Insertion sequences (IS) represent mobile genetic elements that have been shown to be associated with bacterial evolution and adaptation due to their effects on genome plasticity. In Bordetella pertussis, the causative agent of whooping cough, the numerous IS elements induce genomic rearrangements and contribute to the diversity of the global B. pertussis population. Previously, we have shown that the majority of IS-specific endogenous promoters induce the synthesis of alternative transcripts and thereby affect the transcriptional landscape of B. pertussis. Here, we describe the regulatory RNA Rfi2, which is transcribed from the Pout promoter of the IS481 gene BP1118 antisense to the adjacent fim2 gene encoding the major serotype 2 fimbrial subunit of B. pertussis. Among the classical bordetellae, Rfi2 is unique to B. pertussis, suggesting its specific role in virulence. We show that Rfi2 RNA attenuates fim2 transcription and, consequently, the production of the Fim2 protein. Interestingly, the mutant that does not produce Rfi2 displayed significantly increased cytotoxicity towards human macrophages compared to the parental strain. This observation suggests that the Rfi2-mediated reduction in cytotoxicity represents an evolutionary adaptation of B. pertussis that fine-tunes its interaction with the human host. Given the immunogenicity of Fim2, we further hypothesize that Rfi2-mediated modulation of Fim2 production contributes to immune evasion. To our knowledge, Rfi2 represents the first functionally characterized IS element-driven antisense RNA that modulates the expression of a virulence gene.

• Klíčová slova
• Bordetella pertussis, antisense RNA, cytotoxicity towards macrophages, fimbriae serotype 2, insertion sequence, modulation of virulence,
• MeSH
• antisense RNA * genetika   metabolismus   MeSH
• bakteriální fimbrie * genetika   metabolismus   MeSH
• Bordetella pertussis * genetika   patogenita   metabolismus   MeSH
• faktory virulence rodu Bordetella genetika   MeSH
• lidé MeSH
• makrofágy mikrobiologie   MeSH
• pertuse mikrobiologie   MeSH
• promotorové oblasti (genetika) MeSH
• proteiny fimbrií * genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• séroskupina MeSH
• transpozibilní elementy DNA * MeSH
• virulence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny bakteriální MeSH
• antisense RNA * MeSH
• faktory virulence rodu Bordetella MeSH
• fim2 protein, Bordetella MeSH Prohlížeč
• proteiny fimbrií * MeSH
• transpozibilní elementy DNA * MeSH

Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. The requirement of the RNA chaperone Hfq for the virulence of B. pertussis suggests that Hfq-dependent small regulatory RNAs (sRNAs) are involved in the virulence of this pathogen. To identify their potential mRNA targets, we applied a method combining experimental and computational approaches called RIL-seq. The majority of putative mRNA targets, including several virulence factors, interact with two sRNAs, CT_433 and CT_521, suggesting that these sRNAs may represent central riboregulatory nodes of B. pertussis. Furthermore, our data suggest that CT_532 sRNA can base pair with the 5'UTR region of ompA mRNA encoding outer membrane protein BP0943 (OmpA) and that CT_532, RNase III and Hfq are involved in the control of ompA expression. The CT_532 sRNA shares 60% identity with the E. coli sRNA MicA and its expression is also modulated by Hfq and stress conditions such as heat and cold shocks. Overall, these results suggest that CT_532 represents a MicA homolog. Importantly, the mutant lacking the first 22 nucleotides of CT_532 exhibits reduced cytotoxicity towards human macrophages and impaired biofilm production but increased resistance to complement compared to the wild type strain.

• MeSH
• 5' nepřekládaná oblast MeSH
• bakteriální RNA * metabolismus   genetika   MeSH
• biofilmy * růst a vývoj   MeSH
• Bordetella pertussis * genetika   patogenita   fyziologie   MeSH
• lidé MeSH
• malá nekódující RNA * metabolismus   genetika   MeSH
• messenger RNA metabolismus   MeSH
• protein hostitelského faktoru 1 * metabolismus   genetika   MeSH
• proteiny vnější bakteriální membrány genetika   metabolismus   MeSH
• regulace genové exprese u bakterií MeSH
• virulence genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• bakteriální RNA * MeSH
• malá nekódující RNA * MeSH
• messenger RNA MeSH
• OMPA outer membrane proteins MeSH Prohlížeč
• protein hostitelského faktoru 1 * MeSH
• proteiny vnější bakteriální membrány MeSH

Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. Similar to other Gram-negative pathogens, B. pertussis produces the type III secretion system, but its role in the pathogenesis of B. pertussis is enigmatic and yet to be elucidated. Here, we combined RNA-seq, LC-MS/MS, and co-immunoprecipitation techniques to identify and characterize the novel CesT family T3SS chaperone BP2265. We show that this chaperone specifically interacts with the secreted T3SS regulator BtrA and represents the first non-flagellar chaperone required for the secretion of an anti-sigma factor. In its absence, secretion but not production of BtrA and most T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS extends beyond its activity as an antagonist of the sigma factor BtrS. Predictions made by artificial intelligence system AlphaFold support the chaperone function of BP2265 towards BtrA and outline the structural basis for the interaction of BtrA with its target BtrS. We propose to rename BP2265 to BtcB for the Bordetella type III chaperone of BtrA.In addition, the absence of the BtcB chaperone results in increased expression of numerous flagellar genes and several virulence genes. While increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant, enhanced production of virulence factors resulted in increased cytotoxicity towards human macrophages. We hypothesize that these phenotypic traits result indirectly from impaired secretion of BtrA and altered activity of the BtrA/BtrS regulatory node.

• Klíčová slova
• Bordetella pertussis, CesT chaperone, T3SS, anti-sigma factor, biofilm,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• Bordetella pertussis * metabolismus   MeSH
• chromatografie kapalinová MeSH
• lidé MeSH
• pertuse * MeSH
• regulace genové exprese u bakterií MeSH
• sigma faktor genetika   MeSH
• tandemová hmotnostní spektrometrie MeSH
• umělá inteligence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• sigma faktor MeSH