SummaryThe BvgAS signal transduction system in Bordetella spp. mediates a transition between infectious (Bvg þ ) and non-infectious (Bvg ¹ ) phases by sensing environmental conditions and regulating gene expression. Using differential display, arbitrary-primed polymerase chain reaction (PCR), we identified a gene expressed in the Bvg þ phase of Bordetella bronchiseptica that shows a high degree of sequence similarity to a locus involved in providing energy for type III secretion in pathogenic Gram-negative bacteria (yscN in Yersinia spp.). We determined that the expression of this homologue in B. bronchiseptica (

SummaryBordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti-Bordetella antibodies upon infection compared with wild-type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice. These observations suggest that type III-secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF-kB into large complexes in the cytoplasm. NF-kB aggregation was dependent on type III secretion and aggregated NF-kB did not respond to TNFa activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF-kB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.

SummaryThe BvgAS virulence control system regulates the expression of type III secretion genes in Bordetella subspecies that infect humans and other mammals. We have identified five open reading frames, btrS , btrU , btrX , btrW and btrV , that are activated by BvgAS and encode regulatory factors that control type III secretion at the levels of transcription, protein expression and secretion. The btrS gene product bears sequence similarity to ECF (extracytoplasmic function) sigma factors and is required for transcription of the bsc locus. btrU , btrW and btrV encode proteins predicted to contain PP2C-like Ser phosphatase, HPK (His protein kinase)-like Ser kinase and STAS anti-sigma factor antagonist domains, respectively, which are characteristic of Gram-positive partner switching proteins in Bacillus subtilis . BtrU and BtrW are required for secretion of proteins that are exported by the bsc type III secretion system, whereas BtrV is specifically required for protein synthesis and/or stability. Bordetella species have thus evolved a unique cascade to differentially regulate type III secretion that combines a canonical phosphorelay system with an ECF sigma factor and a set of proteins with domain signatures that define partner switchers, which were traditionally thought to function only in Gram-positive bacteria. The presence of multiple layers and mechanisms of regulation most likely reflects the need to integrate multiple signals in controlling type III secretion. The bsc and btr loci are nearly identical between broad-host-range and human-specific Bordetella . Comparative analysis of Bordetella subspecies revealed that, whereas bsc and btr loci were transcribed in all subspecies, only broad-host-range strains expressed a functional type III secretion system in vitro . The block in type III secretion is post-transcriptional in human-adapted strains, and signal recognition appears to be a point of divergence between subspecies.

؉ phase encode known virulence factors, including adhesins such as filamentous hemagglutinin (FHA) and fimbriae, as well as toxins such as the bifunctional adenylate cyclase/hemolysin (ACY). Previous studies showed that in the Bvg i phase, FHA and fimbriae continue to be expressed, but ACY expression is significantly downregulated. In this report, we determine that Bordetella bronchiseptica can form biofilms in vitro and that the generation of biofilm is maximal in the Bvg i phase. We show that FHA is required for maximal biofilm formation and that fimbriae may also contribute to this phenotype. However, expression of ACY inhibits biofilm formation, most likely via interactions with FHA. Therefore, the coordinated regulation of adhesins and ACY expression leads to maximal biofilm formation in the Bvg i phase in B. bronchiseptica.Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are closely related gram-negative coccobacilli that colonize the upper respiratory tract of mammals. B. pertussis and most B. parapertussis strains are obligate human pathogens that usually cause acute respiratory diseases. B. bronchiseptica has a much broader host range and is considered to be representative of the evolutionary progenitor of all Bordetella spp. (10, 27). It naturally infects many laboratory animals, including mice, rats, and rabbits, and thus serves as an ideal model for studying bacterial pathogenesis in a natural infection setting. Although B. bronchiseptica has been associated with various respiratory diseases, infection by this organism generally leads to chronic and asymptomatic colonization in the host. This lifestyle indicates that the bacteria employ specific mechanisms to counteract host immune responses and also implies successful interactions with other commensal bacteria commonly found in the upper respiratory tract.

SummaryBordetella bronchiseptica utilizes a type III secretion system (TTSS) for induction of non-apoptotic cytotoxicity in host cells and modulation of host immunity. The identity of Bordetella TTSS effectors, however, has remained elusive. Here we report a genome-wide screen for TTSS effectors based on shared biophysical and functional characteristics of class I chaperones and their frequent colocalization with TTSS effectors. When applied to B. bronchiseptica , the screen identified the first TTSS chaperone-effector locus, btcA-bteA , and we experimentally confirmed its function. Expression of bteA is co-ordinated with expression of TTSS apparatus genes, BteA is secreted through the TTSS of B. bronchiseptica , it is required for cytotoxicity towards mammalian cells, and it is highly conserved in the human-adapted subspecies B. pertussis and B. parapertussis . Transfection of bteA into epithlieal cells results in rapid cell death, indicating that BteA alone is sufficient to induce potent cytotoxicity. Finally, an in vitro interaction between BteA and BtcA was demonstrated. The search for TTSS chaperones and effectors was then expanded to other bacterial genomes, including mammalian and insect pathogens, where we identified a large number of novel candidate chaperones and effectors. Although the majority of putative effectors are proteins of unknown function, several have similarities to eukaryotic protein domains or previously identified effectors from other species.