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.

• Keywords
• Bordetella pertussis, antisense RNA, cytotoxicity towards macrophages, fimbriae serotype 2, insertion sequence, modulation of virulence,
• MeSH
• Antigens, Bacterial MeSH
• RNA, Antisense * genetics   metabolism   MeSH
• Fimbriae, Bacterial * genetics   metabolism   MeSH
• Bordetella pertussis * genetics   pathogenicity   metabolism   MeSH
• Virulence Factors, Bordetella genetics   MeSH
• Humans MeSH
• Macrophages microbiology   MeSH
• Whooping Cough microbiology   MeSH
• Promoter Regions, Genetic MeSH
• Fimbriae Proteins * genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Serogroup MeSH
• DNA Transposable Elements * MeSH
• Virulence MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Bacterial MeSH
• RNA, Antisense * MeSH
• Virulence Factors, Bordetella MeSH
• fim2 protein, Bordetella MeSH Browser
• Fimbriae Proteins * MeSH
• DNA Transposable Elements * 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.

• Keywords
• Bordetella pertussis, CesT chaperone, T3SS, anti-sigma factor, biofilm,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Bordetella pertussis * metabolism   MeSH
• Chromatography, Liquid MeSH
• Humans MeSH
• Whooping Cough * MeSH
• Gene Expression Regulation, Bacterial MeSH
• Sigma Factor genetics   MeSH
• Tandem Mass Spectrometry MeSH
• Artificial Intelligence MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Sigma Factor MeSH