Bacterial pathogens sense specific cues associated with different host niches and integrate these signals to appropriately adjust the global gene expression. Bordetella pertussis is a Gram-negative, strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Though B. pertussis does not cause invasive infections, previous results indicated that this reemerging pathogen responds to blood exposure. Here, omics RNA-seq and LC-MS/MS techniques were applied to determine the blood-responsive regulon of B. pertussis. These analyses revealed that direct contact with blood rewired global gene expression profiles in B. pertussis as the expression of almost 20% of all genes was significantly modulated. However, upon loss of contact with blood, the majority of blood-specific effects vanished, with the exception of several genes encoding the T3SS-secreted substrates. For the first time, the T3SS regulator BtrA was identified in culture supernatants of B. pertussis. Furthermore, proteomic analysis identified BP2259 protein as a novel secreted T3SS substrate, which is required for T3SS functionality. Collectively, presented data indicate that contact with blood represents an important cue for B. pertussis cells.

• Klíčová slova
• Bordetella pertussis, T3SS, blood exposure, gene expression, omics analyses, protein secretion,
• MeSH
• anotace sekvence MeSH
• bakteriální proteiny metabolismus   MeSH
• Bordetella pertussis fyziologie   MeSH
• chromatografie kapalinová MeSH
• faktory virulence MeSH
• genomika * metody   MeSH
• lidé MeSH
• proteomika * metody   MeSH
• regulace genové exprese u bakterií MeSH
• sekreční systém typu III genetika   metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• tandemová hmotnostní spektrometrie MeSH
• transkriptom MeSH
• virulence MeSH
• výpočetní biologie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• faktory virulence MeSH
• sekreční systém typu III MeSH

Burkholderia cenocepacia causes severe pulmonary infections in cystic fibrosis (CF) patients. Since the bacterium is virtually untreatable by antibiotics, chronic infections persist for years and might develop into fatal septic pneumonia (cepacia syndrome, CS). To devise new strategies to combat chronic B. cenocepacia infections, it is essential to obtain comprehensive knowledge about their pathogenesis. We conducted a comparative genomic analysis of 32 Czech isolates of epidemic clone B. cenocepacia ST32 isolated from various stages of chronic infection in 8 CF patients. High numbers of large-scale deletions were found to occur during chronic infection, affecting preferentially genomic islands and nonessential replicons. Recombination between insertion sequences (IS) was inferred as the mechanism behind deletion formation; the most numerous IS group was specific for the ST32 clone and has undergone transposition burst since its divergence. Genes functionally related to transition metal metabolism were identified as hotspots for deletions and IS insertions. This functional category was also represented among genes where nonsynonymous point mutations and indels occurred parallelly among patients. Another category exhibiting parallel mutations was oxidative stress protection; mutations in catalase KatG resulted in impaired detoxification of hydrogen peroxide. Deep sequencing revealed substantial polymorphism in genes of both categories within the sputum B. cenocepacia ST32 populations, indicating extensive adaptive evolution. Neither oxidative stress response nor transition metal metabolism genes were previously reported to undergo parallel evolution during chronic CF infection. Mutations in katG and copper metabolism genes were overrepresented in patients where chronic infection developed into CS. Among professional phagocytes, macrophages use both hydrogen peroxide and copper for their bactericidal activity; our results thus tentatively point to macrophages as suspects in pathogenesis towards the fatal CS.

• MeSH
• Burkholderia cenocepacia genetika   MeSH
• chronická nemoc MeSH
• cystická fibróza komplikace   mikrobiologie   MeSH
• infekce bakteriemi rodu Burkholderia genetika   MeSH
• infekce dýchací soustavy mikrobiologie   MeSH
• lidé MeSH
• srovnávací genomová hybridizace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence factors implies a genuine pathogenic nature of this novel Bcc species.

• MeSH
• bakteriální proteiny genetika   MeSH
• Burkholderia genetika   izolace a purifikace   patogenita   MeSH
• cystická fibróza komplikace   mikrobiologie   MeSH
• dítě MeSH
• faktory virulence genetika   metabolismus   fyziologie   MeSH
• genom bakteriální MeSH
• infekce bakteriemi rodu Burkholderia komplikace   MeSH
• lidé MeSH
• objevující se infekční nemoci MeSH
• oportunní infekce komplikace   mikrobiologie   MeSH
• quorum sensing MeSH
• regulace genové exprese u bakterií MeSH
• stanovení celkové genové exprese MeSH
• virulence genetika   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• faktory virulence MeSH