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.

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.

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.

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.

Conditional growth defect of Bordetella pertussis and Bordetella bronchiseptica ferric uptake regulator (fur) mutants.

Outer-membrane vesicles (OMVs) are promising tools in the development of novel vaccines against the respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica. Unfortunately, vesiculation by bordetellae is too low for cost-effective vaccine production. In other bacteria, iron limitation or inactivation of the fur gene has been shown to increase OMV production, presumably by downregulation of the mla genes, which encode machinery for maintenance of lipid asymmetry in the outer membrane. Here, we followed a similar approach in bordetellae. Whereas a fur mutant was readily obtained in B. bronchiseptica, a B. pertussis fur mutant could only be obtained in iron-deplete conditions, indicating that a fur mutation is conditionally lethal in this bacterium. The fur mutants displayed a growth defect in iron-replete media, presumably because constitutive expression of iron-uptake systems resulted in iron intoxication. Accordingly, expression of the Escherichia coli ferritin FtnA to sequester intracellularly accumulated iron rescued the growth of the mutants in these media. The fur mutations led to the constitutive expression of novel vaccine candidates, such as the TonB-dependent receptors FauA for the siderophore alcaligin and BhuR for heme. However, neither inactivation of fur nor growth under iron limitation improved vesiculation, presumably because the expression of the mla genes appeared unaffected.

Cyclic di-GMP Regulates the Type III Secretion System and Virulence in Bordetella bronchiseptica.

The second messenger cyclic di-GMP (c-di-GMP) is a ubiquitous molecule in bacteria that regulates diverse phenotypes. Among them, motility and biofilm formation are the most studied. Furthermore, c-di-GMP has been suggested to regulate virulence factors, making it important for pathogenesis. Previously, we reported that c-di-GMP regulates biofilm formation and swimming motility in Bordetella bronchiseptica. Here, we present a multi-omics approach for the study of B. bronchiseptica strains expressing different cytoplasmic c-di-GMP levels, including transcriptome sequencing (RNA-seq) and shotgun proteomics with label-free quantification. We detected 64 proteins significantly up- or downregulated in either low or high c-di-GMP levels and 358 genes differentially expressed between strains with high c-di-GMP levels and the wild-type strain. Among them, we found genes for stress-related proteins, genes for nitrogen metabolism enzymes, phage-related genes, and virulence factor genes. Interestingly, we observed that a virulence factor like the type III secretion system (TTSS) was regulated by c-di-GMP. B. bronchiseptica with high c-di-GMP levels showed significantly lower levels of TTSS components like Bsp22, BopN, and Bcr4. These findings were confirmed by independent methods, such as quantitative reverse transcription-PCR (q-RT-PCR) and Western blotting. Higher intracellular levels of c-di-GMP correlated with an impaired capacity to induce cytotoxicity in a eukaryotic cell in vitro and with attenuated virulence in a murine model. This work presents data that support the role that the second messenger c-di-GMP plays in the pathogenesis of Bordetella.

Two Bordetella bronchiseptica attenuated vaccine candidates confer protection against lethal challenge with B. Bronchiseptica and Pasteurella multocida toxin in mouse models.

Dermonecrotic toxin (DNT) is an important bacterial virulence factor produced by the zoonotic pathogens Bordetella bronchiseptica and Pasteurella multocida. This study aims to explore the possibility of expressing different fragments of P. multocida toxin (PMT) in the chromosome of attenuated B. bronchiseptica to generate single-component mucosal vaccine candidates. To achieve this, a 954-bp fragment (basepairs 301 âˆ¼ 1254) of the B. bronchiseptica aroA gene was replaced with an N-terminal, 930-bp fragment (basepairs 1-930; PMTN) or a C-terminal, 900-bp fragment (base pairs 2959 âˆ¼ 3858; PMTC) of the PMT encoding gene toxA. The resulting strains, denoted as Bb-PMTN or Bb-PMTC, expressed PMTN and PMTC, as evidenced by ELISA using polyclonal against full-length of PMT. Phenotypical analyses revealed that Bb-PMTN and Bb-PMTC grew much slower than wild type strains in tryptic soy broth. These strains also displayed significantly decreased 161-fold-virulence compared to the wildtype strains in mouse models. Intranasal immunization of Bb-PMTN and Bb-PMTC in mice induced high levels of antibodies against B. bronchiseptica and PMT, as well as IFN-γ and IL-10 in mouse sera, and most importantly, high titers of sIgA in mouse lungs. Vaccination with these two engineering strains provided 100% protection of mice against lethal challenge with B. bronchiseptica and 80%∼100% protection against lethal challenge with PMT, with Bb-PMTN exhibiting 1.25-fold greater immunogenic efficacy over Bb-PMTC. This study highlights the use of B. bronchiseptica attenuated strains as live mucosal vectors to deliver heterologous antigens.

Natural History and Ecology of Interactions Between Bordetella Species and Amoeba.

A variety of bacteria have evolved the ability to interact with environmental phagocytic predators such as amoebae, which may have facilitated their subsequent interactions with phagocytes in animal hosts. Our recent study found that the animal pathogen Bordetella bronchiseptica can evade predation by the common soil amoeba Dictyostelium discoideum, survive within, and hijack its complex life cycle as a propagation and dissemination vector. However, it is uncertain whether the mechanisms allowing interactions with predatory amoebae are conserved among Bordetella species, because divergence, evolution, and adaptation to different hosts and ecological niches was accompanied by acquisition and loss of many genes. Here we tested 9 diverse Bordetella species in three assays representing distinct aspects of their interactions with D. discoideum. Several human and animal pathogens retained the abilities to survive within single-celled amoeba, to inhibit amoebic plaque expansion, and to translocate with amoebae to the fruiting body and disseminate along with the fruiting body. In contrast, these abilities were partly degraded for the bird pathogen B. avium, and for the human-restricted species B. pertussis and B. parapertussis. Interestingly, a different lineage of B. parapertussis only known to infect sheep retained the ability to interact with D. discoideum, demonstrating that these abilities were lost in multiple lineages independently, correlating with niche specialization and recent rapid genome decay apparently mediated by insertion sequences. B. petrii has been isolated sporadically from diverse human and environmental sources, has acquired insertion sequences, undergone genome decay and has also lost the ability to interact with amoebae, suggesting some specialization to some unknown niche. A genome-wide association study (GWAS) identified a set of genes that are potentially associated with the ability to interact with D. discoideum. These results suggest that massive gene loss associated with specialization of some Bordetella species to a closed life cycle in a particular host was repeatedly and independently accompanied by loss of the ability to interact with amoebae in an environmental niche.

Pathogenicity of Bordetella bronchiseptica isolated from apparently healthy rabbits in guinea pig, rat, and mouse.

Bordetella bronchiseptica (B. bronchiseptica) is associated with respiratory tract infections in laboratory animals. In our laboratory animal facility, B. bronchiseptica was isolated from 21 of 27 apparently healthy rabbits obtained from a breeding farm contaminated with B. bronchiseptica. Restriction fragment length polymorphism (RFLP) analysis showed that the flagellin genotype of isolates from the laboratory animal facility and breeding farm was type A, which is seen relatively frequently in rabbits in Europe. To examine its pathogenicity, guinea pigs, rats, and mice were inoculated intranasally with a representative strain isolated in the laboratory animal facility. Following inoculation of 107 colony forming unit (cfu), severe inflammation was observed in the lungs of guinea pig and mice, although the inflammation was less severe in rats. The strain was recovered from the trachea and lungs of these species after inoculation with lower dose such as 103 or 104 cfu. These results suggest that the isolated strain causes respiratory tract infection in guinea pigs, rats, and mice, and that its pathogenicity higher in mice than in rats. This study extends our knowledge of interpreting the microbiologic status of laboratory animals, which will contribute to the development of reliable and reproducible animal experiments.

Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins.

Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines  by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.

Pathogenicity and virulence of Bordetella pertussis and its adaptation to its strictly human host.

The highly contagious whooping cough agent Bordetella pertussis has evolved as a human-restricted pathogen from a progenitor which also gave rise to Bordetella parapertussis and Bordetella bronchiseptica. While the latter colonizes a broad range of mammals and is able to survive in the environment, B. pertussis has lost its ability to survive outside its host through massive genome decay. Instead, it has become a highly successful human pathogen by the acquisition of tightly regulated virulence factors and evolutionary adaptation of its metabolism to its particular niche. By the deployment of an arsenal of highly sophisticated virulence factors it overcomes many of the innate immune defenses. It also interferes with vaccine-induced adaptive immunity by various mechanisms. Here, we review data from invitro, human and animal models to illustrate the mechanisms of adaptation to the human respiratory tract and provide evidence of ongoing evolutionary adaptation as a highly successful human pathogen.

Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesic.

Noncoding small RNAs (sRNAs) are crucial for the posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. In the human pathogen Bordetella pertussis, which causes whooping cough, virulence is controlled primarily by the master two-component system BvgA (response regulator)/BvgS (sensor kinase). In this system, BvgA is phosphorylated (Bvg+ mode) or nonphosphorylated (Bvg- mode), with global transcriptional differences between the two. B. pertussis also carries the bacterial sRNA chaperone Hfq, which has previously been shown to be required for virulence. Here, we conducted transcriptomic analyses to identify possible B. pertussis sRNAs and to determine their BvgAS dependence using transcriptome sequencing (RNA-seq) and the prokaryotic sRNA prediction program ANNOgesic. We identified 143 possible candidates (25 Bvg+ mode specific and 53 Bvg- mode specific), of which 90 were previously unreported. Northern blot analyses confirmed all of the 10 ANNOgesic candidates that we tested. Homology searches demonstrated that 9 of the confirmed sRNAs are highly conserved among B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, with one that also has homologues in other species of the Alcaligenaceae family. Using coimmunoprecipitation with a B. pertussis FLAG-tagged Hfq, we demonstrated that 3 of the sRNAs interact directly with Hfq, which is the first identification of sRNA binding to B. pertussis Hfq. Our study demonstrates that ANNOgesic is a highly useful tool for the identification of sRNAs in this system and that its combination with molecular techniques is a successful way to identify various BvgAS-dependent and Hfq-binding sRNAs. IMPORTANCE Noncoding small RNAs (sRNAs) are crucial for posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. We have investigated the presence of sRNAs in the obligate human pathogen B. pertussis, using transcriptome sequencing (RNA-seq) and the recently developed prokaryotic sRNA search program ANNOgesic. This analysis has identified 143 sRNA candidates (90 previously unreported). We have classified their dependence on the B. pertussis two-component system required for virulence, namely, BvgAS, based on their expression in the presence/absence of the phosphorylated response regulator BvgA, confirmed several by Northern analyses, and demonstrated that 3 bind directly to B. pertussis Hfq, the RNA chaperone involved in mediating sRNA effects. Our study demonstrates the utility of combining RNA-seq, ANNOgesic, and molecular techniques to identify various BvgAS-dependent and Hfq-binding sRNAs, which may unveil the roles of sRNAs in pertussis pathogenesis.

DegP Initiates Regulated Processing of Filamentous Hemagglutinin in Bordetella bronchiseptica.

Filamentous hemagglutinin (FhaB) is a critical virulence factor for both Bordetella pertussis, the causal agent of whooping cough, and the closely related species Bordetella bronchiseptica. FhaB is an adhesin, suppresses inflammatory cytokine production, and protects against phagocytic cell clearance during infection. Regulated degradation of the FhaB C-terminal prodomain is required to establish a persistent infection in mice. Two proteases, CtpA in the periplasm and SphB1 on the bacterial surface, are known to mediate FhaB processing, and we recently determined that CtpA functions before, and controls the FhaB cleavage site of, SphB1. However, the data indicate that another periplasmic protease must initiate degradation of the prodomain by removing a portion of the FhaB C terminus that inhibits CtpA-mediated degradation. Using a candidate approach, we identified DegP as the initiating protease. Deletion of degP or substitution of its predicted catalytic residue resulted in reduced creation of FHA' (the main product of FhaB processing) and an accumulation of full-length FhaB in whole-cell lysates. Also, FHA' was no longer released into culture supernatants in degP mutants. Alterations of the FhaB C terminus that relieve inhibition of CtpA abrogate the need for DegP, consistent with DegP functioning prior to CtpA in the processing pathway. DegP is not required for secretion of FhaB through FhaC or for adherence of the bacteria to host cells, indicating that DegP acts primarily as a protease and not a chaperone for FhaB in B. bronchiseptica. Our results highlight a role for HtrA family proteases in activation of virulence factors in pathogenic bacteria. IMPORTANCE Two-partner secretion (TPS) systems are broadly distributed among Gram-negative bacteria and play important roles in bacterial pathogenesis. FhaB-FhaC is the prototypical member of the TPS family and we here identified the protease that initiates a processing cascade that controls FhaB function. Our results are significant because they provide insight into the molecular mechanism underlying the ability of Bordetella species to prevent clearance by phagocytic cells, which is critical for bacterial persistence in the lower respiratory tract. Our findings also highlight an underappreciated role for HtrA family proteases in processing specific bacterial virulence factors.

Reduction of endotoxicity in Bordetella bronchiseptica by lipid A engineering: Characterization of lpxL1 and pagP mutants.

Whole-cell vaccines against Gram-negative bacteria commonly display high reactogenicity caused by the endotoxic activity of lipopolysaccharide (LPS), one of the major components of the bacterial outer membrane. Underacylation of the lipid A moiety of LPS has been related with reduced endotoxicity in several Gram-negative species. Here, we evaluated whether the inactivation of two genes encoding lipid A acylases of Bordetella bronchiseptica, i.e. pagP and lpxL1, could be used for the development of less reactogenic vaccines against this pathogen for livestock and companion animals. Inactivation of pagP resulted in the loss of the secondary palmitate chain at position 3' of lipid A, but hardly affected the potency of the LPS to activate the Toll-like receptor 4 (TLR4). Inactivation of lpxL1 resulted in the loss of the secondary 2-hydroxy laurate group present at position 2 of lipid A and, unexpectedly, in the additional loss of the glucosamines that decorate the phosphate groups at positions 1 and 4' and in an increase in LPS molecules carrying O-antigen. The resulting LPS showed greatly reduced potency to activate TLR4 in HEK-Blue reporter cells expressing human or mouse TLR4 as well as in porcine macrophages. Characterization of the lpxL1 mutant revealed many pleiotropic phenotypes, including increased resistance to SDS and rifampicin, increased susceptibility to cationic antimicrobial peptides, decreased auto-aggregation and biofilm formation, and a tendency to decreased infectivity of macrophages, which are all related to the altered LPS structure. We suggest that the lpxL1 mutant will be useful for the generation of safer vaccines.

Genomic plasticity and antibody response of Bordetella bronchiseptica strain HT200, a natural variant from a thermal spring.

Classical Bordetella species are primarily isolated from animals and humans causing asymptomatic infection to lethal pneumonia. However, isolation of these bacteria from any extra-host environmental niche has not been reported so far. Here, we have characterized the genomic plasticity and antibody response of Bordetella bronchiseptica strain HT200, isolated from a thermal spring. Genomic ANI value and SNPs-based phylogenetic tree suggest a divergent evolution of strain HT200 from a human-adapted lineage of B. bronchiseptica. Growth and survivability assay showed strain HT200 retained viability for more than 5 weeks in the filter-sterilized spring water. In addition, genes or loci encoding the Bordetella virulence factors such as DNT, ACT and LPS O-antigen were absent in strain HT200, while genes encoding other virulence factors were highly divergent. Phenotypically, strain HT200 was non-hemolytic and showed weak hemagglutination activity, but was able to colonize in the respiratory organs of mice. Further, both infection and vaccination with strain HT200 induced protective antibody response in mouse against challenge infection with virulent B. bronchiseptica strain RB50. In addition, genome of strain HT200 (DSM 26023) showed presence of accessory genes and operons encoding predicted metabolic functions pertinent to the ecological conditions of the thermal spring.

Lipid binding by the N-terminal motif mediates plasma membrane localization of Bordetella effector protein BteA.

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.

Bordetella bronchiseptica promotes adherence, colonization, and cytotoxicity of Streptococcus suis in a porcine precision-cut lung slice model.

Bordetella (B.) bronchiseptica and Streptococcus (S.) suis are major pathogens in pigs, which are frequently isolated from co-infections in the respiratory tract and contribute to the porcine respiratory disease complex (PRDC). Despite the high impact of co-infections on respiratory diseases of swine (and other hosts), very little is known about pathogen-pathogen-host interactions and the mechanisms of pathogenesis. In the present study, we established a porcine precision-cut lung slice (PCLS) model to analyze the effects of B. bronchiseptica infection on adherence, colonization, and cytotoxic effects of S. suis. We hypothesized that induction of ciliostasis by a clinical isolate of B. bronchiseptica may promote subsequent infection with a virulent S. suis serotype 2 strain. To investigate this theory, we monitored the ciliary activity by light microscopy, measured the release of lactate dehydrogenase, and calculated the number of PCLS-associated bacteria. To study the role of the pore-forming toxin suilysin (SLY) in S. suis-induced cytotoxicity, we included a SLY-negative isogenic mutant and the complemented mutant strain. Furthermore, we analyzed infected PCLS by histopathology, immunofluorescence microscopy, and field emission scanning electron microscopy. Our results showed that pre-infection with B. bronchiseptica promoted adherence, colonization, and, as a consequence of the increased colonization, the cytotoxic effects of S. suis, probably by reduction of the ciliary activity. Moreover, cytotoxicity induced by S. suis is strictly dependent on the presence of SLY. Though the underlying molecular mechanisms remain to be fully clarified, our results clearly support the hypothesis that B. bronchiseptica paves the way for S. suis infection.

Counter-Selection Method for Markerless Allelic Exchange in Bordetella bronchiseptica Based on sacB Gene From Bacillus subtilis.

Bordetella bronchiseptica is a gram-negative bacterium that causes respiratory tract infections. It is a natural pathogen of a wide variety of mammals, including some used as laboratory models. This makes B. bronchiseptica an ideal organism to study pathogen-host interactions in order to unveil molecular mechanisms behind pathogenic processes. Even though genetic engineering is an essential tool in this area, there are just a few reports about genome manipulation techniques in this organism. In this article we describe an allelic exchange protocol based on double crossover recombination facilitated by the Bacillus subtilis sacB gene that can be applied for partial or complete gene knockouts, single-nucleotide mutations, or even introduction of coding sequences for transcriptional fusions. In contrast to previously employed techniques, this protocol renders genetically manipulated chromosomes without foreign DNA and enables the construction of successive genome manipulation using the same vector backbone. The entire procedure has been developed for fast and reliable manipulations with a total duration of 2 weeks. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Setting up strains Basic Protocol 2: Homologous recombination (first crossing-over) Alternate Protocol: B. bronchiseptica electroporation Basic Protocol 3: Screening for sucrose-sensitive clones Basic Protocol 4: Homologous recombination (second crossing-over) Basic Protocol 5: PCR screening of putative marker-exchange mutants Support Protocol: Electrocompetent cell preparation.