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' Untranslated Regions MeSH
• RNA, Bacterial * metabolism   genetics   MeSH
• Biofilms * growth & development   MeSH
• Bordetella pertussis * genetics   pathogenicity   physiology   MeSH
• Humans MeSH
• RNA, Small Untranslated * metabolism   genetics   MeSH
• RNA, Messenger metabolism   MeSH
• Host Factor 1 Protein * metabolism   genetics   MeSH
• Bacterial Outer Membrane Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial MeSH
• Virulence genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 5' Untranslated Regions MeSH
• RNA, Bacterial * MeSH
• RNA, Small Untranslated * MeSH
• RNA, Messenger MeSH
• OMPA outer membrane proteins MeSH Browser
• Host Factor 1 Protein * MeSH
• Bacterial Outer Membrane Proteins MeSH

Pertussis, or whooping cough, is a highly contagious and acute respiratory illness caused primarily by the gram-negative coccobacillus Bordetella pertussis. Despite near-universal vaccination, pertussis remains one of the least-controlled vaccine-preventable infectious diseases. Since 2023, pertussis incidence has been rising, and widespread pertussis outbreaks have resurged in many countries. In response to these emerging challenges, almost 300 experts from institutions across 24 countries convened at the 14th International Bordetella Symposium in Prague, Czech Republic, from 24 to 28 June 2024 to discuss pertussis epidemiology and research and strategies to mitigate the global pertussis burden. We present here the highlights of the symposium, comprising epidemiological and clinical aspects of Bordetella infections, results of clinical trials of pertussis vaccination in pregnant women and effectiveness of maternal vaccination in protecting newborn infants in Africa and Europe, the controlled human infection model (CHIM), and the latest insights into the biology, immunology, and pathogenesis of B. pertussis infection.

• Keywords
• Bordetella pertussis, epidemiology, pathogenesis, toxins, vaccines, virulence,
• MeSH
• Bordetella pertussis * immunology   MeSH
• Global Health MeSH
• Congresses as Topic MeSH
• Humans MeSH
• Infant, Newborn MeSH
• Whooping Cough * prevention & control   epidemiology   microbiology   MeSH
• Pertussis Vaccine administration & dosage   immunology   MeSH
• Pregnancy MeSH
• Vaccination MeSH
• Check Tag
• Humans MeSH
• Infant, Newborn MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Congress MeSH
• Review MeSH
• Names of Substances
• Pertussis Vaccine 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

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.

• Keywords
• Bordetella pertussis, BvgAS, adaptation to stress, avirulent phase, cysteine toxicity, intramacrophage environment,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Bordetella pertussis * metabolism   MeSH
• Phenotype MeSH
• Humans MeSH
• Macrophages metabolism   MeSH
• Whooping Cough * MeSH
• Gene Expression Regulation, Bacterial MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH

The ability of bacterial pathogens to acquire essential micronutrients is critical for their survival in the host environment. Manganese plays a complex role in the virulence of a variety of pathogens due to its function as an antioxidant and enzymatic cofactor. Therefore, host cells deprive pathogens of manganese to prevent or attenuate infection. Here, we show that evolution of the human-restricted pathogen Bordetella pertussis has selected for an inhibitory duplication within a manganese exporter of the calcium:cation antiporter superfamily. Intriguingly, upon exposure to toxic levels of manganese, the nonfunctional exporter becomes operative in resister cells due to a unique reverse adaptation mechanism. However, compared with wild-type (wt) cells, the resisters carrying a functional copy of the exporter displayed strongly reduced intracellular levels of manganese and impaired growth under oxidative stress. Apparently, inactivation of the manganese exporter and the resulting accumulation of manganese in the cytosol benefited the pathogen by improving its survival under stress conditions. The inhibitory duplication within the exporter gene is highly conserved among B. pertussis strains, absent from all other Bordetella species and from a vast majority of organisms across all kingdoms of life. Therefore, we conclude that inactivation of the exporter gene represents an exceptional example of a flexible genome decay strategy employed by a human pathogen to adapt to its exclusive host. IMPORTANCE Bordetella pertussis, a respiratory pathogen restricted to humans, continuously adapts its genome to its exclusive host. We show that speciation of this reemerging pathogen was accompanied by loss of function of the manganese exporter. Intriguingly, the functionality of the exporter can be restored in the presence of toxic levels of manganese by a unique genetic modification. However, compared with the wt strain, the strain carrying the functional exporter failed to resist the oxidative stress in vitro. Thus, our data demonstrate that inactivation of the exporter resulting in manganese accumulation assists B. pertussis in adaptation to oxidative stress. We conclude that this sophisticated process of reverse adaptation enables B. pertussis to adjust to rapidly changing environments by facilitating its resistance to both manganese toxicity and manganese scarcity.

• Keywords
• Bordetella, Bordetella pertussis, genome decay, manganese, oxidative stress, pathogen adaptation,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Bordetella pertussis drug effects   genetics   pathogenicity   MeSH
• Virulence Factors genetics   MeSH
• Humans MeSH
• Manganese toxicity   MeSH
• Oxidative Stress MeSH
• Whooping Cough prevention & control   MeSH
• Gene Expression Regulation, Bacterial drug effects   MeSH
• Virulence drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Virulence Factors MeSH
• Manganese 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.

• Keywords
• Bordetella pertussis, T3SS, blood exposure, gene expression, omics analyses, protein secretion,
• MeSH
• Molecular Sequence Annotation MeSH
• Bacterial Proteins metabolism   MeSH
• Bordetella pertussis physiology   MeSH
• Chromatography, Liquid MeSH
• Virulence Factors MeSH
• Genomics * methods   MeSH
• Humans MeSH
• Proteomics * methods   MeSH
• Gene Expression Regulation, Bacterial MeSH
• Type III Secretion Systems genetics   metabolism   MeSH
• Gene Expression Profiling MeSH
• Tandem Mass Spectrometry MeSH
• Transcriptome MeSH
• Virulence MeSH
• Computational Biology methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Virulence Factors MeSH
• Type III Secretion Systems 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.

• Keywords
• Bordetella pertussis, host-pathogen interactions, intracellular bacteria, macrophages, two-component regulatory systems, virulence regulation,
• Publication type
• Journal Article 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.

• Keywords
• Bordetella pertussis, host-pathogen interaction, infection, intracellular survival, macrophage,
• MeSH
• Bordetella pertussis physiology   MeSH
• Cell Line MeSH
• Gene Ontology MeSH
• Gene Regulatory Networks MeSH
• Host-Pathogen Interactions genetics   immunology   MeSH
• Cells, Cultured MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Humans MeSH
• Macrophages immunology   metabolism   microbiology   MeSH
• Whooping Cough genetics   immunology   virology   MeSH
• Gene Expression Regulation MeSH
• Reproducibility of Results MeSH
• Gene Expression Profiling * methods   MeSH
• Transcriptome * MeSH
• Computational Biology methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Bordetella pertussis is a Gram-negative strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Previously, we have shown that RNA chaperone Hfq is required for virulence of B. pertussis. Furthermore, microarray analysis revealed that a large number of genes are affected by the lack of Hfq. This study represents the first attempt to characterize the Hfq regulon in bacterial pathogen using an integrative omics approach. Gene expression profiles were analyzed by RNA-seq and protein amounts in cell-associated and cell-free fractions were determined by LC-MS/MS technique. Comparative analysis of transcriptomic and proteomic data revealed solid correlation (r2 = 0.4) considering the role of Hfq in post-transcriptional control of gene expression. Importantly, our study confirms and further enlightens the role of Hfq in pathogenicity of B. pertussis as it shows that Δhfq strain displays strongly impaired secretion of substrates of Type III secretion system (T3SS) and substantially reduced resistance to serum killing. On the other hand, significantly increased production of proteins implicated in transport of important metabolites and essential nutrients observed in the mutant seems to compensate for the physiological defect introduced by the deletion of the hfq gene.

• Keywords
• Bordetella pertussis, Hfq, T3SS, omics analysis, serum resistance, solute-binding proteins,
• MeSH
• Bordetella pertussis genetics   metabolism   MeSH
• Chromatography, Liquid MeSH
• Gene Ontology MeSH
• Humans MeSH
• Host Factor 1 Protein genetics   metabolism   MeSH
• Proteome MeSH
• Proteomics * methods   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Regulon * MeSH
• Type III Secretion Systems genetics   metabolism   MeSH
• Gene Expression Profiling * methods   MeSH
• Tandem Mass Spectrometry MeSH
• Transcriptome MeSH
• Computational Biology methods   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Host Factor 1 Protein MeSH
• Proteome MeSH
• Type III Secretion Systems 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.

• Keywords
• Bordetella, riboregulation, sRNA, signal transduction, translational repression,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Bordetella pertussis genetics   metabolism   MeSH
• Glutamates metabolism   MeSH
• Humans MeSH
• RNA, Small Untranslated genetics   MeSH
• Gene Expression Regulation, Bacterial MeSH
• Signal Transduction * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Glutamates MeSH
• RNA, Small Untranslated MeSH