Host Alternation Is Necessary to Maintain the Genome Stability of Rift Valley Fever Virus

Arthropod-borne viruses are transmitted among vertebrate hosts by insect vectors. Unusually, Rift Valley fever virus (RVFV) can also be transmitted by direct contacts of animals/humans with infectious tissues. What are the molecular mechanisms and evolutionary events leading to adopt one mode of transmission rather than the other? Viral replication is implied to be different in a vertebrate host and an invertebrate host. The alternating host cycle tends to limit virus evolution by adopting a compromise fitness level for replication in both hosts. To test this hypothesis, we used a cell culture model system to study the evolution of RVFV. We found that freeing RVFV from alternating replication in mammalian and mosquito cells led to large deletions in the NSs gene carrying the virulence factor. Resulting NSs-truncated viruses were able to protect mice from a challenge with a virulent RVFV. Thus, in nature, virulence is likely maintained by continuous alternating passages between vertebrates and insects. Thereby, depending on the mode of transmission adopted, the evolution of RVFV will be of major importance to predict the outcome of outbreaks

Genomic structure and insertion sites of Helicobacter pylori prophages from various geographical origins

We present the full genomic sequences, insertion sites and phylogenetic analysis of 28 prophages found in H. pylori isolates from patients of distinct disease types, ranging from gastritis to gastric cancer, and geographic origins, covering most continents. The gentic diversity of H pylori is known to be influenced by these genomic elements including prophages who’s geneomes range from 22.6 to 33.0 Kbp. There was a high conservation of integration site shared in over 50% of cases with greater than 40% or prophage genomes harbouring insertion sequences (IS). Furthermore prophage genomes present a robust phylogeographic pattern, revealing four distinct clusters: one African, one Asian and two European prophage populations. There was evidence of recombination within the genome of some prophages, which resulted in genome mosaics composed by different populations, which may yield additional H. pylori phenotypes

New Implications on Genomic Adaptation Derived from the Helicobacter pylori Genome Comparison

BACKGROUND: Helicobacter pylori has a reduced genome and lives in a tough environment for long-term persistence. It evolved with its particular characteristics for biological adaptation. Because several H. pylori genome sequences are available, comparative analysis could help to better understand genomic adaptation of this particular bacterium. PRINCIPAL FINDINGS: We analyzed nine H. pylori genomes with emphasis on microevolution from a different perspective. Inversion was an important factor to shape the genome structure. Illegitimate recombination not only led to genomic inversion but also inverted fragment duplication, both of which contributed to the creation of new genes and gene family, and further, homological recombination contributed to events of inversion. Based on the information of genomic rearrangement, the first genome scaffold structure of H. pylori last common ancestor was produced. The core genome consists of 1186 genes, of which 22 genes could particularly adapt to human stomach niche. H. pylori contains high proportion of pseudogenes whose genesis was principally caused by homopolynucleotide (HPN) mutations. Such mutations are reversible and facilitate the control of gene expression through the change of DNA structure. The reversible mutations and a quasi-panmictic feature could allow such genes or gene fragments frequently transferred within or between populations. Hence, pseudogenes could be a reservoir of adaptation materials and the HPN mutations could be favorable to H. pylori adaptation, leading to HPN accumulation on the genomes, which corresponds to a special feature of Helicobacter species: extremely high HPN composition of genome. CONCLUSION: Our research demonstrated that both genome content and structure of H. pylori have been highly adapted to its particular life style

A cyclic GMP signalling module that regulates gliding motility in a malaria parasite.

The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3', 5'-cyclic monophosphate (cGMP) as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase delta (PDEdelta), unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase beta (GCbeta), showing that in ookinetes GCbeta is an important source for cGMP, and that PDEdelta is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epifluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.IST/Imaging Science and TechnologyApplied Science

Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells

The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results