Iron starvation regulates the type III secretion system in Bordetella bronchiseptica

The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron-depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron-replete conditions. Furthermore, it was confirmed that induction of T3SS-dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron-depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron-depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.     

Btc22 chaperone is required for secretion and stability of the type III secreted protein Bsp22 in Bordetella bronchiseptica

The type III secretion system (T3SS) is a sophisticated protein secretion machinery that delivers bacterial virulence proteins into host cells. A needle-tip protein, Bsp22 , is one of the secreted substrates of the T3SS and plays an essential role in the full function of the T3SS in Bordetella bronchiseptica. In this study, we found that BB1618 functions as a chaperone for Bsp22 . The deletion of BB1618 resulted in a dramatic impairment of Bsp22 secretion into the culture supernatants and Bsp22 stability in the bacterial cytosol. In contrast, the secretion of other type III secreted proteins was not affected by the BB1618 mutation. Furthermore, the BB1618 mutant strain could not induce cytotoxicity and displayed the same phenotypes as the Bsp22 mutant strain. An immunoprecipitation assay demonstrated that BB1618 interacts with Bsp22 , but not with BopB and BopD . Thus, we identified BB1618 as a specific type III chaperone for Bsp22 . Therefore, we propose that BB1618 be renamed Btc22 for the Bordetella type III chaperone for Bsp22 .     

The Bordetella type III secretion system effector BteA contains a conserved N-terminal motif that guides bacterial virulence factors to lipid rafts

The Bordetella type III secretion system (T3SS) effector protein BteA is necessary and sufficient for rapid cytotoxicity in a wide range of mammalian cells. We show that BteA is highly conserved and functionally interchangeable between Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis . The identification of BteA sequences required for cytotoxicity allowed the construction of non-cytotoxic mutants for localization studies. BteA derivatives were targeted to lipid rafts and showed clear colocalization with cortical actin, ezrin and the lipid raft marker GM1. We hypothesized that BteA associates with the cytoplasmic face of lipid rafts to locally modulate host cell responses to Bordetella attachment. B. bronchiseptica adhered to host cells almost exclusively to GM1-enriched lipid raft microdomains and BteA colocalized to these same sites following T3SS-mediated translocation. Disruption of lipid rafts with methyl-β-cyclodextrin protected cells from T3SS-induced cytotoxicity. Localization to lipid rafts was mediated by a 130-amino-acid lipid raft targeting domain at the N-terminus of BteA, and homologous domains were identified in virulence factors from other bacterial species. Lipid raft targeting sequences from a T3SS effector (Plu4750) and an RTX-type toxin (Plu3217) from Photorhabdus luminescens directed fusion proteins to lipid rafts in a manner identical to the N-terminus of BteA.     

Comparison of the genome sequence of the poultry pathogen Bordetella avium with those of B. bronchiseptica, B. pertussis, and B. parapertussis reveals extensive diversity in surface structures associated with host interaction

Bordetella avium is a pathogen of poultry and is phylogenetically distinct from Bordetella bronchiseptica, Bordetella pertussis, and Bordetella parapertussis, which are other species in the Bordetella genus that infect mammals. In order to understand the evolutionary relatedness of Bordetella species and further the understanding of pathogenesis, we obtained the complete genome sequence of B. avium strain 197N, a pathogenic strain that has been extensively studied. With 3,732,255 base pairs of DNA and 3,417 predicted coding sequences, it has the smallest genome and gene complement of the sequenced bordetellae. In this study, the presence or absence of previously reported virulence factors from B. avium was confirmed, and the genetic bases for growth characteristics were elucidated. Over 1,100 genes present in B. avium but not in B. bronchiseptica were identified, and most were predicted to encode surface or secreted proteins that are likely to define an organism adapted to the avian rather than the mammalian respiratory tracts. These include genes coding for the synthesis of a polysaccharide capsule, hemagglutinins, a type I secretion system adjacent to two very large genes for secreted proteins, and unique genes for both lipopolysaccharide and fimbrial biogenesis. Three apparently complete prophages are also present. The BvgAS virulence regulatory system appears to have polymorphisms at a poly(C) tract that is involved in phase variation in other bordetellae. A number of putative iron-regulated outer membrane proteins were predicted from the sequence, and this regulation was confirmed experimentally for five of these.     

Genetics of pertussis toxin

Pertussis toxin (PT) is the major virulence factor of Bordetella pertussis. The cloning and nucleotide sequencing of the PT genes from B. pertussis, Bordetella parapertussis and Bordetella bronchiseptica has elucidated the evolution of the Bordetella species and allowed considerable advances towards the understanding of their gene expression and the development of safer vaccines against pertussis.     

Bordetella parapertussis and Bordetella bronchiseptica contain transcriptionally silent pertussis toxin genes

Pertussis toxin, the major virulence factor of Bordetella pertussis, is not produced by the closely related species Bordetella parapertussis and Bordetella bronchiseptica. It is shown here that these two species possess but do not express the complete toxin operon. Nucleotide sequencing of an EcoRI fragment of 5 kilobases comprising the regions homologous to the pertussis toxin genes shows that in this region, B. parapertussis and B. bronchiseptica are 98.5% and 96% homologous, respectively, to B. pertussis. The changes (mostly base pair substitutions) in many cases are identical in B. parapertussis and B. bronchiseptica, suggesting that these two species derive from a common ancestor. Many of the mutations common to B. parapertussis and B. bronchiseptica involve the promoter region, which becomes very inefficient. The S1 subunits of both species, when expressed in Escherichia coli, have the same ADP-ribosylating activity as the S1 subunit from B. pertussis, indicating that the mutations in the S1 gene described here do not affect its function.     

Adaptive evolution of the Bordetella autotransporter pertactin

The virulence factor pertactin is expressed by the closely related pathogens Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Pertactin is an autotransporter involved in adherence of Bordetella species to the lung epithelium of mammalian hosts, and it is an important component of most current acellular pertussis vaccines. These three species produce immunologically distinct pertactin molecules, resulting in a lack of cross-protection against B. parapertussis and probably also against B. bronchiseptica. Variation in pertactin is not only inter-specific, but also occurs between isolates from the same species. Knowledge about codons that are under positive selection could facilitate the development of more broadly protective vaccines. Using different nucleotide substitution models, pertactin genes from B. bronchiseptica, B. parapertussis and B. pertussis were compared, and positively selected codons were identified using an empirical Bayesian approach. This approach yielded 15 codons predicted to be under diversifying selection pressure. These results were interpreted in an immunological context and may help in improving future pertussis vaccines.     

Evolutionary relationships in the genus Bordetella

The nucleotide sequence of the pertussis toxin operon of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica, has shown that the last two species contain many common mutations and are likely to derive from a common ancestor (Aricò and Rappuoli, 1987). To elucidate further the evolutionary relationships between the Bordetella species, we have cloned and sequenced the promoter region and the gene coding for the S1 subunit of pertussis toxin from additional B. pertussis strains, such as the type strain BP 18323 and two recent clinical isolates, namely strain BP 13456 from Sweden and strain BP SA1 from Italy. While the strains BP SA1 and BP 13456 are shown to differ from the published B. pertussis sequences by only one base pair, the type strain BP 18323 contains a total of 11 base-pair substitutions. Remarkably, 9 of the 11 substitutions found in BP 18323 are also common to B. parapertussis and B. bronchiseptica, strongly suggesting that this strain derives from the same ancestor as B. parapertussis and B. bronchiseptica. Computer analysis of the sequence data allows the construction of an evolutionary 'tree' showing that the B. pertussis strains are very homogeneous and significantly distant from B. parapertussis and B. bronchiseptica. Therefore the proposed conversion from B. parapertussis to B. pertussis appears highly improbable.     

BopC is a novel type III effector secreted by Bordetella bronchiseptica and has a critical role in type III-dependent necrotic cell death

In Bordetella bronchiseptica, the functional type III secretion system (TTSS) is required for the induction of necrotic cell death in infected mammalian cells. To identify the factor(s) involved in necrotic cell death, type III-secreted proteins from B. bronchiseptica were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry. We identified a 69-kDa secreted protein designated BopC. The gene encoding BopC is located outside of the TTSS locus and is also highly conserved in both Bordetella parapertussis and Bordetella pertussis. The results of a lactate dehydrogenase release assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay demonstrated that BopC is required for necrotic cell death. It has been reported that tyrosine-phosphorylated proteins (PY) of host cells are dephosphorylated during B. bronchiseptica infection in a TTSS-dependent manner. We found that BopC is also involved in PY dephosphorylation in infected host cells. It appears that the necrotic cell death triggered by BopC occurs prior to the PY reduction in host cells, because Bordetella-induced cell death was not affected even in the presence of a dephosphorylation inhibitor. Furthermore, a translocation assay showed that the signal sequence for both secretion into culture supernatant and translocation into the host cell is located in 48 amino acid residues of the BopC N terminus. This report reveals for the first time that a novel type III effector, BopC, is required for the induction of necrotic cell death during Bordetella infection.     

Bordetella bronchiseptica expresses the fimbrial structural subunit gene fimA.

The differential host species specificities of Bordetella pertussis, B. parapertussis, and B. bronchiseptica might be explained by polymorphisms in adherence factor genes. We have found that B. parapertussis and B. bronchiseptica, unlike B. pertussis, contain a full-length gene for the fimbrial subunit FimA. B. bronchiseptica expresses fimA in a BvgAS-dependent fashion.     

BopB is a type III secreted protein in Bordetella bronchiseptica and is required for cytotoxicity against cultured mammalian cells

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although the precise mechanism of the type III secretion system is unknown, BopN, BopD and Bsp22 have been identified as type III secreted proteins. In order to identify other proteins secreted via the type III secretion machinery in Bordetella, a type III mutant was generated, and its secretion profile was compared with that of the wild-type strain. The results showed that the wild-type strain, but not the type III mutant, secreted a 40-kDa protein into the culture supernatant. This protein was identified as BopB by the analysis of its N-terminal amino acid sequence. Severe cytotoxicity such as necrosis was induced in L2 cells by infection with the wild-type B. bronchiseptica. In contrast, this effect was not observed by the BopB mutant infection. The haemolytic activity of the BopB mutant was greatly impaired compared with that of the wild-type strain. The results of a digitonin assay strongly suggested that BopB was translocated into HeLa cells infected with the wild-type strain. Taken together, our results demonstrate that Bordetella secretes BopB via a type III secretion system during infection. BopB may play a role in the formation of pores in the host plasma membrane which serve as a conduit for the translocation of effector proteins into host cells.     

Molecular aspects of Bordetella pertussis pathogenesis.

The molecular mechanisms of Bordetella virulence are now well understood, and many virulence factors have been identified and characterized at the molecular level. These virulence factors can be grouped into two major categories: adhesins, such as filamentous hemagglutinin, pertactin and fimbriae, and toxins, such as pertussis toxin, adenylate cyclase, dermonecrotic toxin and tracheal cytotoxin. The production of most virulence factors is coordinately regulated by a two-component signal transduction system composed of the regulator BvgA and the sensor protein BvgS. The adhesins and toxins act in concert to establish infection. Some adhesins exert their effects synergically or are redundant functioning only in the absence of another adhesin, illustrating the importance of adhesion in infection. Most virulence factors are secreted into the culture supernatant or exposed at the surface of the bacterial cell. A notable exception is dermonecrotic toxin, which remains in the cytoplasmic compartment of bacterial cells. Most virulence factors are produced by all of the three major Bordetella species, B. pertussis, B. parapertussis and B. bronchiseptica. However, some, such as pertussis toxin and the tracheal colonization factor, are only produced by B. pertussis. Our understanding of Bordetella virulence at the molecular level has led to the development of new acellular vaccines against whooping cough, and of genetically attenuated B. pertussis strains to be used as recombinant live bacterial vaccine vectors for homologous and heterologous protection.     

Adenylate cyclase activity of Bordetella organisms. I. Its production in liquid medium

Abundant adenylate cyclase activity was found in the phase I cultures not only of Bordetella pertussis but also fo B. parapertussis and B. bronchiseptica. The enzyme activity in the culture fluid increased rapidly and reached a peak during the logarithmic growth phase. B. parapertussis and B. bronchiseptica especially produced a high activity of the enzyme in the culture fluid during the logarithmic phase, but little or no activity was detected in the cells throughout the growth period. In the culture of B. pertussis, the intracellular activity was higher than that in the culture fluid. Phase III cultures of these species lacked both the extracellular and intracellular enzyme activities throughout their growth. In the culture of B. parapertussis, accumulation of cyclic AMP was parallel to that of adenylate cyclase activity through the growth periods, but in B. pertussis there was no parallelism from the stationary through the declining phases. The difference in production patterns of the enzyme activity among the species is discussed.     

Limitation of phage multiplication by microbes of the genus Bordetella

Possible causes limiting the multiplication of Bordetella phages or inducing their restriction, such as the influence of lysogenic immunity and the restriction-modification (R-M) system or the incompatibility of the receptor apparatus, have been studied. The limitation of the multiplication of phages by some B. bronchiseptica and B. pertussis strains has been shown to be due to the presence of the R-M system and lysogenic immunity. In five B. bronchiseptica strains and two B. pertussis strains site-specific endonucleases (restrictases) with Hind III specificity have been detected. One B. bronchiseptica strain without the R-M system has been detected. B. bronchiseptica strains producing site-specific endonucleases are practically nonpathogenic for humans, grow in common culture media and selectively produce only one restrictase, type Hind III, which guarantees from the admixture of other specific endonucleases. The B. parapertussis strains under study (altogether 100 strains) have not been found to limit the multiplication of Bordetella test phages. The absence of site-specific endonucleases has also been confirmed biochemically. These strains are recommended as indicator strains for the multiplication of Bordetella phages.     

Biochemical and immunological comparison of lipopolysaccharides from Bordetella species

Lipopolysaccharides (LPS) isolated from Bordetella pertussis, B. parapertussis and B. bronchiseptica were analysed for their chemical composition, molecular heterogeneity and immunological properties. All the LPS preparations contained heptose, 3-deoxy-D-manno-2-octulosonic acid, glucosamine, uronic acid, phosphate and fatty acids. The fatty acids C14:0, C16:0 and beta OHC14:0 were common to all the LPS preparations. LPS from B. pertussis strains additionally contained isoC16:0, those from B. parapertussis contained isoC14:0 and isoC16:0, and those from B. bronchiseptica contained C16:1. By SDS-PAGE, LPS from B. pertussis had two bands of low molecular mass, and the LPS from B. parapertussis and B. bronchiseptica showed low molecular mass bands together with a ladder arrangement of high molecular mass bands. Immunodiffusion, quantitative agglutination and ELISA demonstrated that the LPS from B. pertussis strains reacted with antisera prepared against whole cells of B. pertussis and B. bronchiseptica; LPS from B. parapertussis reacted with antisera to B. parapertussis and B. bronchiseptica, and LPS from B. bronchiseptica reacted with anti-whole cell serum raised against any of the three species. From these results, it is concluded that LPS from B. bronchiseptica has structures in common with LPS from B. pertussis and B. parapertussis, while the LPS from B. pertussis and B. parapertussis are serologically entirely different from each other.     

Structural and genetic analysis of the bvg locus in Bordetella species

The bvg locus contains two genes, bvgA and bvgS, which control the expression of the virulence-associated genes in Bordetella species by a system similar to the two-component systems used by a variety of bacterial species to respond to environmental stimuli. We determined the nucleotide sequence of the bvg loci of Bordetella parapertussis and Bordetella bronchiseptica and compared them with the previously determined sequence of Bordetella pertussis. The nucleotide and amino acid sequences of the bvg loci of these species are well conserved in those regions coding for the protein domains which have putative kinase and DNA-binding activities. In marked contrast, the region of BvgS that codes for the protein domain with putative sensor activity shows a high degree of variability. In total, we find 198 base-pair changes in the bvg loci of B. parapertussis and B. bronchiseptica relative to the bvg locus of B. pertussis. One hundred and seventy-three of these base-pair changes are identical in B. parapertussis and B. bronchiseptica. This confirms our previous observation that B. parapertussis and B. bronchiseptica are more related to each other than to B. pertussis. We have mapped the mutations that cause phase changes in B. bronchiseptica and we have found that in three cases these are due to spontaneous deletions in the bvgS gene. The wild-type bvg locus present on a multicopy plasmid cannot complement avirulent derivatives of B. bronchiseptica to wild-type levels, but it can do so when the bvgA gene on the plasmid is inactivated. This suggests that hyperexpression of bvgA down-regulates the bvg system.     

A genome-wide screen identifies a Bordetella type III secretion effector and candidate effectors in other species

Bordetella 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.