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
• antigeny bakteriální 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 coccobacillus that causes whooping cough or pertussis, a respiratory disease that has recently experienced a resurgence. Upon entering the respiratory tract, B. pertussis colonizes the airway epithelium and attaches to ciliated cells. Here, we used primary human nasal epithelial cells (hNECs) cultured at the air-liquid interface and investigated their interaction with B. pertussis B1917, focusing on the role of the type III secretion system effector protein BteA. In this model, which resembles the epithelial cells of nasal epithelium in vivo, B. pertussis B1917 localized predominantly in the overlying mucus and scarcely colonized the cell cilia. The colonization led to a gradual decline in epithelial barrier function, as shown by measurements of transepithelial electrical resistance (TEER) and staining of the tight junction protein zonula occludens 1. The decrease in TEER occurred independently of the cytotoxic effector protein BteA. Transcriptomic and proteomic analyses of hNECs showed only moderate changes following infection, primarily characterized by increased mucus production, including upregulation of mucin MUC5AC. No profound response to BteA was detected. Furthermore, the infection did not induce production of inflammatory cytokines, suggesting that B. pertussis B1917 evades recognition by hNECs in this model system. These results suggest that the mucus may serve as a niche that allows B. pertussis B1917 to minimize epithelial recognition and damage. The lack of a robust immune response further indicates that additional components of the nasal mucosa, such as innate immune cells, are likely required to initiate an effective host defense.IMPORTANCEThe nasal epithelium is the initial site where Bordetella pertussis comes into contact with the host during respiratory tract infection. In this study, human nasal epithelial cells cultured at the air-liquid interface were established as an in vitro model to investigate the early stages of B. pertussis infection. We showed that the clinical isolate B. pertussis B1917 resides in the mucus during the early stages of colonization without disrupting the epithelial barrier function. Infection results in moderate transcriptomic and proteomic changes, characterized by increased mucus production and minimal inflammatory signaling. These results suggest that B. pertussis B1917 may evade early host recognition by residing in mucus and avoiding direct interaction with epithelial cells. They also highlight the importance of other components of the mucosal immune system, such as resident immune cells, for the initiation of an effective defense.

• Klíčová slova
• Bordetella pertussis, BteA effector, air-liquid interface culture, airway epithelium, human nasal epithelial cell, type III secretion system,
• MeSH
• bakteriální proteiny metabolismus   genetika   MeSH
• Bordetella pertussis * patogenita   genetika   fyziologie   MeSH
• epitelové buňky * mikrobiologie   imunologie   metabolismus   MeSH
• faktory virulence rodu Bordetella metabolismus   MeSH
• hlen mikrobiologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mucin 5AC metabolismus   genetika   MeSH
• nosní sliznice * mikrobiologie   cytologie   imunologie   MeSH
• pertuse * mikrobiologie   imunologie   MeSH
• proteomika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• faktory virulence rodu Bordetella MeSH
• mucin 5AC 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

Bordetella pertussis, the causative agent of whooping cough, is an extracellular, strictly human pathogen. However, it has been shown that B. pertussis cells can escape phagocytic killing and survive in macrophages upon internalization. Our time-resolved RNA-seq data suggest that B. pertussis efficiently adapts to the intramacrophage environment and responds to host bactericidal activities. We show that this adaptive response is multifaceted and, surprisingly, related to the BvgAS two-component system, a master regulator of virulence. Our results show that the expression of this regulatory circuit is downregulated upon internalization. Moreover, we demonstrate that the switch to the avirulent Bvg- phase augments a very complex process based on the adjustment of central and energy metabolism, cell wall reinforcement, maintenance of appropriate redox and metal homeostasis, and repair of damaged macromolecules. Nevertheless, not all observed effects could be simply attributed to the transition to Bvg- phase, suggesting that additional regulators are involved in the adaptation to the intramacrophage environment. Interestingly, a large number of genes required for the metabolism of sulphur were strongly modulated within macrophages. In particular, the mutant lacking two genes encoding cysteine dioxygenases displayed strongly attenuated cytotoxicity toward THP-1 cells. Collectively, our results suggest that intracellular B. pertussis cells have adopted the Bvg- mode to acclimate to the intramacrophage environment and respond to antimicrobial activities elicited by THP-1 cells. Therefore, we hypothesize that the avirulent phase represents an authentic phenotype of internalized B. pertussis cells.

• Klíčová slova
• Bordetella pertussis, BvgAS, adaptation to stress, avirulent phase, cysteine toxicity, intramacrophage environment,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• Bordetella pertussis * metabolismus   MeSH
• fenotyp MeSH
• lidé MeSH
• makrofágy metabolismus   MeSH
• pertuse * MeSH
• regulace genové exprese u bakterií MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH

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

The BvgS/BvgA two-component system controls expression of ∼550 genes of Bordetella pertussis, of which, ∼245 virulence-related genes are positively regulated by the BvgS-phosphorylated transcriptional regulator protein BvgA (BvgA∼P). We found that a single G-to-T nucleotide transversion in the 5'-untranslated region (5'-UTR) of the rplN gene enhanced transcription of the ribosomal protein operon and of the rpoA gene and provoked global dysregulation of B. pertussis genome expression. This comprised overproduction of the alpha subunit (RpoA) of the DNA-dependent RNA polymerase, downregulated BvgA and BvgS protein production, and impaired production and secretion of virulence factors by the mutant. Nonetheless, the mutant survived like the parental bacteria for >2 weeks inside infected primary human macrophages and persisted within infected mouse lungs for a longer period than wild-type B. pertussis These observations suggest that downregulation of virulence factor production by bacteria internalized into host cells may enable persistence of the whooping cough agent in the airways.IMPORTANCE We show that a spontaneous mutation that upregulates transcription of an operon encoding ribosomal proteins and causes overproduction of the downstream-encoded α subunit (RpoA) of RNA polymerase causes global effects on gene expression levels and proteome composition of Bordetella pertussis Nevertheless, the resulting important downregulation of the BvgAS-controlled expression of virulence factors of the whooping cough agent did not compromise its capacity to persist for prolonged periods inside primary human macrophage cells, and it even enhanced its capacity to persist in infected mouse lungs. These observations suggest that the modulation of BvgAS-controlled expression of virulence factors may occur also during natural infections of human airways by Bordetella pertussis and may possibly account for long-term persistence of the pathogen within infected cells of the airways.

• Klíčová slova
• Bordetella pertussis, host-pathogen interactions, intracellular bacteria, macrophages, two-component regulatory systems, virulence regulation,
• Publikační typ
• časopisecké články MeSH

Pertussis, also known as whooping cough, is a resurging acute respiratory disease of humans primarily caused by the Gram-negative coccobacilli Bordetella pertussis, and less commonly by the human-adapted lineage of B. parapertussisHU. The ovine-adapted lineage of B. parapertussisOV infects only sheep, while B. bronchiseptica causes chronic and often asymptomatic respiratory infections in a broad range of mammals but rarely in humans. A largely overlapping set of virulence factors inflicts the pathogenicity of these bordetellae. Their genomes also harbor a pathogenicity island, named bsc locus, that encodes components of the type III secretion injectosome, and adjacent btr locus with the type III regulatory proteins. The Bsc injectosome of bordetellae translocates the cytotoxic BteA effector protein, also referred to as BopC, into the cells of the mammalian hosts. While the role of type III secretion activity in the persistent colonization of the lower respiratory tract by B. bronchiseptica is well recognized, the functionality of the type III secretion injectosome in B. pertussis was overlooked for many years due to the adaptation of laboratory-passaged B. pertussis strains. This review highlights the current knowledge of the type III secretion system in the so-called classical Bordetella species, comprising B. pertussis, B. parapertussis, and B. bronchiseptica, and discusses its functional divergence. Comparison with other well-studied bacterial injectosomes, regulation of the type III secretion on the transcriptional and post-transcriptional level, and activities of BteA effector protein and BopN protein, homologous to the type III secretion gatekeepers, are addressed.

• Klíčová slova
• BopN, Bordetella, BteA/BopC, effector protein, pertussis, type III secretion system,
• MeSH
• bakteriální proteiny genetika   MeSH
• Bordetella bronchiseptica * MeSH
• Bordetella pertussis genetika   MeSH
• faktory virulence genetika   MeSH
• infekce bakteriemi rodu Bordetella * MeSH
• ovce MeSH
• sekreční systém typu III genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• bakteriální proteiny MeSH
• faktory virulence MeSH
• sekreční systém typu III MeSH