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.

• Klíčová slova
• Bordetella, calcium homeostasis, effector protein BteA, host cell death mechanism, type III secretion system (T3SS),
• MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• Bordetella bronchiseptica genetika   metabolismus   účinky léků   MeSH
• Bordetella pertussis genetika   patogenita   metabolismus   účinky léků   MeSH
• buněčná smrt * účinky léků   MeSH
• endoplazmatické retikulum metabolismus   účinky léků   MeSH
• homeostáza * MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• mitochondrie metabolismus   účinky léků   MeSH
• sekreční systém typu III metabolismus   genetika   MeSH
• vápník * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• sekreční systém typu III MeSH
• vápník * 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.

• Klíčová slova
• BopN, Bordetella, gatekeeper, type III secretion system,
• MeSH
• bakteriální proteiny metabolismus   MeSH
• Bordetella pertussis metabolismus   MeSH
• faktory virulence metabolismus   MeSH
• lidé MeSH
• pertuse * MeSH
• savci MeSH
• sekreční systém typu III * metabolismus   MeSH
• vápník MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• faktory virulence MeSH
• sekreční systém typu III * MeSH
• vápník 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

The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice.

• MeSH
• antigeny bakteriální imunologie   MeSH
• bakteriální proteiny imunologie   MeSH
• Bordetella pertussis imunologie   MeSH
• ELISA MeSH
• infekce bakteriemi rodu Bordetella imunologie   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• pertuse imunologie   MeSH
• protilátky bakteriální krev   imunologie   MeSH
• tvorba protilátek imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH
• bakteriální proteiny MeSH
• protilátky bakteriální MeSH