DAT, deacylating autotransporter toxin, from Bordetella parapertussis demyristoylates Gαi GTPases and contributes to cough.

The pathogenic bacteria Bordetella pertussis and Bordetella parapertussis cause pertussis (whooping cough) and pertussis-like disease, respectively, both of which are characterized by paroxysmal coughing. We previously reported that pertussis toxin (PTx), which inactivates heterotrimeric GTPases of the Gi family through ADP-ribosylation of their α subunits, causes coughing in combination with Vag8 and lipid A in B. pertussis infection. In contrast, the mechanism of cough induced by B. parapertussis, which produces Vag8 and lipopolysaccharide (LPS) containing lipid A, but not PTx, remained to be elucidated. Here, we show that a toxin we named deacylating autotransporter toxin (DAT) of B. parapertussis inactivates heterotrimeric Gi GTPases through demyristoylation of their α subunits and contributes to cough production along with Vag8 and LPS. These results indicate that DAT plays a role in B. parapertussis infection in place of PTx.

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.

Immunization with autotransporter Vag8 prevents coughing induced by Bordetella pertussis infection in mice.

Bordetella pertussis causes pertussis, which is characterized by paroxysmal coughing. This disease is generally prevented through vaccination; however, the number of pertussis cases is increasing worldwide despite high vaccination coverage. We previously reported that an autotransporter of B. pertussis, virulence-associated gene 8 (Vag8), causes coughing in combination with pertussis toxin and lipooligosaccharide. Here, we show that immunization with Vag8 protected mice from coughing after B. pertussis infection and enhanced the efficacy of a current pertussis vaccine containing pertussis toxoid against the cough. Our findings indicate that Vag8 could be a vaccine antigen to prevent pertussis cough.

Expression of small RNAs of Bordetella pertussis colonizing murine tracheas.

We performed RNA sequencing on Bordetella pertussis, the causative agent of whooping cough, and identified nine novel small RNAs (sRNAs) that were transcribed during the bacterial colonization of murine tracheas. Among them, four sRNAs were more strongly expressed in vivo than in vitro. Moreover, the expression of eight sRNAs was not regulated by the BvgAS two-component system, which is the master regulator for the expression of genes contributing to the bacterial infection. The present results suggest a BvgAS-independent gene regulatory system involving the sRNAs that is active during B. pertussis infection.

Identification of the minimum region of Bordetella pertussis Vag8 required for interaction with C1 inhibitor.

An autotransporter of Bordetella pertussis, virulence-associated gene 8 (Vag8), binds and inactivates the complement regulator, C1 inhibitor (C1-Inh), and plays a role in evasion of the complement system. However, the molecular interaction between Vag8 and C1-Inh remains unclear. Here, we localized the minimum region of Vag8 required for interaction with C1-Inh by examining the differently truncated Vag8 derivatives for the ability to bind and inactivate C1-Inh. The truncated Vag8 containing amino-acid residues 102-548, but not 102-479 and 202-648, showed the full activity of intact Vag8, suggesting that the separate 102-202 and 548-648 amino-acid regions of Vag8 mediate the interaction with C1-Inh.

Bordet-Gengou agar medium supplemented with albumin-containing biologics for cultivation of bordetellae.

Bordetella pertussis, B. parapertussis, and B. bronchiseptica cause respiratory infections in mammals, including humans, and are generally cultivated on Bordet-Gengou (BG) agar plates in laboratories. The medium requires animal blood as a supplement for better bacterial growth. However, using blood is problematic, as its constant supply is occasionally difficult because of the limited shelf-life. This study proposes modified BG agar plates supplemented with bovine serum albumin and fetal bovine serum as a simple and convenient medium that confers sufficient growth of bordetellae.

Lactobacillus plantarum induces genomic DNA-dependent and TLR9-mediated elafin secretion from Caco-2 cells.

BACKGROUND: Lactobacilli show anti-inflammatory effects in the human intestine, and their genomic DNA was identified as one of the anti-inflammatory components. Increased levels of the natural protease inhibitor elafin in the intestine plays an important role in protection against intestinal inflammation. However, there have been no previous reports regarding whether lactobacilli increase elafin levels. OBJECTIVE: This study was performed to investigate whether Lactobacillus plantarum induces elafin secretion from the human epithelial colorectal adenocarcinoma cell line, Caco-2. Moreover, we examined the roles of bacterial genomic DNA and toll-like receptor 9 (TLR9), a specific receptor of bacterial DNA, in this effect. METHODS: Elafin secretion from Caco-2 cells by live and heat-killed L. plantarum was measured. The analysis was also performed using DNase-treated L. plantarum and genomic DNA extracted from L. plantarum. We examined the role of TLR9 in elafin secretion by L. plantarum and its genomic DNA by suppressing TLR9 expression using RNAi in Caco-2 cells. RESULTS: Heat-killed L. plantarum time- and dose-dependently increased elafin secretion, whereas live L. plantarum had no such effect. The elafin secretion by heat-killed L. plantarum was partially abolished by DNase treatment of the bacterium. In addition, L. plantarum genomic DNA also increased elafin secretion. Furthermore, suppression of TLR9 expression partially or completely abolished elafin secretion by heat-killed L. plantarum and its genomic DNA. CONCLUSION: Our results indicated that heat-killed L. plantarum induced genomic DNA-dependent and TLR9-mediated elafin secretion. The anti-inflammatory effects of lactobacilli may be mediated by increases in the levels of elafin in the intestine.

Significant Decrease in Pertactin-Deficient Bordetella pertussis Isolates, Japan.

Prevalence of pertactin-lacking Bordetella pertussis isolates has been observed worldwide. In Japan, however, we found that the frequency of pertactin-deficient isolates in 2014-2016 (8%) was significantly lower than the frequency in 2005-2007 (41%), 2008-2010 (35%), and 2011-2013 (25%). This reduction was closely associated with changes in genotypes.

Enhancement and regulation effect of myrcene on antibody response in immunization with ovalbumin and Ag85B in mice.

BACKGROUND: MF59, which is an adjuvant belonging to C30 member of the terpene family, is a T helper type-2 (Th2)-biased immune enhancer. Our previous studies showed that pyriproxyfen, a member of the terpene family with fewer carbon atoms (C20) than MF59, enhanced active T helper type-1 (Th1)-biased immune responses. OBJECTIVE: This study was performed to investigate the enhancement of antigen-specific immune responses by myrcene, a member of the terpene family with fewer carbon atoms (C10) than pyriproxyfen. METHOD: Ovalbumin (OVA) was used as an antigen to determine the effects of myrcene on the immune response. The IgG subtypes and cytokines induced by immunization of OVA with or without myrcene were monitored. Thereafter, we determined the effects of myrcene in the immune response against Ag85B, which is a dominant protective antigen for tuberculosis. RESULTS: The results showed that 0.8 mg/dose of myrcene enhanced antigen-specific total IgG immune response to OVA. Direct mixing of the antigen with myrcene was required for the enhancement of antibody production. Myrcene increased OVA-specific IgG2a titer, suggesting induction of Th1-immune response. The level of Th1 cytokines, IFN-γ was increased at 8 weeks after immunization, although IL-13 was also increased at the same time point. However, finally myrcene was found to increase Ag85B-specific total IgG titers at 5 weeks and specific IgG2a titer was increased at both 5 and 8 weeks. The results suggested that myrcene could enhance Th1 immune response. CONCLUSIONS: Myrcene enhanced specific immune responses against OVA and Ag85B. This study suggested the tendency of the enhancement of Th1 immune response by myrcene.

Recombinant fusion protein of cholera toxin B subunit with YVAD secreted by Lactobacillus casei inhibits lipopolysaccharide-induced caspase-1 activation and subsequent IL-1 beta secretion in Caco-2 cells.

BACKGROUND: Lactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB-YVAD). YVAD is a tetrapeptide (tyrosine-valine-alanine-aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1ß, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB-YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs. RESULTS: We constructed the rCTB-YVAD secretion vector pSCTB-YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB-YVAD was generated by transformation with pSCTB-YVAD. Both the culture supernatant of pSCTB-YVAD-transformed L. casei and purified rCTB-YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB-YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB-YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1ß secretion in Caco-2 cells, without affecting cell viability. CONCLUSIONS: The rCTB protein fused to a functional peptide secreted by L. casei can bind to GM1 ganglioside, like rCTB, and recombinant YVAD secreted by L. casei may exert anti-inflammatory effects in the intestine. Therefore, rCTB secreted by L. casei has potential utility as a vector for the delivery of YVAD to IECs.

Characterization of Dak Nong virus, an insect nidovirus isolated from Culex mosquitoes in Vietnam.

In this study, we isolated and characterized an insect nidovirus from the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae) in Vietnam, as an additional member of the new family Mesoniviridae in the order Nidovirales. The virus, designated "Dak Nong virus (DKNV)," shared many characteristics with Cavally virus and Nam Dinh virus, which have also been discovered recently in mosquitoes, and these viruses should be considered members of a single virus species, Alphamesonivirus 1. DKNV grew in cultured mosquito cells but could not replicate in the cultured vertebrate cells tested. N-terminal sequencing of the DKNV structural proteins revealed two posttranslational cleavage sites in the spike glycoprotein precursor. DKNV is assumed to be a new member of the species Alphamesonivirus 1, and the current study provides further understanding of viruses belonging to the new family Mesoniviridae.

Pyriproxyfen enhances the immunoglobulin G immune response in mice.

Pyriproxyfen is a juvenile hormone mimic of vital importance for insect development with little risk to humans. This study was performed to investigate whether large doses of pyriproxyfen affect the immune response in mammals. Mice were immunized thrice with ovalbumin in 5% ethanol, with or without pyriproxyfen or alum. Large doses of pyriproxyfen (9 or 15 mM) significantly enhanced specific total IgG immune response. This enhancement was no longer present 24 hr after treatment with pyriproxyfen. These results suggest that pyriproxyfen is a safe chemical. Moreover, pyriproxyfen induced higher titers of IgG2a and enhanced tumor necrosis factor-alpha and gamma-interferon responses whereas alum induced IgG1 with enhanced interleukin-4 and -10. These observations indicate that the mechanism of immune enhancement by pyriproxyfen may differ from that of alum.

Expression and secretion of cholera toxin B subunit in lactobacilli.

Lactic acid bacteria (LAB) are used in various fields, including in food and medical supplies. There has been a great deal of research into vaccine development using LAB as carriers due to their "generally recognized as safe" status. Cholera is an infectious disease that causes diarrhea due to cholera toxin (CT) produced by Vibrio cholerae. The pentameric cholera toxin B (CTB) subunit has no toxicity, and is used as an antigen in cholera vaccines and as a delivery molecule in vaccines to various diseases. In this study, we generated recombinant LAB expressing and secreting CTB. Here, we first report that CTB expressed and secreted from LAB bound to GM1 ganglioside. The secreted CTB was purified, and its immunogenicity was determined by intranasal administration into mice. The results of the present study suggested that it may be useful as the basis of a new oral cholera vaccine combining LAB and CTB.

Complete genome sequences of nine Burkholderia pseudomallei strains.

We report the complete genome sequences of nine Burkholderia pseudomallei strains preserved in research facilities in Japan; GTC3P0019, GTC3P0050, GTC3P0054, GTC3P0254T (type strain), Kanagawa, Tokushima, KM376, KM390, and KM391. The genomic information of these strains may provide references for comparative studies of B. pseudomallei pathogenicity.

Survival of Bordetella bronchiseptica in Acanthamoeba castellanii.

The respiratory pathogenic bacterium Bordetella bronchiseptica can persistently survive in terrestrial and aquatic environments, providing a source of infection. However, the environmental lifestyle of the bacterium is poorly understood. In this study, expecting repeated encounters of the bacteria with environmental protists, we explored the interaction between B. bronchiseptica and a representative environmental amoeba, Acanthamoeba castellanii, and found that the bacteria resisted amoeba digestion and entered contractile vacuoles (CVs), which are intracellular compartments involved in osmoregulation, to escape amoeba cells. In prolonged coculture, A. castellanii supported the proliferation of B. bronchiseptica. The avirulent Bvg- phase, but not the virulent Bvg+ phase, of the bacteria was advantageous for survival in the amoebae. We further demonstrate that two Bvg+ phase-specific virulence factors, filamentous hemagglutinin and fimbriae, were targeted for predation by A. castellanii. These results are evidence that the BvgAS two-component system, the master regulator for Bvg phase conversion, plays an indispensable role in the survival of B. bronchiseptica in amoebae. IMPORTANCE The pathogenic bacterium Bordetella bronchiseptica, which causes respiratory diseases in various mammals, exhibits distinct Bvg+ and Bvg- phenotypes. The former represents the virulent phase, in which the bacteria express a set of virulence factors, while the role of the latter in the bacterial life cycle remains to be understood. In this study, we demonstrate that B. bronchiseptica in the Bvg- phase, but not the Bvg+ phase, survives and proliferates in coculture with Acanthamoeba castellanii, an environmental amoeba. Two Bvg+ phase-specific virulence factors, filamentous hemagglutinin and fimbriae, were targeted by A. castellanii predation. B. bronchiseptica turns into the Bvg- phase at temperatures in which the bacteria normally encounter these amoebae. These findings demonstrate that the Bvg- phase of B. bronchiseptica is advantageous for survival outside mammalian hosts and that the bacteria can utilize protists as transient hosts in natural environments.

Interference of flagellar rotation up-regulates the expression of small RNA contributing to Bordetella pertussis infection.

Bacterial small RNAs (sRNAs) posttranscriptionally regulate gene expressions involved in various biological processes, including pathogenicity. Our previous study identified sRNAs, the expression of which was up-regulated in Bordetella pertussis, the causative agent of whooping cough, upon tracheal colonization of the bacteria; however, their roles in bacterial infection remain unknown. Here, we found that one sRNA, Bpr4, contributes to B. pertussis infection by posttranscriptionally up-regulating filamentous hemagglutinin (FHA), a major adhesin of the bacteria. Bpr4 bound to the 5' untranslated region of fhaB mRNA encoding FHA and inhibited its degradation mediated by RNaseE. Our results demonstrated that Bpr4 up-regulation was triggered by the interference of flagellar rotation, which caused the disengagement of MotA, a flagellar stator. Subsequently, MotA activated a diguanylate cyclase to generate cyclic di-GMP, which plays a role in Bpr4 up-regulation through the RisK/RisA two-component system. Our findings indicate that a flagellum-triggered sensory system contributes to B. pertussis infection.

The Mechanism of Pertussis Cough Revealed by the Mouse-Coughing Model.

Pertussis, also known as whooping cough, is a contagious respiratory disease caused by the Gram-negative bacterium Bordetella pertussis. This disease is characterized by severe and uncontrollable coughing, which imposes a significant burden on patients. However, its etiological agent and the mechanism are totally unknown because of a lack of versatile animal models that reproduce the cough. Here, we present a mouse model that reproduces coughing after intranasal inoculation with the bacterium or its components and demonstrate that lipooligosaccharide (LOS), pertussis toxin (PTx), and Vag8 of the bacterium cooperatively function to cause coughing. Bradykinin induced by LOS sensitized a transient receptor potential ion channel, TRPV1, which acts as a sensor to evoke the cough reflex. Vag8 further increased bradykinin levels by inhibiting the C1 esterase inhibitor, the major downregulator of the contact system, which generates bradykinin. PTx inhibits intrinsic negative regulation systems for TRPV1 through the inactivation of Gi GTPases. Our findings provide a basis to answer long-standing questions on the pathophysiology of pertussis cough. IMPORTANCE The Gram-negative bacterium Bordetella pertussis causes a respiratory disease called whooping cough, or pertussis. This disease is characterized by paroxysmal coughing, the mechanism of which has not been intensively studied because of a lack of versatile animal models that reproduce the cough. In this study, we present a mouse model that reproduces coughing after intranasal inoculation with the bacterium or its components. Using this model, we demonstrate that lipooligosaccharide, Vag8, and pertussis toxin of the bacteria cooperatively function to cause coughing. Our results also indicate that bradykinin, an inflammatory mediator, and TRPV1, an ion channel linked to nociceptive signaling, are host factors involved in the coughing mechanism.

Melanin Produced by Bordetella parapertussis Confers a Survival Advantage to the Bacterium during Host Infection.

Bordetella parapertussis causes respiratory infection in humans, with a mild pertussis (whooping cough)-like disease. The organism produces a brown pigment, the nature and biological significance of which have not been elucidated. Here, by screening a transposon library, we demonstrate that the gene encoding 4-hydroxyphenylpyruvate dioxygenase (HppD) is responsible for production of this pigment. Our results also indicate that the brown pigment produced by the bacterium is melanin, because HppD is involved in the biosynthesis of a type of melanin called pyomelanin, and homogentisic acid, the monomeric precursor of pyomelanin, was detected by high-performance liquid chromatography-mass spectrometry analyses. In an infection assay using macrophages, the hppD-deficient mutant was internalized by THP-1 macrophage-like cells, similar to the wild-type strain, but was less able to survive within the cells, indicating that melanin protects B. parapertussis from intracellular killing in macrophages. Mouse infection experiments also showed that the hppD-deficient mutant was eliminated from the respiratory tract more rapidly than the wild-type strain, although the initial colonization levels were comparable between the two strains. In addition, melanin production by B. parapertussis was not regulated by the BvgAS two-component system, which is the master regulator for the expression of genes contributing to the bacterial infection. Taken together, our findings indicate that melanin produced by B. parapertussis in a BvgAS-independent manner confers a survival advantage to the bacterium during host infection. IMPORTANCE In addition to the Gram-negative bacterium Bordetella pertussis, the etiological agent of pertussis, Bordetella parapertussis also causes respiratory infection in humans, with a mild pertussis-like disease. These bacteria are genetically closely related and share many virulence factors, including adhesins and toxins. However, B. parapertussis is clearly distinguished from B. pertussis by its brown pigment production, the bacteriological significance of which remains unclear. Here, we demonstrate that this pigment is melanin, which is known to be produced by a wide range of organisms from prokaryotes to humans and helps the organisms to survive under various environmental stress conditions. Our results show that melanin confers a survival advantage to B. parapertussis within human macrophages through its protective effect against reactive oxygen species and eventually contributes to respiratory infection of the bacterium in mice. This study proposes melanin as a virulence factor involved in the increased survival of B. parapertussis during host infection.

Bordetella Dermonecrotic Toxin Is a Neurotropic Virulence Factor That Uses CaV3.1 as the Cell Surface Receptor.

Dermonecrotic toxin (DNT) is one of the representative toxins produced by Bordetella pertussis, but its role in pertussis, B. pertussis infection, remains unknown. In this study, we identified the T-type voltage-gated Ca2+ channel CaV3.1 as the DNT receptor by CRISPR-Cas9-based genome-wide screening. As CaV3.1 is highly expressed in the nervous system, the neurotoxicity of DNT was examined. DNT affected cultured neural cells and caused flaccid paralysis in mice after intracerebral injection. No neurological symptoms were observed by intracerebral injection with the other major virulence factors of the organisms, pertussis toxin and adenylate cyclase toxin. These results indicate that DNT has aspects of the neurotropic virulence factor of B. pertussis The possibility of the involvement of DNT in encephalopathy, which is a complication of pertussis, is also discussed.IMPORTANCEBordetella pertussis, which causes pertussis, a contagious respiratory disease, produces three major protein toxins, pertussis toxin, adenylate cyclase toxin, and dermonecrotic toxin (DNT), for which molecular actions have been elucidated. The former two toxins are known to be involved in the emergence of some clinical symptoms and/or contribute to the establishment of bacterial infection. In contrast, the role of DNT in pertussis remains unclear. Our study shows that DNT affects neural cells through specific binding to the T-type voltage-gated Ca2+ channel that is highly expressed in the central nervous system and leads to neurological disorders in mice after intracerebral injection. These data raise the possibility of DNT as an etiological agent for pertussis encephalopathy, a severe complication of B. pertussis infection.

BspR/BtrA, an Anti-σ Factor, Regulates the Ability of Bordetella bronchiseptica To Cause Cough in Rats.

Bordetella pertussis, B. parapertussis, and B. bronchiseptica cause respiratory infections, many of which are characterized by coughing of the infected hosts. The pathogenesis of the coughing remains to be analyzed, mainly because there were no convenient infection models of small animals that replicate coughing after Bordetella infection. Here, we present a coughing model of rats infected with B. bronchiseptica Rats, which are one of natural hosts of B. bronchiseptica, were readily infected with the organisms and showed frequent coughing. B. pertussis also caused coughing in rats, which is consistent with previous reports, but the cough response was less apparent than the B. bronchiseptica-induced cough. By using the rat model, we demonstrated that adenylate cyclase toxin, dermonecrotic toxin, and the type III secretion system are not involved in cough production, but BspR/BtrA (different names for the same protein), an anti-σ factor, regulates the production of unknown factor(s) to cause coughing. Rat coughing was observed by inoculation of not only the living bacteria but also the bacterial lysates. Infection with bspR (btrA)-deficient strains caused significantly less frequent coughing than the wild type; however, intranasal inoculation of the lysates from a bspR (btrA)-deficient strain caused coughing similarly to the wild type, suggesting that BspR/BtrA regulates the production of the cough factor(s) only when the bacteria colonize host bodies. Moreover, the cough factor(s) was found to be heat labile and produced by B. bronchiseptica in the Bvg+ phase. We consider that our rat model provides insight into the pathogenesis of cough induced by the Bordetella infection.IMPORTANCE Whooping cough is a contagious respiratory disease caused by Bordetella pertussis This disease is characterized by severe paroxysmal coughing, which becomes a heavy burden for patients and occasionally results in death; however, its pathogenesis remains largely unknown. The major obstacle to analyzing Bordetella-induced coughing is the lack of conventional animal models that replicate coughing. As Bordetella pertussis is highly adapted to humans, infection models in experimental animals are not considered to be well established. In the present study, we examined coughing in rats infected with B. bronchiseptica, which shares many virulence factors with B. pertussis Using this rat model, we demonstrated that some of the major virulence factors of Bordetella are not involved in cough production, but an anti-σ factor, BspR/BtrA, of B. bronchiseptica regulates the production of unknown cough-causing bacterial factor(s). Our results provide important clues to understand the mechanism by which Bordetella induces cough.