Bordetella pertussis is the causative agent of human whooping cough (pertussis), a highly contagious respiratory disease that remains to be the least controlled vaccine-preventable infectious disease. Despite generalized vaccination, pertussis is again on the rise in highly vaccinated populations including Czech Republic. The increase in pertussis cases has been mostly attributed to short-lived immunity induced by the acellular vaccine and escape from immunity due to pathogen adaptation and antigen variation. Therefore, using comparative genomics we will analyze Czech clinical isolates of B. pertussis collected during the recent period 2008 - 2015. Importantly, genomic sequences, expression and production of virulence factors will be compared with those of B. pertussis vaccine strains. In addition, using proteomics and immunoprecipitation we will apply recent human sera of infected patients for identification of novel immunogenic antigens of B. pertussis.

Bordetella pertussis je lidský patogen, který je původcem černého kašle, vysoce nakažlivé respirační choroby, jejíž výskyt je v současné době na vzestupu a to i v ekonomicky vyspělých zemích s vysokou proočkovaností jako je Česká republika. Tento vzestup je přičítán zejména přechodu z celobuněčných vakcín na vakcíny acelulární. Acelulární vakcíny jsou sice ve srovnání s celobuněčnými méně reaktogenní, nicméně poskytují sníženou a kratší ochranu před nákazou. Navíc došlo u cirkulujících kmenů B. pertussis v reakci na antigeny obsažené ve vakcínách ke genotypovým změnám. Naším cílem je analyzovat celogenomové sekvence a produkci faktorů virulence v klinických izolátech získaných na území České republiky v letech 2008 – 2015. Pro srovnávací analýzu budou využity i původní vakcinační kmeny používané k výrobě celobuněčné vakcíny. Dále pomocí proteomiky a imunoprecipitace použijeme séra pacientů nakažených černým kašlem k identifikaci nových imunogenních antigenů produkovaných současnými kmeny B. pertussis.

• MeSH
• Bordetella pertussis genetika   patogenita   MeSH
• faktory virulence rodu Bordetella analýza   MeSH
• lidé MeSH
• pertuse genetika   prevence a kontrola   MeSH
• sekvenování celého genomu metody   MeSH
• virulence MeSH
• Check Tag
• lidé MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• genetika, lékařská genetika
• pneumologie a ftizeologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Bordetella pertussis is the causative agent of whooping cough, a respiratory disease still considered as a major public health threat and for which recent re-emergence has been observed. Constant reshuffling of Bordetella pertussis genome organization was observed during evolution. These rearrangements are essentially mediated by Insertion Sequences (IS), a mobile genetic elements present in more than 230 copies in the genome, which are supposed to be one of the driving forces enabling the pathogen to escape from vaccine-induced immunity. Here we use high-throughput sequencing approaches (RNA-seq and differential RNA-seq), to decipher Bordetella pertussis transcriptome characteristics and to evaluate the impact of IS elements on transcriptome architecture. Transcriptional organization was determined by identification of transcription start sites and revealed also a large variety of non-coding RNAs including sRNAs, leaderless mRNAs or long 3' and 5'UTR including seven riboswitches. Unusual topological organizations, such as overlapping 5'- or 3'-extremities between oppositely orientated mRNA were also unveiled. The pivotal role of IS elements in the transcriptome architecture and their effect on the transcription of neighboring genes was examined. This effect is mediated by the introduction of IS harbored promoters or by emergence of hybrid promoters. This study revealed that in addition to their impact on genome rearrangements, most of the IS also impact on the expression of their flanking genes. Furthermore, the transcripts produced by IS are strain-specific due to the strain to strain variation in IS copy number and genomic context.

• MeSH
• 3' nepřekládaná oblast MeSH
• 5' nepřekládaná oblast MeSH
• bakteriální RNA genetika   MeSH
• Bordetella pertussis genetika   MeSH
• genetická transkripce * MeSH
• genom bakteriální genetika   MeSH
• messenger RNA genetika   MeSH
• nekódující RNA genetika   MeSH
• počátek transkripce MeSH
• stanovení celkové genové exprese * MeSH
• transpozibilní elementy DNA genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Changes in environmental temperature represent one of the major stresses faced by microorganisms as they affect the function of the cytoplasmic membrane. In this study, we have analyzed the thermal adaptation in two closely related respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica Although B. pertussis represents a pathogen strictly adapted to the human body temperature, B. bronchiseptica causes infection in a broad range of animals and survives also outside of the host. We applied GC-MS to determine the fatty acids of both Bordetella species grown at different temperatures and analyzed the membrane fluidity by fluorescence anisotropy measurement. In parallel, we also monitored the effect of growth temperature changes on the expression and production of several virulence factors. In response to low temperatures, B. pertussis adapted its fatty acid composition and membrane fluidity to a considerably lesser extent when compared with B. bronchiseptica Remarkably, B. pertussis maintained the production of virulence factors at 24 °C, whereas B. bronchiseptica cells resumed the production only upon temperature upshift to 37 °C. This growth temperature-associated differential modulation of virulence factor production was linked to the phosphorylation state of transcriptional regulator BvgA. The observed differences in low-temperature adaptation between B. pertussis and B. bronchiseptica may result from selective adaptation of B. pertussis to the human host. We propose that the reduced plasticity of the B. pertussis membranes ensures sustained production of virulence factors at suboptimal temperatures and may play an important role in the transmission of the disease.

• MeSH
• aklimatizace * MeSH
• anizotropie MeSH
• bakteriální proteiny metabolismus   MeSH
• Bordetella bronchiseptica cytologie   fyziologie   MeSH
• Bordetella pertussis cytologie   fyziologie   MeSH
• buněčná membrána metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• druhová specificita MeSH
• faktory virulence metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• fosforylace MeSH
• lidé MeSH
• mastné kyseliny chemie   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• signální transdukce MeSH
• tělesná teplota MeSH
• teplota * MeSH
• transkripční faktory metabolismus   MeSH
• virulence MeSH
• životní prostředí MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Bordetella pertussis is a strictly human pathogen causing the respiratory infectious disease called whooping cough or pertussis. B. pertussis adaptation to acellular pertussis vaccine pressure has been repeatedly highlighted, but recent data indicate that adaptation of circulating strains started already in the era of the whole cell pertussis vaccine (wP) use. We sequenced the genomes of five B. pertussis wP vaccine strains isolated in the former Czechoslovakia in the pre-wP (1954-1957) and early wP (1958-1965) eras, when only limited population travel into and out of the country was possible. Four isolates exhibit a similar genome organization and form a distinct phylogenetic cluster with a geographic signature. The fifth strain is rather distinct, both in genome organization and SNP-based phylogeny. Surprisingly, despite isolation of this strain before 1966, its closest sequenced relative appears to be a recent isolate from the US. On the genome content level, the five vaccine strains contained both new and already described regions of difference. One of the new regions contains duplicated genes potentially associated with transport across the membrane. The prevalence of this region in recent isolates indicates that its spread might be associated with selective advantage leading to increased strain fitness.

• MeSH
• Bordetella pertussis genetika   izolace a purifikace   MeSH
• genetická variace MeSH
• genomika * MeSH
• lidé MeSH
• pertusová vakcína genetika   MeSH
• pořadí genů MeSH
• sekvenování celého genomu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Československo MeSH

Bordetella pertussis is the causative agent of human whooping cough, a highly contagious respiratory disease which despite vaccination programs remains the major cause of infant morbidity and mortality. The requirement of the RNA chaperone Hfq for virulence of B. pertussis suggested that Hfq-dependent small regulatory RNAs are involved in the modulation of gene expression. High-throughput RNA sequencing revealed hundreds of putative noncoding RNAs including the RgtA sRNA. Abundance of RgtA is strongly decreased in the absence of the Hfq protein and its expression is modulated by the activities of the two-component regulatory system BvgAS and another response regulator RisA. Whereas RgtA levels were elevated under modulatory conditions or in the absence of bvg genes, deletion of the risA gene completely abolished RgtA expression. Profiling of the ΔrgtA mutant in the ΔbvgA genetic background identified the BP3831 gene encoding a periplasmic amino acid-binding protein of an ABC transporter as a possible target gene. The results of site-directed mutagenesis and in silico analysis indicate that RgtA base-pairs with the region upstream of the start codon of the BP3831 mRNA and thereby weakens the BP3831 protein production. Furthermore, our data suggest that the function of the BP3831 protein is related to transport of glutamate, an important metabolite in the B. pertussis physiology. We propose that the BvgAS/RisA interplay regulates the expression of RgtA which upon infection, when glutamate might be scarce, attenuates translation of the glutamate transporter and thereby assists in adaptation of the pathogen to other sources of energy.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• Bordetella pertussis genetika   metabolismus   MeSH
• glutamáty metabolismus   MeSH
• lidé MeSH
• malá nekódující RNA genetika   MeSH
• regulace genové exprese u bakterií MeSH
• signální transdukce * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Despite high vaccination coverage, pertussis is increasing in many industrialized countries, including the Czech Republic. To better understand Bordetella pertussis resurgence, we analyzed historic strains and recent clinical isolates by using a comparative omics approach. Whole-genome sequencing showed that historic and recent isolates of B. pertussis have substantial variation in genome organization and form separate phylogenetic clusters. Subsequent RNA sequence analysis and liquid chromatography with mass tandem spectrometry analyses showed that these variations translated into discretely separated transcriptomic and proteomic profiles. When compared with historic strains, recent isolates showed increased expression of flagellar genes and genes involved in lipopolysaccharide biosynthesis and decreased expression of polysaccharide capsule genes. Compared with reference strain Tohama I, all strains had increased expression and production of the type III secretion system apparatus. We detected the potential link between observed effects and insertion sequence element-induced changes in gene context only for a few genes.

• MeSH
• Bordetella pertussis * genetika   MeSH
• fylogeneze MeSH
• lidé MeSH
• pertuse * epidemiologie   MeSH
• pertusová vakcína MeSH
• proteomika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

Bordetella pertussis, a strictly human re-emerging pathogen and the causative agent of whooping cough, exploits a broad variety of virulence factors to establish efficient infection. Here, we used RNA sequencing to analyse the changes in gene expression profiles of human THP-1 macrophages resulting from B. pertussis infection. In parallel, we attempted to determine the changes in intracellular B. pertussis-specific transcriptomic profiles resulting from interaction with macrophages. Our analysis revealed that global gene expression profiles in THP-1 macrophages are extensively rewired 6 h post-infection. Among the highly expressed genes, we identified those encoding cytokines, chemokines, and transcription regulators involved in the induction of the M1 and M2 macrophage polarization programmes. Notably, several host genes involved in the control of apoptosis and inflammation which are known to be hijacked by intracellular bacterial pathogens were overexpressed upon infection. Furthermore, in silico analyses identified large temporal changes in expression of specific gene subsets involved in signalling and metabolic pathways. Despite limited numbers of the bacterial reads, we observed reduced expression of majority of virulence factors and upregulation of several transcriptional regulators during infection suggesting that intracellular B. pertussis cells switch from virulent to avirulent phase and actively adapt to intracellular environment, respectively.

• MeSH
• Bordetella pertussis fyziologie   MeSH
• buněčné linie MeSH
• genová ontologie MeSH
• genové regulační sítě MeSH
• interakce hostitele a patogenu genetika   imunologie   MeSH
• kultivované buňky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• makrofágy imunologie   metabolismus   mikrobiologie   MeSH
• pertuse genetika   imunologie   virologie   MeSH
• regulace genové exprese MeSH
• reprodukovatelnost výsledků MeSH
• stanovení celkové genové exprese * metody   MeSH
• transkriptom * MeSH
• výpočetní biologie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH