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.

• Keywords
• Bordetella pertussis, BteA effector, air-liquid interface culture, airway epithelium, human nasal epithelial cell, type III secretion system,
• MeSH
• Bacterial Proteins metabolism   genetics   MeSH
• Bordetella pertussis * pathogenicity   genetics   physiology   MeSH
• Epithelial Cells * microbiology   immunology   metabolism   MeSH
• Virulence Factors, Bordetella metabolism   MeSH
• Mucus microbiology   metabolism   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mucin 5AC metabolism   genetics   MeSH
• Nasal Mucosa * microbiology   cytology   immunology   MeSH
• Whooping Cough * microbiology   immunology   MeSH
• Proteomics MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Virulence Factors, Bordetella MeSH
• Mucin 5AC MeSH

Bordetella pertussis is the causative agent of whooping cough in humans, a disease that has recently experienced a resurgence. In contrast, Bordetella bronchiseptica infects the respiratory tract of various mammalian species, causing a range of symptoms from asymptomatic chronic carriage to acute illness. Both pathogens utilize type III secretion system (T3SS) to deliver the effector protein BteA into host cells. Once injected, BteA triggers a cascade of events leading to caspase 1-independent necrosis through a mechanism that remains incompletely understood. We demonstrate that BteA-induced cell death is characterized by the fragmentation of the cellular endoplasmic reticulum and mitochondria, the formation of necrotic balloon-like protrusions, and plasma membrane permeabilization. Importantly, genome-wide CRISPR-Cas9 screen targeting 19,050 genes failed to identify any host factors required for BteA cytotoxicity, suggesting that BteA does not require a single nonessential host factor for its cytotoxicity. We further reveal that BteA triggers a rapid and sustained influx of calcium ions, which is associated with organelle fragmentation and plasma membrane permeabilization. The sustained elevation of cytosolic Ca2+ levels results in mitochondrial calcium overload, mitochondrial swelling, cristolysis, and loss of mitochondrial membrane potential. Inhibition of calcium channels with 2-APB delays both the Ca2+ influx and BteA-induced cell death. Our findings indicate that BteA exploits essential host processes and/or redundant pathways to disrupt calcium homeostasis and mitochondrial function, ultimately leading to host cell death.IMPORTANCEThe respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica exhibit cytotoxicity toward a variety of mammalian cells, which depends on the type III secretion effector BteA. Moreover, the increased virulence of B. bronchiseptica is associated with enhanced expression of T3SS and BteA. However, the molecular mechanism underlying BteA cytotoxicity is elusive. In this study, we performed a CRISPR-Cas9 screen, revealing that BteA-induced cell death depends on essential or redundant host processes. Additionally, we demonstrate that BteA disrupts calcium homeostasis, which leads to mitochondrial dysfunction and cell death. These findings contribute to closing the gap in our understanding of the signaling cascades targeted by BteA.

• Keywords
• Bordetella, calcium homeostasis, effector protein BteA, host cell death mechanism, type III secretion system (T3SS),
• MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• Bordetella bronchiseptica genetics   metabolism   drug effects   MeSH
• Bordetella pertussis genetics   pathogenicity   metabolism   drug effects   MeSH
• Cell Death * drug effects   MeSH
• Endoplasmic Reticulum metabolism   drug effects   MeSH
• Homeostasis * MeSH
• Host-Pathogen Interactions MeSH
• Humans MeSH
• Mitochondria metabolism   drug effects   MeSH
• Type III Secretion Systems metabolism   genetics   MeSH
• Calcium * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins * MeSH
• Type III Secretion Systems MeSH
• Calcium * 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

The classical Bordetella species infect the respiratory tract of mammals. While B. bronchiseptica causes rather chronic respiratory infections in a variety of mammals, the human-adapted species B. pertussis and B. parapertussisHU cause an acute respiratory disease known as whooping cough or pertussis. The virulence factors include a type III secretion system (T3SS) that translocates effectors BteA and BopN into host cells. However, the regulatory mechanisms underlying the secretion and translocation activity of T3SS in bordetellae are largely unknown. We have solved the crystal structure of BopN of B. pertussis and show that it is similar to the structures of gatekeepers that control access to the T3SS channel from the bacterial cytoplasm. We further found that BopN accumulates at the cell periphery at physiological concentrations of calcium ions (2 mM) that inhibit the secretion of BteA and BopN. Deletion of the bopN gene in B. bronchiseptica increased secretion of the BteA effector into calcium-rich medium but had no effect on secretion of the T3SS translocon components BopD and BopB. Moreover, the ΔbopN mutant secreted approximately 10-fold higher amounts of BteA into the medium of infected cells than the wild-type bacteria, but it translocated lower amounts of BteA into the host cell cytoplasm. These data demonstrate that BopN is a Bordetella T3SS gatekeeper required for regulated and targeted translocation of the BteA effector through the T3SS injectisome into host cells. IMPORTANCE The T3SS is utilized by many Gram-negative bacteria to deliver effector proteins from bacterial cytosol directly into infected host cell cytoplasm in a regulated and targeted manner. Pathogenic bordetellae use the T3SS to inject the BteA and BopN proteins into infected cells and upregulate the production of the anti-inflammatory cytokine interleukin-10 (IL-10) to evade host immunity. Previous studies proposed that BopN acted as an effector in host cells. In this study, we report that BopN is a T3SS gatekeeper that regulates the secretion and translocation activity of Bordetella T3SS.

• Keywords
• BopN, Bordetella, gatekeeper, type III secretion system,
• MeSH
• Bacterial Proteins metabolism   MeSH
• Bordetella pertussis metabolism   MeSH
• Virulence Factors metabolism   MeSH
• Humans MeSH
• Whooping Cough * MeSH
• Mammals MeSH
• Type III Secretion Systems * metabolism   MeSH
• Calcium MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Virulence Factors MeSH
• Type III Secretion Systems * MeSH
• Calcium MeSH

The respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector protein into host cells. This effector is known to contain two functional domains, including an N-terminal lipid raft targeting (LRT) domain and a cytotoxic C-terminal domain that induces nonapoptotic and caspase-1-independent host cell death. However, the exact molecular mechanisms underlying the interaction of BteA with plasma membrane (PM) as well as its cytotoxic activity in the course of Bordetella infections remain poorly understood. Using a protein-lipid overlay assay and surface plasmon resonance, we show here that the recombinant LRT domain binds negatively charged membrane phospholipids. Specifically, we determined that the dissociation constants of the LRT domain-binding liposomes containing phosphatidylinositol 4,5-bisphosphate, phosphatidic acid, and phosphatidylserine were ∼450 nM, ∼490 nM, and ∼1.2 μM, respectively. Both phosphatidylserine and phosphatidylinositol 4,5-bisphosphate were required to target the LRT domain and/or full-length BteA to the PM of yeast cells. The membrane association further involved electrostatic and hydrophobic interactions of LRT and depended on a leucine residue in the L1 loop between the first two helices of the four-helix bundle. Importantly, charge-reversal substitutions within the L1 region disrupted PM localization of the BteA effector without hampering its cytotoxic activity during B. bronchiseptica infection of HeLa cells. The LRT-mediated targeting of BteA to the cytosolic leaflet of the PM of host cells is, therefore, dispensable for effector cytotoxicity.

• Keywords
• Bordetella pertussis, BteA effector protein, Saccharomyces cerevisiae, lipid–protein interaction, membrane localization domain, plasma membrane, protein motif, surface plasmon resonance, type III secretion system, virulence factor,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Bordetella bronchiseptica genetics   growth & development   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Phospholipids metabolism   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Lipid Bilayers metabolism   MeSH
• Membrane Microdomains metabolism   MeSH
• Protein Domains MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Phospholipids MeSH
• Lipid Bilayers 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