reagent based on recombinant Luciola mingrelica luciferase. To simplify the assay, special luminometer microcuvette Filtravette (New Horizons Diagnostics Corp., USA) are used. A good correlation (R=0.98) between ATP concentration measured after 6 h incubation and initial bacterial titre in DW was observed. Semi-quantitative TBC assay of DW is also available. The TBC value in DW is assessed by the fixation of incubation time required to detect a measurable bioluminescent signal: 3, 4 and 6 h corresponds to 100-1000, 10-100 and 1-10 CFU/mL, respectively.     

Splice variants of mda-7/IL-24 differentially affect survival and induce apoptosis in U2OS cells

Interleukin-24 (mda-7/IL-24) is a cytokine in the IL-10 family that has received a great deal of attention for its properties as a tumor suppressor and as a potential treatment for cancer. In this study, we have identified and characterized five alternatively spliced isoforms of this gene. Several, but not all of these isoforms induce apoptosis in the osteosarcoma cell line U2OS, while none affect the survival of the non-cancerous NOK cell line. One of these isoforms, lacking three exons and encoding the N-terminal end of the mda-7/IL-24 protein sequence, caused levels of apoptosis that were higher than those caused by the full-length mda-7/IL-24 variant. Additionally, we found that the ratio of isoform expression can be modified by the splice factor SRp55. This regulation suggests that alternative splicing of mda-7/IL-24 is under tight control in the cell, and can be modified under various cellular conditions, such as DNA damage. In addition to providing new insights into the function of an important tumor suppressor gene, these findings may also point toward new avenues for cancer treatment.     

Oxidation and removal of industrial textile dyes by a novel peroxidase extracted from post-harvest lentil (Lens culinaris L.) stubble

The degradation and removal of a series of dyes used in the textile industry for polyester/wool (PES/WO) blends and present in effluents, such as Green, Ash-Grey, Black, Navy Blue, Red and Yellow Domalan, and Orange and Red Bemacid, by catalytic action, in the presence of H₂O₂, of extracts of a novel peroxidase from postharvest lentil stubble was investigated. The extracts of this peroxidase (LSP) were effective in degrading these lastgeneration textile dyes, especially Green Domalan, Orange Bemacid, Grey and Black Domalan. A sensitivity study was carried out for Green Domalan biodegradation to determine the effects of process parameters such as pH, H₂O₂, enzyme and dye concentrations, contact and centrifugation times, and temperature. Standard ecotoxicity studies performed with Vibrio fischeri revealed that the dye solutions treated with peroxidase and H₂O₂ were less ecotoxic than the untreated ones.     

Fluorescence imaging to quantify the fluorescent microspheres in cardiac tissue

To quantify the fluorescent microsphere (FM) content in cardiac tissue, which is an indicative of blood flow, fluorescence imaging of both sides of the pig heart slice was employed. Despite the light scattering inside the tissue and contributions from multiple tissue layers to the total emission, it is shown that the fluorescence intensity at any pixel is proportional to the FM content and the fluorescence image may be transformed to the image of the FM concentration. A convenient standard for the emission‐FM concentration transformation is proposed. The approach has several advantages in comparison with the traditional “digestion & extraction” method such as: non‐destructiveness, high spatial resolution, high throughput, repeatability and simplicity of operation. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)