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1.
Front Cell Infect Microbiol ; 12: 960065, 2022.
Article in English | MEDLINE | ID: mdl-36405967

ABSTRACT

Known SARS-CoV-2 variants of concern (VOCs) can be detected and differentiated using an RT-PCR-based genotyping approach, which offers quicker time to result, lower cost, higher flexibility, and use of the same laboratory instrumentation for detection of SARS-CoV-2 when compared with whole genome sequencing (WGS). In the current study, we demonstrate how we applied a genotyping approach for identification of all VOCs and that such technique can offer comparable performance to WGS for identification of known SARS-CoV-2 VOCs, including more recent strains, Omicron BA.1 and BA.2.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Genotype , Whole Genome Sequencing
2.
Appl Environ Microbiol ; 85(21)2019 11 01.
Article in English | MEDLINE | ID: mdl-31471303

ABSTRACT

As for many opportunistic pathogens, the virulence potential of Listeria monocytogenes is highly heterogeneous between isolates and correlated, to some extent, with phylogeny and gene repertoires. In sharp contrast with copious data on intraspecies genome diversity, little is known about transcriptome diversity despite the role of complex genetic regulation in pathogenicity. The current study implemented RNA sequencing to characterize the transcriptome profiles of 33 isolates under optimal in vitro growth conditions. Transcript levels of conserved single-copy genes were comprehensively explored from several perspectives, including phylogeny, in silico-predicted virulence category based on epidemiological multilocus sequence typing (MLST) data, and in vivo virulence phenotype assessed in Galleria mellonella Comparing baseline transcriptomes between isolates was intrinsically more complex than standard genome comparison because of the inherent plasticity of gene expression in response to environmental conditions. We show that the relevance of correlation analyses and their statistical power can be enhanced by using principal-component analysis to remove the first level of irrelevant, highly coordinated changes linked to growth phase. Our results highlight the major contribution of transcription factors with key roles in virulence to the diversity of transcriptomes. Divergence in the basal transcript levels of a substantial fraction of the transcriptome was observed between lineages I and II, echoing previously reported epidemiological differences. Correlation analysis with in vivo virulence identified numerous sugar metabolism-related genes, suggesting that specific pathways might play roles in the onset of infection in G. mellonellaIMPORTANCEListeria monocytogenes is a multifaceted bacterium able to proliferate in a wide range of environments from soil to mammalian host cells. The accumulated genomic data underscore the contribution of intraspecies variations in gene repertoire to differential adaptation strategies between strains, including infection and stress resistance. It seems very likely that the fine-tuning of the transcriptional regulatory network is also a key component of the phenotypic diversity, albeit more difficult to investigate than genome content. Some studies reported incongruity in the basal transcriptome between isolates, suggesting a putative relationship with phenotypes, but small isolate numbers hampered proper correlation analyses with respect to their characteristics. The present study is the embodiment of the promising approach that consists of analyzing correlations between transcriptomes and various isolate characteristics. Statistically significant correlations were found with phylogenetic groups, epidemiological evidence of virulence potential, and virulence in Galleria mellonella larvae used as an in vivo model.


Subject(s)
Listeria monocytogenes/genetics , Listeria monocytogenes/pathogenicity , Listeriosis/microbiology , Moths/microbiology , Transcriptome , Animals , Disease Models, Animal , Gene Expression Profiling , Genome, Bacterial/genetics , Genomics , Humans , Larva/microbiology , Listeria monocytogenes/classification , Listeria monocytogenes/isolation & purification , Multilocus Sequence Typing , Phenotype , Phylogeny , Regulon , Virulence/genetics , Whole Genome Sequencing
3.
Genome Biol Evol ; 10(6): 1403-1415, 2018 06 01.
Article in English | MEDLINE | ID: mdl-29788048

ABSTRACT

The merging of two divergent genomes in a hybrid is believed to trigger a "genomic shock", disrupting gene regulation and transposable element (TE) silencing. Here, we tested this expectation by comparing the pattern of expression of transposable elements in their native and hybrid genomic context. For this, we sequenced the transcriptome of the Arabidopsis thaliana genotype Col-0, the A. lyrata genotype MN47 and their F1 hybrid. Contrary to expectations, we observe that the level of TE expression in the hybrid is strongly correlated to levels in the parental species. We detect that at most 1.1% of expressed transposable elements belonging to two specific subfamilies change their expression level upon hybridization. Most of these changes, however, are of small magnitude. We observe that the few hybrid-specific modifications in TE expression are more likely to occur when TE insertions are close to genes. In addition, changes in epigenetic histone marks H3K9me2 and H3K27me3 following hybridization do not coincide with TEs with changed expression. Finally, we further examined TE expression in parents and hybrids exposed to severe dehydration stress. Despite the major reorganization of gene and TE expression by stress, we observe that hybridization does not lead to increased disorganization of TE expression in the hybrid. Although our study did not examine TE transposition activity in hybrids, the examination of the transcriptome shows that TE expression is globally robust to hybridization. The term "genomic shock" is perhaps not appropriate to describe transcriptional modification in a viable hybrid merging divergent genomes.


Subject(s)
Arabidopsis/genetics , DNA Transposable Elements/genetics , Gene Expression Regulation, Plant/genetics , Genome, Plant/genetics , Epigenesis, Genetic/genetics , Genomics/methods , Histones/genetics , Hybridization, Genetic , Transcription, Genetic/genetics
4.
Biosens Bioelectron ; 80: 418-425, 2016 Jun 15.
Article in English | MEDLINE | ID: mdl-26874109

ABSTRACT

A surface plasmon resonance (SPR)-based SELEX approach has been used to raise RNA aptamers against a structured RNA, derived from XBP1 pre-mRNA, that folds as two contiguous hairpins. Thanks to the design of the internal microfluidic cartridge of the instrument, the selection was performed during the dissociation phase of the SPR analysis by recovering the aptamer candidates directly from the target immobilized onto the sensor chip surface. The evaluation of the pools was performed by SPR, simultaneously, during the association phase, each time the amplified and transcribed candidates were injected over the immobilized target. SPR coupled with SELEX from the first to the last round allowed identifying RNA aptamers that formed highly stable loop-loop complexes (KD equal to 8nM) with the hairpin located on the 5' side of the target. High throughput sequencing of two key rounds confirmed the evolution observed by SPR and also revealed the selection of hairpins displaying a loop not fully complementary to the loop of its target. These candidates were selected mainly because they bound 79 times faster to the target than those having a complementary loop. SELEX coupled with SPR is expected to speed up the selection process because selection and evaluation are performed simultaneously.


Subject(s)
Aptamers, Nucleotide/chemistry , RNA Precursors/chemistry , SELEX Aptamer Technique/methods , Surface Plasmon Resonance/methods , Base Sequence , Kinetics , Microfluidic Analytical Techniques/methods , RNA Precursors/genetics , X-Box Binding Protein 1/genetics
5.
PLoS One ; 7(11): e49455, 2012.
Article in English | MEDLINE | ID: mdl-23166675

ABSTRACT

BACKGROUND: Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages. METHODOLOGY/PRINCIPAL FINDINGS: We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes. CONCLUSIONS/SIGNIFICANCE: Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of genomic content. Differences in gene content likely contribute to differences in the clinical and environmental distribution of species and sequence types.


Subject(s)
Cronobacter/genetics , Evolution, Molecular , Genome, Bacterial/genetics , Phylogeny , Bacterial Secretion Systems/genetics , Base Sequence , Cronobacter/pathogenicity , Fimbriae, Bacterial/genetics , Likelihood Functions , Models, Genetic , Molecular Sequence Data , Multigene Family/genetics , Sequence Analysis, DNA , Species Specificity , Virulence Factors/genetics
6.
PLoS One ; 6(7): e22751, 2011.
Article in English | MEDLINE | ID: mdl-21799941

ABSTRACT

An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.


Subject(s)
Disease Outbreaks , Enterohemorrhagic Escherichia coli/genetics , Enterohemorrhagic Escherichia coli/pathogenicity , Escherichia coli Infections/epidemiology , Genomics/methods , Sequence Analysis, DNA/methods , Adult , Evolution, Molecular , Germany/epidemiology , Humans , Phylogeny , Prospective Studies , Time Factors
7.
Virol J ; 8: 99, 2011 Mar 04.
Article in English | MEDLINE | ID: mdl-21375749

ABSTRACT

BACKGROUND: Mimivirus, a giant dsDNA virus infecting Acanthamoeba, is the prototype of the mimiviridae family, the latest addition to the family of the nucleocytoplasmic large DNA viruses (NCLDVs). Its 1.2 Mb-genome was initially predicted to encode 917 genes. A subsequent RNA-Seq analysis precisely mapped many transcript boundaries and identified 75 new genes. FINDINGS: We now report a much deeper analysis using the SOLiD™ technology combining RNA-Seq of the Mimivirus transcriptome during the infectious cycle (202.4 Million reads), and a complete genome re-sequencing (45.3 Million reads). This study corrected the genome sequence and identified several single nucleotide polymorphisms. Our results also provided clear evidence of previously overlooked transcription units, including an important RNA polymerase subunit distantly related to Euryarchea homologues. The total Mimivirus gene count is now 1018, 11% greater than the original annotation. CONCLUSIONS: This study highlights the huge progress brought about by ultra-deep sequencing for the comprehensive annotation of virus genomes, opening the door to a complete one-nucleotide resolution level description of their transcriptional activity, and to the realistic modeling of the viral genome expression at the ultimate molecular level. This work also illustrates the need to go beyond bioinformatics-only approaches for the annotation of short protein and non-coding genes in viral genomes.


Subject(s)
Gene Expression Profiling , Genome, Viral , Mimiviridae/genetics , Amino Acid Sequence , High-Throughput Nucleotide Sequencing , Mimiviridae/chemistry , Molecular Sequence Data , Sequence Alignment , Viral Proteins/chemistry , Viral Proteins/genetics
8.
Mem. Inst. Oswaldo Cruz ; 95(3): 345-52, May-Jun. 2000. ilus, tab
Article in English | LILACS | ID: lil-258188

ABSTRACT

The PyAG1 gene, identified by the screening of a Plasmodium yoelii genomic DNA library with a rhoptry-specific Mab, encodes a protein with a zinc finger structure immediately followed by the consensus sequence of the Arf GAP catalytic site. The serum of mice immunized with the recombinant protein recognized specifically the rhoptries of the late infected erythrocytic stages. Blast analysis using the Genbank database gave the highest scores with four proteins presenting an Arf1 GAP activity. If presenting also this activity, the PyAG1 protein could be involved in the regulation of the secreted protein vesicular transport and, consequently, in the rhoptry biogenesis.


Subject(s)
Animals , Female , Mice , Rats , ADP-Ribosylation Factor 1/genetics , Genes, Protozoan , GTPase-Activating Proteins/genetics , Plasmodium yoelii/genetics , ADP-Ribosylation Factor 1/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Base Sequence , Drosophila melanogaster/genetics , Fluorescent Antibody Technique , Genomic Library , GTPase-Activating Proteins/metabolism , Immunoblotting , Mice, Inbred BALB C , Plasmodium yoelii/immunology , Protozoan Proteins/genetics , Rats/genetics , Saccharomyces cerevisiae/genetics , Zinc Fingers
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