Bordetella filamentous hemagglutinin and adenylate cyclase toxin interactions on the bacterial surface are consistent with FhaB-mediated delivery of ACT to phagocytic cells.

Bordetella species that cause respiratory infections in mammals include B. pertussis, which causes human whooping cough, and B. bronchiseptica, which infects nearly all mammals. Both bacterial species produce filamentous hemagglutinin (FhaB) and adenylate cyclase toxin (ACT), prominent surface-associated and secreted virulence factors that contribute to persistence in the lower respiratory tract by inhibiting clearance by phagocytic cells. FhaB and ACT proteins interact with themselves, each other, and host cells. Using immunoblot analyses, we showed that ACT binds to FhaB on the bacterial surface before it can be detected in culture supernatants. We determined that SphB1, a surface protease identified based on its requirement for FhaB cleavage, is also required for ACT cleavage, and we determined that the presence of ACT blocks SphB1-dependent and -independent cleavage of FhaB, but the presence of FhaB does not affect SphB1-dependent cleavage of ACT. The primary SphB1-dependent cleavage site on ACT is proximal to ACT's active site, in a region that is critical for ACT activity. We also determined that FhaB-bound ACT on the bacterial surface can intoxicate host cells producing CR3, the receptor for ACT. In addition to increasing our understanding of FhaB, ACT, and FhaB-ACT interactions on the Bordetella surface, our data are consistent with a model in which FhaB functions as a novel toxin delivery system by binding to ACT and allowing its release upon binding of ACT to its receptor, CR3, on phagocytic cells.IMPORTANCEBacteria need to control the variety, abundance, and conformation of proteins on their surface to survive. Members of the Gram-negative bacterial genus Bordetella include B. pertussis, which causes whooping cough in humans, and B. bronchiseptica, which causes respiratory infections in a broad range of mammals. These species produce two prominent virulence factors, the two-partner secretion (TPS) effector FhaB and adenylate cyclase toxin (ACT), that interact with themselves, each other, and host cells. Here, we determined that ACT binds FhaB on the bacterial surface before being detected in culture supernatants and that ACT bound to FhaB can be delivered to eukaryotic cells. Our data are consistent with a model in which FhaB delivers ACT specifically to phagocytic cells. This is the first report of a TPS system facilitating the delivery of a separate polypeptide toxin to target cells and expands our understanding of how TPS systems contribute to bacterial pathogenesis.

RpoN (sigma factor 54) contributes to bacterial fitness during tracheal colonization of Bordetella bronchiseptica.

The Gram-negative pathogenic bacterium Bordetella bronchiseptica is a respiratory pathogen closely related to Bordetella pertussis, the causative agent of whooping cough. Despite sharing homologous virulence factors, B. bronchiseptica infects a broad range of mammalian hosts, including some experimental animals, whereas B. pertussis is strictly adapted to humans. Therefore, B. bronchiseptica is often used as a representative model to explore the pathogenicity of Bordetella in infection experiments with laboratory animals. Although Bordetella virulence factors, including toxins and adhesins have been studied well, our recent study implied that unknown virulence factors are involved in tracheal colonization and infection. Here, we investigated bacterial genes contributing to tracheal colonization by high-throughput transposon sequencing (Tn-seq). After the screening, we picked up 151 candidate genes of various functions and found that a rpoN-deficient mutant strain was defective in tracheal colonization when co-inoculated with the wild-type strain. rpoN encodes σ54 , a sigma factor that regulates the transcription of various genes, implying its contribution to various bacterial activities. In fact, we found RpoN of B. bronchiseptica is involved in bacterial motility and initial biofilm formation. From these results, we propose that RpoN supports bacterial colonization by regulating various bacteriological functions.

Anti-Bordetella bronchiseptica effects of targeted bacteriophages via microbiome and metabolic mediated mechanisms.

Bordetella bronchiseptica poses a significant challenge in the context of respiratory infections, particularly in weanling pigs. In this study, we investigated the impact of a novel targeted bacteriophage in controlling B. bronchiseptica challenge (BBC) in an experimental design involving five distinct treatment groups: NC (no challenge), PC (BBC challenge), BF (108 pfu bacteriophage/kg diet + BBC), BN (2 × 107 pfu/day bacteriophage by nasal spray + BBC), and AT (antibiotic + BBC). The experiment was conducted for 2 weeks. The highest turbinate score was observed in the PC. The BF treatment showed higher plasma IL (interleukine)-1ß and IL-6 compared with the BN and AT treatments. Plasma concentrations of IL-1ß were increased in the BF pigs compared with the BN, AT, and NC. Among the BBC groups, the PC treatment exhibited a higher abundance of Staphylococcus. aureus and B. bronchiseptica in the lung. A lower S. aureus, Streptococcus. suis, and B. bronchiseptica colonization was detected in the AT compared with the BF and BN treatments. The BF showed lower plasma zonulin compared with the BN and AT. A higher plasma concentration of superoxide dismutase was observed in the BF and AT compared with PC and BN. The BN influenced the glycine, serine-threonine metabolism; glycerolipid metabolism; glyoxylate-dicarboxylate metabolism; and arachidonic acid metabolism compared with the NC. In conclusion, nasal-sprayed bacteriophage effectively controlled B. bronchiseptica infection, however, their efficiency was lower than the antibiotic.

Adaptive immune protection of the middle ears differs from that of the respiratory tract.

The efficacy of the adaptive immune system in the middle ear (ME) is well established, but the mechanisms are not as well defined as those of gastrointestinal or respiratory tracts. While cellular elements of the adaptive response have been detected in the MEs following infections (or intranasal immunizations), their specific contributions to protecting the organ against reinfections are unknown. How immune protection mechanisms of the MEs compares with those in the adjacent and attached upper and lower respiratory airways remains unclear. To address these knowledge gaps, we used an established mouse respiratory infection model that we recently showed also involves ME infections. Bordetella bronchiseptica delivered to the external nares of mice in tiny numbers very efficiently infects the respiratory tract and ascends the Eustachian tube to colonize and infect the MEs, where it causes severe but acute inflammation resembling human acute otitis media (AOM). Since this AOM naturally resolves, we here examine the immunological mechanisms that clear infection and protect against subsequent infection, to guide efforts to induce protective immunity in the ME. Our results show that once the MEs are cleared of a primary B. bronchiseptica infection, the convalescent organ is strongly protected from reinfection by the pathogen despite its persistence in the upper respiratory tract, suggesting important immunological differences in these adjacent and connected organs. CD4+ and CD8+ T cells trafficked to the MEs following infection and were necessary to robustly protect against secondary challenge. Intranasal vaccination with heat killed B. bronchiseptica conferred robust protection against infection to the MEs, even though the nasopharynx itself was only partially protected. These data establish the MEs as discrete effector sites of adaptive immunity and shows that effective protection in the MEs and the respiratory tract is significantly different. This model system allows the dissection of immunological mechanisms that can prevent bacteria in the nasopharynx from ascending the ET to colonize the ME.

Bordetella spp. block eosinophil recruitment to suppress the generation of early mucosal protection.

Bordetella spp. are respiratory pathogens equipped with immune evasion mechanisms. We previously characterized a Bordetella bronchiseptica mutant (RB50ΔbtrS) that fails to suppress host responses, leading to rapid clearance and long-lasting immunity against reinfection. This work revealed eosinophils as an exclusive requirement for RB50ΔbtrS clearance. We also show that RB50ΔbtrS promotes eosinophil-mediated B/T cell recruitment and inducible bronchus-associated lymphoid tissue (iBALT) formation, with eosinophils being present throughout iBALT for Th17 and immunoglobulin A (IgA) responses. Finally, we provide evidence that XCL1 is critical for iBALT formation but not maintenance, proposing a novel role for eosinophils as facilitators of adaptive immunity against B. bronchiseptica. RB50ΔbtrS being incapable of suppressing eosinophil effector functions illuminates active, bacterial targeting of eosinophils to achieve successful persistence and reinfection. Overall, our discoveries contribute to understanding cellular mechanisms for use in future vaccines and therapies against Bordetella spp. and extension to other mucosal pathogens.

Expression, purification, crystallization of a ZIP metal transporter from Bordetella bronchiseptica (BbZIP).

Structural studies of the ZIPs have greatly improved the understanding of the working mechanism for this functionally important metal transporter family. In this chapter, we describe the procedures to overexpress, purify, and crystallize a representative bacterial ZIP from Bordetella bronchiseptica (BbZIP), the structure of which was the first one that revealed the common structural framework of the transmembrane domain conserved within the entire ZIP family. We also discuss the considerations when we designed these experiments and compare the approaches used in this study with those commonly used in other works. The protocols provided in this chapter will facilitate structural and biochemical studies of other members of the ZIP family.

Genomic evidence and virulence properties decipher the extra-host origin of Bordetella bronchiseptica.

Until recently, members of the classical Bordetella species comprised only pathogenic bacteria that were thought to live exclusively in warm-blooded animals. The close phylogenetic relationship of Bordetella with Achromobacter and Alcaligenes, which include primarily environmental bacteria, suggests that the ancestral Bordetellae were probably free-living. Eventually, the Bordetella species evolved to infect and live within warm-blooded animals. The modern history of pathogens related to the genus Bordetella started towards the end of the 19th century when it was discovered in the infected respiratory epithelium of mammals, including humans. The first identified member was Bordetella pertussis, which causes whooping cough, a fatal disease in young children. In due course, B. bronchiseptica was recovered from the trachea and bronchi of dogs with distemper. Later, a second closely related human pathogen, B. parapertussis, was described as causing milder whooping cough. The classical Bordetellae are strictly host-associated pathogens transmitted via the host-to-host aerosol route. Recently, the B. bronchiseptica strain HT200 has been reported from a thermal spring exhibiting unique genomic features that were not previously observed in clinical strains. Therefore, it advocates that members of classical Bordetella species have evolved from environmental sources. This organism can be transmitted via environmental reservoirs as it can survive nutrient-limiting conditions and possesses a motile flagellum. This study aims to review the molecular basis of origin and virulence properties of obligate host-restricted and environmental strains of classical Bordetella.

Prior infection with Bordetella bronchiseptica enhanced colonization but not disease with Streptococcus suis.

Bordetella bronchiseptica and Streptococcus suis are widely distributed swine pathogens. B. bronchiseptica is a primary pathogen and causes atrophic rhinitis and bronchopneumonia. S. suis is a contributing agent to porcine respiratory disease complex and causes systemic diseases including arthritis, meningitis, polyserositis, and septicemia. Colonization with B. bronchiseptica has been associated with increased colonization by other pathogenic bacteria and increased disease severity with viral and bacterial pathogens. It has also been reported to predispose cesarean derived, colostrum deprived (CDCD) piglets to S. suis systemic disease. Here, we evaluated the role of B. bronchiseptica colonization on S. suis colonization, dissemination, and disease in one study using conventional pigs and another using CDCD pigs. Pigs were challenged with S. suis, B. bronchiseptica, or B. bronchiseptica followed by S. suis. Incidence of S. suis disease was not increased in either study for animals pre-inoculated with B. bronchiseptica. Nasal colonization with S. suis was increased in coinfected animals, while B. bronchiseptica was similar between mono- and co-infected animals. Although increased S. suis disease was not seen in coinfected pigs, there is evidence that B. bronchiseptica can increase colonization with S. suis, which may contribute to enhanced disease when animals are stressed or immunocompromised.

Immunostimulatory activity and safety evaluation of Bordetella bronchiseptica-derived lipopolysaccharide, a new vaccine adjuvant candidate.

Adjuvants are used to elicit strong immune responses for vaccines that show poor immunogenicity. Previously, we demonstrated that a sonicated bacterin of Bordetella bronchiseptica can be used as a safe adjuvant that enhances the antigen-presenting capability of dendritic cells (DCs). In this study, we purified the lipopolysaccharide (LPS) of B. bronchiseptica (Bb-LPS) and investigated its immunogenic effects on DCs compared to those of Escherichia coli O26:B6 (O26)-derived LPS (O26-LPS), a positive control. Bb-LPS was purified using an LPS extraction kit. Limulus amebocyte lysate assay was performed to determine the optimal concentration of Bb-LPS and O26-LPS for treatment. Bb-LPS increased the metabolic activity of DCs at a concentration of 0 to 250 EU/mL, similar to that of O26-LPS. Bb-LPS significantly increased the expression level of CD40 and CD54, related to the immune responses of DCs. Bb-LPS enhanced the antigen-presenting capability of DCs and significantly increased the interferon-gamma/interleukin-4 ratio of CD4+ T cells co-cultured with DCs to 0.95 (p < 0.05). Moreover, Bb-LPS increased the production of pro-inflammatory cytokines in a safer manner than that obtained by O26-LPS. In vivo safety tests revealed that Bb-LPS was less toxic than O26-LPS in mice. This study demonstrated that Bb-LPS showed unique immune characteristics and immunogenic effects on the antigen-presenting capability of DCs, which differed from those of O26-LPS. This study provides valuable information for basic and clinical research for developing safe vaccine adjuvants.

Enhancing effect of chitosan nanoparticles on the immune efficacy of Bordetella bronchiseptica outer membrane vesicles.

The outer membrane vesicle (OMV) of bacteria is a bilayer membrane vesicle with a diameter of about 10-300 nm that is secreted during the growth of Gram-negative bacteria. OMV is considered as a high-quality vaccine candidate antigen because of its natural immunogenicity and non-replicability. Although the excellent antigenicity of OMV has been widely confirmed, its instability and heterogeneity greatly affect its immune effect. Many studies have demonstrated that in combination with nanoparticles can enhance the stability of OMV. In this study, OMVs were used to coat chitosan nanoparticles (CNPs) and obtain a stable OMV vaccine. The characteristics, including morphology, hydrodynamic size, and zeta potential were evaluated. The immune protection of CNP-OMV and anti-infection efficacy were examined and compared in vivo and in vitro. The results showed that the CNP-OMV were homogenous with a size of 139 nm and a stable core-shell structure. And CNP-OMV could significantly increase the cell proliferation, phagocytosis and TNF-α, IL-6 and IL-10 secretion of RAW264.7 in vitro. In vivo, CNP-OMV could significantly increase the levels of anti-Bb and OMV IgG antibodies. Levels of blood lymphocyte, and Th1 (IFN-γ, IL-12), Th2 (IL-4, IL-5), and Th17 (IL-17, TNF-α) type cytokines in the serum were all significantly increased. At the same time, CNP-OMV could significantly reduce the bacterial invading the lungs of challenged rabbits. And CNP-OMV could largely protect the lungs from injury. The above results showed that CNP-OMV had a good immune efficacy and could resist the infection of Bordetella bronchiseptica. This study provided a scientific basis for the development of novel effective and safe vaccine against Bordetella bronchiseptica, and also provided a new idea for the development of new bacterial vaccine.

Adjuvant Efficacy of the ECMS-Oil on Immune Responses against Bordetella bronchiseptica in Mice through the TLR2/MyD88/NF-κB Pathway.

Bordetella infection can be efficiently prevented through vaccination. The current study investigated the effects of an extract of Cochinchina momordica seed (ECMS) combined with oil on the immune responses to the inactivated Bordetella vaccine in mice. Serum IgG and IgG1 level was significantly increased in ECMS-oil group compared to any other group (P < 0.05) 2 weeks after immunization, while groups ECMS200 µg/400 µg-oil had a markedly higher level of serum IgG2b and IgG3 than any other groups (P < 0.05). Moreover, lipopolysaccharide/ConA-stimulated proliferation of splenocytes was significantly enhanced in ECMS 400 µg-oil immunized mice in comparison with mice in any other group (P < 0.05). RT-PCR assay revealed that while ECMS800 µg-oil group had significantly higher levels of serum IL-4, IL-10, Toll-like receptor (TLR)2, and IL-1 beta than any other group (P < 0.05), the levels of serum IL-2, IL-4, and IL-10 were markedly increased in ECMS 400 µg-oil group as compared to any other groups (P < 0.05). Blood analysis showed that ECMS800 µg-oil and oil groups had a significantly higher number of immunocytes than any other groups (P < 0.05). There were significant differences in the number of IgG+, IgG2b+, and IgA+ cells in the lung between ECMS800 µg-oil group and any other groups (P < 0.05). Western blot analysis demonstrated that stimulation with ECMS 25 µg/mL or 50 ng/mL led to a significant increase in the expression of TLR2, MyD88, and NF-κB in Raw264.7 cells (P < 0.05). Compared with any other group, the expression of MyD88 was markedly increased in the cells stimulated with ECMS 50 ng/mL, as indicated by the RT-PCR analysis (P < 0.05). Overall, we observed that ECMS-oil efficiently enhanced the humoral or cellular immune responses against Bordetella and suggested that the mechanism of adjuvant activity of ECMS-oil might involve TLR2/MyD88/NF-κB signaling pathway.

Bordetella bronchiseptica and Bordetella pertussis: Similarities and Differences in Infection, Immuno-Modulation, and Vaccine Considerations.

Bordetella pertussis and Bordetella bronchiseptica belong to the genus Bordetella, which comprises 14 other species. B. pertussis is responsible for whooping cough in humans, a severe infection in children and less severe or chronic in adults. These infections are restricted to humans and currently increasing worldwide. B. bronchiseptica is involved in diverse respiratory infections in a wide range of mammals. For instance, the canine infectious respiratory disease complex (CIRDC), characterized by a chronic cough in dogs. At the same time, it is increasingly implicated in human infections, while remaining an important pathogen in the veterinary field. Both Bordetella can evade and modulate host immune responses to support their persistence, although it is more pronounced in B. bronchiseptica infection. The protective immune responses elicited by both pathogens are comparable, while there are important characteristics in the mechanisms that differ. However, B. pertussis pathogenesis is more difficult to decipher in animal models than those of B. bronchiseptica because of its restriction to humans. Nevertheless, the licensed vaccines for each Bordetella are different in terms of formulation, route of administration and immune responses induced, with no known cross-reaction between them. Moreover, the target of the mucosal tissues and the induction of long-lasting cellular and humoral responses are required to control and eliminate Bordetella. In addition, the interaction between both veterinary and human fields are essential for the control of this genus, by preventing the infections in animals and the subsequent zoonotic transmission to humans.

Bordetella bronchiseptica diguanylate cyclase BdcB inhibits the type three secretion system and impacts the immune response.

Bordetella bronchiseptica is a gram-negative bacterium that causes respiratory diseases in different animals, including mice, making B. bronchiseptica the gold-standard model to investigate host-pathogen interaction at the molecular level. B. bronchiseptica utilizes many different mechanisms to precisely regulate the expression of virulence factors. Cyclic di-GMP is a second messenger synthesized by diguanylate cyclases and degraded by phosphodiesterases that regulates the expression of multiple virulence factors including biofilm formation. As in other bacteria, we have previously shown that c-di-GMP regulates motility and biofilm formation in B. bronchiseptica. This work describes the diguanylate cyclase BdcB (Bordetella diguanylate cyclase B) as an active diguanylate cyclase that promotes biofilm formation and inhibits motility in B. bronchiseptica. The absence of BdcB increased macrophage cytotoxicity in vitro and induced a greater production of TNF-α, IL-6, and IL-10 by macrophages. Our study reveals that BdcB regulates the expression of components of T3SS, an important virulence factor of B. bronchiseptica. The Bb∆bdcB mutant presented increased expression of T3SS-mediated toxins such as bteA, responsible for cytotoxicity. Our in vivo results revealed that albeit the absence of bdcB did not affect the ability of B. bronchiseptica to infect and colonize the respiratory tract of mice, mice infected with Bb∆bdcB presented a significantly higher pro-inflammatory response than those infected with wild type B. bronchiseptica.

Physiological consequences of inactivation of lgmB and lpxL1, two genes involved in lipid A synthesis in Bordetella bronchiseptica.

To develop a Bordetella bronchiseptica vaccine with reduced endotoxicity, we previously inactivated lpxL1, the gene encoding the enzyme that incorporates a secondary 2-hydroxy-laurate in lipid A. The mutant showed a myriad of phenotypes. Structural analysis showed the expected loss of the acyl chain but also of glucosamine (GlcN) substituents, which decorate the phosphates in lipid A. To determine which structural change causes the various phenotypes, we inactivated here lgmB, which encodes the GlcN transferase, and lpxL1 in an isogenic background and compared the phenotypes. Like the lpxL1 mutation, the lgmB mutation resulted in reduced potency to activate human TLR4 and to infect macrophages and in increased susceptibility to polymyxin B. These phenotypes are therefore related to the loss of GlcN decorations. The lpxL1 mutation had a stronger effect on hTLR4 activation and additionally resulted in reduced murine TLR4 activation, surface hydrophobicity, and biofilm formation, and in a fortified outer membrane as evidenced by increased resistance to several antimicrobials. These phenotypes, therefore, appear to be related to the loss of the acyl chain. Moreover, we determined the virulence of the mutants in the Galleria mellonella infection model and observed reduced virulence of the lpxL1 mutant but not of the lgmB mutant.

Outbreak management of multidrug-resistant Bordetella bronchiseptica in 16 shelter-housed cats.

CASE SERIES SUMMARY: This case series describes an outbreak of multidrug-resistant (MDR) Bordetella bronchiseptica in 16 shelter-housed cats with infectious respiratory disease. Four cats presented with acute dyspnea on the same day, each with a history of previous upper respiratory disease that had resolved with treatment. Early diagnostic testing and culture and sensitivity allowed for targeted antimicrobial therapy and environmental interventions. A case definition based on exposure and clinical signs identified 12 additional presumptive cases, including the likely index case. Comprehensive outbreak management included diagnostic testing, risk assessment, vaccination, use of isolation and quarantine, increased surveillance and review of biosecurity practices. The outbreak resolved in 26 days. RELEVANCE AND NOVEL INFORMATION: Management of an MDR B bronchiseptica outbreak in shelter-housed cats has not been previously described. Along with standard population and environmental measures, early and appropriate use of necropsy, PCR and bacterial culture allowed rapid and appropriate use of effective, second-line antibiotics. Shelters are resource-challenged population centers. Veterinarians working in animal shelters can play an important role in helping to develop cost-efficient and effective antimicrobial stewardship practices for companion animal settings. Outbreak management expertise and funding for diagnostic testing, as well as application of the principles of antimicrobial stewardship, are essential components of shelter medicine practice.

Phosphorylation chemistry of the Bordetella PlrSR TCS and its contribution to bacterial persistence in the lower respiratory tract.

Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against disease but does not prevent transmission or colonization. Cases of pertussis are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past 3 days in the lung, suggesting PlrSR works in a phosphorylation-dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 was essential and Glu522 was critical for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS could both phosphotransfer to and exert phosphatase activity toward PlrR. Unexpectedly, PlrR formed a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics.

Gastrointestinal helminths increase Bordetella bronchiseptica shedding and host variation in supershedding.

Co-infected hosts, individuals that carry more than one infectious agent at any one time, have been suggested to facilitate pathogen transmission, including the emergence of supershedding events. However, how the host immune response mediates the interactions between co-infecting pathogens and how these affect the dynamics of shedding remains largely unclear. We used laboratory experiments and a modeling approach to examine temporal changes in the shedding of the respiratory bacterium Bordetella bronchiseptica in rabbits with one or two gastrointestinal helminth species. Experimental data showed that rabbits co-infected with one or both helminths shed significantly more B. bronchiseptica, by direct contact with an agar petri dish, than rabbits with bacteria alone. Co-infected hosts generated supershedding events of higher intensity and more frequently than hosts with no helminths. To explain this variation in shedding an infection-immune model was developed and fitted to rabbits of each group. Simulations suggested that differences in the magnitude and duration of shedding could be explained by the effect of the two helminths on the relative contribution of neutrophils and specific IgA and IgG to B. bronchiseptica neutralization in the respiratory tract. However, the interactions between infection and immune response at the scale of analysis that we used could not capture the rapid variation in the intensity of shedding of every rabbit. We suggest that fast and local changes at the level of respiratory tissue probably played a more important role. This study indicates that co-infected hosts are important source of variation in shedding, and provides a quantitative explanation into the role of helminths to the dynamics of respiratory bacterial infections.

Bcr4 Is a Chaperone for the Inner Rod Protein in the Bordetella Type III Secretion System.

Bordetella bronchiseptica injects virulence proteins called effectors into host cells via a type III secretion system (T3SS) conserved among many Gram-negative bacteria. Small proteins called chaperones are required to stabilize some T3SS components or localize them to the T3SS machinery. In a previous study, we identified a chaperone-like protein named Bcr4 that regulates T3SS activity in B. bronchiseptica. Bcr4 does not show strong sequence similarity to well-studied T3SS proteins of other bacteria, and its function remains to be elucidated. Here, we investigated the mechanism by which Bcr4 controls T3SS activity. A pulldown assay revealed that Bcr4 interacts with BscI, based on its homology to other bacterial proteins, to be an inner rod protein of the T3SS machinery. An additional pulldown assay using truncated Bcr4 derivatives and secretion profiles of B. bronchiseptica producing truncated Bcr4 derivatives showed that the Bcr4 C-terminal region is necessary for the interaction with BscI and activation of the T3SS. Moreover, the deletion of BscI abolished the secretion of type III secreted proteins from B. bronchiseptica and the translocation of a cytotoxic effector into cultured mammalian cells. Finally, we show that BscI is unstable in the absence of Bcr4. These results suggest that Bcr4 supports the construction of the T3SS machinery by stabilizing BscI. This is the first demonstration of a chaperone for the T3SS inner rod protein among the virulence bacteria possessing the T3SS. IMPORTANCE The type III secretion system (T3SS) is a needle-like complex that projects outward from bacterial cells. Bordetella bronchiseptica uses the T3SS to inject virulence proteins into host cells. Our previous study reported that a protein named Bcr4 is essential for the secretion of virulence proteins from B. bronchiseptica bacterial cells and delivery through the T3SS. Because other bacteria lack a Bcr4 homologue, the function of Bcr4 has not been elucidated. In this study, we discovered that Bcr4 interacts with BscI, a component of the T3SS machinery. We show that a B. bronchiseptica BscI-deficient strain was unable to secrete type III secreted proteins. Furthermore, in a B. bronchiseptica strain that overproduces T3SS component proteins, Bcr4 is required to maintain BscI in bacterial cells. These results suggest that Bcr4 stabilizes BscI to allow construction of the T3SS in B. bronchiseptica.