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

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

Bystander activation of Bordetella pertussis-induced nasal tissue-resident memory CD4 T cells confers heterologous immunity to Klebsiella pneumoniae.

Tissue-resident memory CD4 T (TRM ) cells induced by infection with Bordetella pertussis persist in respiratory tissues and confer long-term protective immunity against reinfection. However, it is not clear how they are maintained in respiratory tissues. Here, we demonstrate that B. pertussis-specific CD4 TRM cells produce IL-17A in response to in vitro stimulation with LPS or heat-killed Klebsiella pneumoniae (HKKP) in the presence of dendritic cells. Furthermore, IL-17A-secreting CD4 TRM cells expand in the lung and nasal tissue of B. pertussis convalescent mice following in vivo administration of LPS or HKKP. Bystander activation of CD4 TRM cells was suppressed by anti-IL-12p40 but not by anti-MHCII antibodies. Furthermore, purified respiratory tissue-resident, but not circulating, CD4 T cells from convalescent mice produced IL-17A following direct stimulation with IL-23 and IL-1ß or IL-18. Intranasal immunization of mice with a whole-cell pertussis vaccine induced respiratory CD4 TRM cells that were reactivated following stimulation with K. pneumoniae. Furthermore, the nasal pertussis vaccine conferred protective immunity against B. pertussis but also attenuated infection with K. pneumoniae. Our findings demonstrate that CD4 TRM cells induced by respiratory infection or vaccination can undergo bystander activation and confer heterologous immunity to an unrelated respiratory pathogen.

Cytotoxicity of the effector protein BteA was attenuated in Bordetella pertussis by insertion of an alanine residue.

Bordetella bronchiseptica and Bordetella pertussis are closely related respiratory pathogens that evolved from a common bacterial ancestor. While B. bronchiseptica has an environmental reservoir and mostly establishes chronic infections in a broad range of mammals, B. pertussis is a human-specific pathogen causing acute pulmonary pertussis in infants and whooping cough illness in older humans. Both species employ a type III secretion system (T3SS) to inject a cytotoxic BteA effector protein into host cells. However, compared to the high BteA-mediated cytotoxicity of B. bronchiseptica, the cytotoxicity induced by B. pertussis BteA (Bp BteA) appears to be quite low and this has been attributed to the reduced T3SS gene expression in B. pertussis. We show that the presence of an alanine residue inserted at position 503 (A503) of Bp BteA accounts for its strongly attenuated cytotoxic potency. The deletion of A503 from Bp BteA greatly enhanced the cytotoxic activity of B. pertussis B1917 on mammalian HeLa cells and expression of Bp BteAΔA503 was highly toxic to Saccharomyces cerevisiae cells. Vice versa, insertion of A503 into B. bronchiseptica BteA (Bb BteA) strongly decreased its cytotoxicity to yeast and HeLa cells. Moreover, the production of Bp BteAΔA503 increased virulence of B. pertussis B1917 in the mouse model of intranasal infection (reduced LD50) but yielded less inflammatory pathology in infected mouse lungs at sublethal infectious doses. This suggests that A503 insertion in the T3SS effector Bp BteA may represent an evolutionary adaptation that fine-tunes B. pertussis virulence and host immune response.

Reinvestigating the Coughing Rat Model of Pertussis To Understand Bordetella pertussis Pathogenesis.

Bordetella pertussis is a highly contagious bacterium that is the causative agent of whooping cough (pertussis). Currently, acellular pertussis vaccines (aP, DTaP, and Tdap) are used to prevent pertussis disease. However, it is clear that the aP vaccine efficacy quickly wanes, resulting in the reemergence of pertussis. Furthermore, recent work performed by the CDC suggest that current circulating strains are genetically distinct from strains of the past. The emergence of genetically diverging strains, combined with waning aP vaccine efficacy, calls for reevaluation of current animal models of pertussis. In this study, we used the rat model of pertussis to compare two genetically divergent strains Tohama 1 and D420. We intranasally challenged 7-week-old Sprague-Dawley rats with 108 viable Tohama 1 and D420 and measured the hallmark signs/symptoms of B. pertussis infection such as neutrophilia, pulmonary inflammation, and paroxysmal cough using whole-body plethysmography. Onset of cough occurred between 2 and 4 days after B. pertussis challenge, averaging five coughs per 15 min, with peak coughing occurring at day 8 postinfection, averaging upward of 13 coughs per 15 min. However, we observed an increase of coughs in rats infected with clinical isolate D420 through 12 days postchallenge. The rats exhibited increased bronchial restriction following B. pertussis infection. Histology of the lung and flow cytometry confirm both cellular infiltration and pulmonary inflammation. D420 infection induced higher production of anti-B. pertussis IgM antibodies compared to Tohama 1 infection. The coughing rat model provides a way of characterizing disease manifestation differences between B. pertussis strains.

Immunogenicity of a new enhanced tetanus-reduced dose diphtheria-acellular pertussis (Tdap) vaccine against Bordetella pertussis in a murine model.

BACKGROUND: The necessity of the tetanus-reduced dose diphtheria-acellular pertussis (Tdap) vaccine in adolescence and adults has been emphasized since the resurgence of small-scale pertussis in Korea and worldwide due to the waning effect of the vaccine and variant pathogenic stains in the late 1990s. GreenCross Pharma (GC Pharma), a Korean company, developed the Tdap vaccine GC3111 in 2010. Recently, they enhanced the vaccine, GC3111, produced previously in 2010 to reinforce the antibody response against filamentous hemagglutinin (FHA). In this study, immunogenicity and efficacy of the enhanced Tdap vaccine compared and evaluated with two Tdap vaccines, GC3111 vaccine produced in 2010 previously and commercially available Tdap vaccine in a murine model. METHODS: Two tests groups and positive control group of Balb/c mice were primed with two doses of the diphtheria-tetanus-acellular pertussis (DTaP) vaccine followed by a single booster Tdap vaccine at 9 week using the commercially available Tdap vaccine or 2 Tdap vaccines from GC Pharma (GC3111, enhanced GC3111). Humoral response was assessed 1 week before and 2 and 4 weeks after Tdap booster vaccination. The enhanced GC3111 generated similar humoral response compare to the commercial vaccine for filamentous hemagglutinin (FHA). The interferon gamma (IFN-γ) (Th1), interleukin 5 (IL-5) (Th2) and interleukin 17 (IL-17) (Th17) cytokines were assessed 4 weeks after booster vaccination by stimulation with three simulators: heat inactivated Bordetella pertussis (hBp), vaccine antigens, and hBp mixed with antigens (hBp + antigen). A bacterial challenge test was performed 4 weeks after booster vaccination. RESULTS: Regarding cell-mediated immunity, cytokine secretion differed among the three simulators. However, no difference was found between two test groups and positive control group. All the vaccinated groups indicated a Th1 or Th1/Th2 response. On Day 5 post-bacterial challenge, B. pertussis colonies were absent in the lungs in two test groups and positive control group. CONCLUSIONS: Our results confirmed the immunogenicity of GC Pharma's Tdap vaccine; enhanced GC3111 was equivalent to the presently used commercial vaccine in terms of humoral response as well as cell-mediated cytokine expression.

Lack of evidence supporting a role of IFN-ß and TGF-ß in differential polarization of Bordetella pertussis specific-T cell responses.

Bordetella Pertussis (BP) vaccine-induced immunity is waning worldwide despite excellent vaccine coverage. Replacement of the whole-cell inactivated vaccine (wP) by an acellular subunit vaccine (aP) is thought to play a major role and to be associated with the recurrence of whooping cough. Previously, we detected that the polarization towards a Th2 and Th1/Th17 response in aP and wP vaccinees, respectively, persists upon aP boosting in adolescents and adults. Additionally, IL-9 and TGF-ß were found to be up-regulated in aP-primed donors and network analysis further identified IFN-ß as a potential upstream regulator of IL-17 and IL-9. Based on these findings, we hypothesized that IFN-ß produced following aP vaccination may lead to increased IL-9 and decreased IL-17 production. Also, due to the well characterized role of TGF-ß in both Th17 and Th9 differentiation, we put forth that TGF-ß addition to BP-stimulated CD4 + T cells might modulate IL-17 and IL-9 production. To test this hypothesis, we stimulated in vitro cultures of PBMC or isolated naive CD4 + T cells from aP vs wP donors with a pool of BP epitopes and assessed the effect of IFN-ß or TGF-ß in proliferative responses as well as in the cytokine secretion of IL-4, IL-9, IL-17, and IFN-γ. IFN-ß reduced BP-specific proliferation in PBMC as well as cytokine production but increased IL-9, IL-4, and IFN-γ cytokines in naïve CD4 + T cells. These effects were independent of the childhood vaccination received by the donors. Similarly, TGF-ß reduced BP-specific proliferation in PBMC but induced proliferation in naïve CD4 + T cells. However, stimulation was associated with a generalized inhibition of cytokine production regardless of the original aP or wP vaccination received by the donors. Our study suggests that key T cell functions such as cytokine secretion are under the control of antigen stimulation and environmental cues but molecular pathways different than the ones investigated here might underlie the long-lasting differential cytokine production associated with aP- vs wP-priming in childhood vaccination.

Omics Analysis of Blood-Responsive Regulon in Bordetella pertussis Identifies a Novel Essential T3SS Substrate.

Bacterial pathogens sense specific cues associated with different host niches and integrate these signals to appropriately adjust the global gene expression. Bordetella pertussis is a Gram-negative, strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Though B. pertussis does not cause invasive infections, previous results indicated that this reemerging pathogen responds to blood exposure. Here, omics RNA-seq and LC-MS/MS techniques were applied to determine the blood-responsive regulon of B. pertussis. These analyses revealed that direct contact with blood rewired global gene expression profiles in B. pertussis as the expression of almost 20% of all genes was significantly modulated. However, upon loss of contact with blood, the majority of blood-specific effects vanished, with the exception of several genes encoding the T3SS-secreted substrates. For the first time, the T3SS regulator BtrA was identified in culture supernatants of B. pertussis. Furthermore, proteomic analysis identified BP2259 protein as a novel secreted T3SS substrate, which is required for T3SS functionality. Collectively, presented data indicate that contact with blood represents an important cue for B. pertussis cells.

Selective Enhancement of the Cell-Permeabilizing Activity of Adenylate Cyclase Toxin Does Not Increase Virulence of Bordetella pertussis.

The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) that catalyzes the conversion of intracellular ATP to cAMP and through its signaling annihilates the bactericidal activities of host sentinel phagocytes. In parallel, CyaA permeabilizes host cells by the formation of cation-selective membrane pores that account for the hemolytic activity of CyaA. The pore-forming activity contributes to the overall cytotoxic effect of CyaA in vitro, and it has previously been proposed to synergize with the cAMP-elevating activity in conferring full virulence on B. pertussis in the mouse model of pneumonic infection. CyaA primarily targets myeloid phagocytes through binding of their complement receptor 3 (CR3, integrin αMß2, or CD11b/CD18). However, with a reduced efficacy, the toxin can promiscuously penetrate and permeabilize the cell membrane of a variety of non-myeloid cells that lack CR3 on the cell surface, including airway epithelial cells or erythrocytes, and detectably intoxicates them by cAMP. Here, we used CyaA variants with strongly and selectively enhanced or reduced pore-forming activity that, at the same time, exhibited a full capacity to elevate cAMP concentrations in both CR3-expressing and CR3-non-expressing target cells. Using B. pertussis mutants secreting such CyaA variants, we show that a selective enhancement of the cell-permeabilizing activity of CyaA does not increase the overall virulence and lethality of pneumonic B. pertussis infection of mice any further. In turn, a reduction of the cell-permeabilizing activity of CyaA did not reduce B. pertussis virulence any importantly. These results suggest that the phagocyte-paralyzing cAMP-elevating capacity of CyaA prevails over the cell-permeabilizing activity of CyaA that appears to play an auxiliary role in the biological activity of the CyaA toxin in the course of B. pertussis infections in vivo.

Transcriptional profiling of human macrophages during infection with Bordetella pertussis.

Bordetella pertussis, a strictly human re-emerging pathogen and the causative agent of whooping cough, exploits a broad variety of virulence factors to establish efficient infection. Here, we used RNA sequencing to analyse the changes in gene expression profiles of human THP-1 macrophages resulting from B. pertussis infection. In parallel, we attempted to determine the changes in intracellular B. pertussis-specific transcriptomic profiles resulting from interaction with macrophages. Our analysis revealed that global gene expression profiles in THP-1 macrophages are extensively rewired 6 h post-infection. Among the highly expressed genes, we identified those encoding cytokines, chemokines, and transcription regulators involved in the induction of the M1 and M2 macrophage polarization programmes. Notably, several host genes involved in the control of apoptosis and inflammation which are known to be hijacked by intracellular bacterial pathogens were overexpressed upon infection. Furthermore, in silico analyses identified large temporal changes in expression of specific gene subsets involved in signalling and metabolic pathways. Despite limited numbers of the bacterial reads, we observed reduced expression of majority of virulence factors and upregulation of several transcriptional regulators during infection suggesting that intracellular B. pertussis cells switch from virulent to avirulent phase and actively adapt to intracellular environment, respectively.

Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain.

Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacterium Bordetella pertussis Secreted as soluble protein, it targets myeloid cells expressing the CD11b/CD18 integrin and on delivery of its N-terminal adenylate cyclase catalytic domain (AC domain) into the cytosol, generates uncontrolled toxic levels of cAMP that ablates bactericidal capacities of phagocytes. Our study deciphers the fundamentals of the heretofore poorly understood molecular mechanism by which the ACT enzyme domain directly crosses the host cell membrane. By combining molecular biology, biochemistry, and biophysics techniques, we discover that ACT has intrinsic phospholipase A (PLA) activity, and that such activity determines AC translocation. Moreover, we show that elimination of the ACT-PLA activity abrogates ACT toxicity in macrophages, particularly at toxin concentrations close to biological reality of bacterial infection. Our data support a molecular mechanism in which in situ generation of nonlamellar lysophospholipids by ACT-PLA activity into the cell membrane would form, likely in combination with membrane-interacting ACT segments, a proteolipidic toroidal pore through which AC domain transfer could directly take place. Regulation of ACT-PLA activity thus emerges as novel target for therapeutic control of the disease.

Genotypic and phenotypic adaptation of pathogens: lesson from the genus Bordetella.

PURPOSE OF REVIEW: To relate genomic changes to phenotypic adaptation and evolution from environmental bacteria to obligate human pathogens, focusing on the examples within Bordetella species. RECENT FINDINGS: Recent studies showed that animal-pathogenic and human-pathogenic Bordetella species evolved from environmental ancestors in soil. The animal-pathogenic Bordetella bronchiseptica can hijack the life cycle of the soil-living amoeba Dictyostelium discoideum, surviving inside single-celled trophozoites, translocating to the fruiting bodies and disseminating along with amoeba spores. The association with amoeba may have been a 'training ground' for bacteria during the evolution to pathogens. Adaptation to an animal-associated life style was characterized by decreasing metabolic versatility and genome size and by acquisition of 'virulence factors' mediating the interaction with the new animal hosts. Subsequent emergence of human-specific pathogens, such as Bordetella pertussis from zoonoses of broader host range progenitors, was accompanied by a dramatic reduction in genome size, marked by the loss of hundreds of genes. SUMMARY: The evolution of Bordetella from environmental microbes to animal-adapted and obligate human pathogens was accompanied by significant genome reduction with large-scale gene loss during divergence.

Proteomic Adaptation of Australian Epidemic Bordetella pertussis.

Bordetella pertussis causes whooping cough. The predominant strains in Australia changed to single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele ptxP3/pertactin gene allele prn2) from cluster II (non-ptxP3/non-prn2). Cluster I was mostly responsible for the 2008-2012 Australian epidemic and was found to have higher fitness compared to cluster II using an in vivo mouse competition assay, regardless of host's immunization status. This study aimed to identify proteomic differences that explain higher fitness in cluster I using isobaric tags for relative and absolute quantification (iTRAQ), and high-resolution multiple reaction monitoring (MRM-hr). A few key differences in the whole cell and secretome were identified between the cluster I and II strains tested. In the whole cell, nine proteins were upregulated (>1.2 fold change, q < 0.05) and three were downregulated (<0.8 fold change, q < 0.05) in cluster I. One downregulated protein was BP1569, a TLR2 agonist for Th1 immunity. In the secretome, 12 proteins were upregulated and 1 was downregulated which was Bsp22, a type III secretion system (T3SS) protein. Furthermore, there was a trend of downregulation in three T3SS effectors and other virulence factors. Three proteins were upregulated in both whole cell and supernatant: BP0200, molybdate ABC transporter (ModB), and tracheal colonization factor A (TcfA). Important expression differences in lipoprotein, T3SS, and transport proteins between the cluster I and II strains were identified. These differences may affect immune evasion, virulence and metabolism, and play a role in increased fitness of cluster I.

The extent of the temperature-induced membrane remodeling in two closely related Bordetella species reflects their adaptation to diverse environmental niches.

Changes in environmental temperature represent one of the major stresses faced by microorganisms as they affect the function of the cytoplasmic membrane. In this study, we have analyzed the thermal adaptation in two closely related respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica Although B. pertussis represents a pathogen strictly adapted to the human body temperature, B. bronchiseptica causes infection in a broad range of animals and survives also outside of the host. We applied GC-MS to determine the fatty acids of both Bordetella species grown at different temperatures and analyzed the membrane fluidity by fluorescence anisotropy measurement. In parallel, we also monitored the effect of growth temperature changes on the expression and production of several virulence factors. In response to low temperatures, B. pertussis adapted its fatty acid composition and membrane fluidity to a considerably lesser extent when compared with B. bronchiseptica Remarkably, B. pertussis maintained the production of virulence factors at 24 °C, whereas B. bronchiseptica cells resumed the production only upon temperature upshift to 37 °C. This growth temperature-associated differential modulation of virulence factor production was linked to the phosphorylation state of transcriptional regulator BvgA. The observed differences in low-temperature adaptation between B. pertussis and B. bronchiseptica may result from selective adaptation of B. pertussis to the human host. We propose that the reduced plasticity of the B. pertussis membranes ensures sustained production of virulence factors at suboptimal temperatures and may play an important role in the transmission of the disease.

The Adjuvant Bordetella Colonization Factor A Attenuates Alum-Induced Th2 Responses and Enhances Bordetella pertussis Clearance from Mouse Lungs.

The reemergence of pertussis or whooping cough in several countries highlights the need for better vaccines. Acellular pertussis vaccines (aPV) contain alum as the adjuvant and elicit Th2-biased immune responses that are less effective in protecting against infection than the reactogenic whole-cell pertussis vaccines (wPV), which elicit primarily a Th1/Th17 response. An important goal for the field is to devise aPV that will induce immune responses similar to those of wPV. We show that Bordetella colonization factor A (BcfA), an outer membrane protein from Bordetella bronchiseptica, has strong adjuvant function and elicits cellular and humoral immune responses to heterologous and Bordetella pertussis antigens. Addition of BcfA to a commercial aPV resulted in greater reduction of B. pertussis numbers from the lungs than that elicited by aPV alone. The more-efficient pathogen clearance was accompanied by increased interleukin-17 (IL-17) and reduced IL-5 and an increased ratio of IgG2/IgG1 antibodies. Thus, our results suggest that BcfA improves aPV-induced responses by modifying the alum-induced Th2-biased aPV response toward Th1/Th17. A redesigned aPV containing BcfA may allow better control of pertussis reemergence by reshaping immune responses to resemble those elicited by wPV immunization.

Bordetella adenylate cyclase toxin interacts with filamentous haemagglutinin to inhibit biofilm formation in vitro.

Bordetella pertussis, the causative agent of whooping cough, secretes and releases adenylate cyclase toxin (ACT), which is a protein bacterial toxin that targets host cells and disarms immune defenses. ACT binds filamentous haemagglutinin (FHA), a surface-displayed adhesin, and until now, the consequences of this interaction were unknown. A B. bronchiseptica mutant lacking ACT produced more biofilm than the parental strain; leading Irie et al. to propose the ACT-FHA interaction could be responsible for biofilm inhibition. Here we characterize the physical interaction of ACT with FHA and provide evidence linking that interaction to inhibition of biofilm in vitro. Exogenous ACT inhibits biofilm formation in a concentration-dependent manner and the N-terminal catalytic domain of ACT (AC domain) is necessary and sufficient for this inhibitory effect. AC Domain interacts with the C-terminal segment of FHA with ∼650 nM affinity. ACT does not inhibit biofilm formation by Bordetella lacking the mature C-terminal domain (MCD), suggesting the direct interaction between AC domain and the MCD is required for the inhibitory effect. Additionally, AC domain disrupts preformed biofilm on abiotic surfaces. The demonstrated inhibition of biofilm formation by a host-directed protein bacterial toxin represents a novel regulatory mechanism and identifies an unprecedented role for ACT.

Hyperbiofilm Formation by Bordetella pertussis Strains Correlates with Enhanced Virulence Traits.

Pertussis, or whooping cough, caused by the obligate human pathogen Bordetella pertussis is undergoing a worldwide resurgence. The majority of studies of this pathogen are conducted with laboratory-adapted strains which may not be representative of the species as a whole. Biofilm formation by B. pertussis plays an important role in pathogenesis. We conducted a side-by-side comparison of the biofilm-forming abilities of the prototype laboratory strains and the currently circulating isolates from two countries with different vaccination programs. Compared to the reference strain, all strains examined herein formed biofilms at high levels. Biofilm structural analyses revealed country-specific differences, with strains from the United States forming more structured biofilms. Bacterial hyperaggregation and reciprocal expression of biofilm-promoting and -inhibitory factors were observed in clinical isolates. An association of increased biofilm formation with augmented epithelial cell adhesion and higher levels of bacterial colonization in the mouse nose and trachea was detected. To our knowledge, this work links for the first time increased biofilm formation in bacteria with a colonization advantage in an animal model. We propose that the enhanced biofilm-forming capacity of currently circulating strains contributes to their persistence, transmission, and continued circulation.

Oxygen and Bis(3',5')-cyclic Dimeric Guanosine Monophosphate Binding Control Oligomerization State Equilibria of Diguanylate Cyclase-Containing Globin Coupled Sensors.

Bacteria sense their environment to alter phenotypes, including biofilm formation, to survive changing conditions. Heme proteins play important roles in sensing the bacterial gaseous environment and controlling the switch between motile and sessile (biofilm) states. Globin coupled sensors (GCS), a family of heme proteins consisting of a globin domain linked by a central domain to an output domain, are often found with diguanylate cyclase output domains that synthesize c-di-GMP, a major regulator of biofilm formation. Characterization of diguanylate cyclase-containing GCS proteins from Bordetella pertussis and Pectobacterium carotovorum demonstrated that cyclase activity is controlled by ligand binding to the heme within the globin domain. Both O2 binding to the heme within the globin domain and c-di-GMP binding to a product-binding inhibitory site (I-site) within the cyclase domain control oligomerization states of the enzymes. Changes in oligomerization state caused by c-di-GMP binding to the I-site also affect O2 kinetics within the globin domain, suggesting that shifting the oligomer equilibrium leads to broad rearrangements throughout the protein. In addition, mutations within the I-site that eliminate product inhibition result in changes to the accessible oligomerization states and decreased catalytic activity. These studies provide insight into the mechanism by which ligand binding to the heme and I-site controls activity of GCS proteins and suggests a role for oligomerization-dependent activity in vivo.

Pertussis in Young Infants Throughout the World.

In typical pertussis in young infants, the child will appear deceptively well; he or she will have coryza, sneezing, and a mild cough. There is no fever. This progresses to gagging, gasping, eye bulging, bradycardia, cyanosis, and vomiting. There is leukocytosis with lymphocytosis and apneic episodes. Deaths relate to leukocytosis, pulmonary hypertension, and pneumonia. The source of pertussis in young infants is most often a family member with cough illness that is not recognized as pertussis. Diagnosis is based on culture/polymerase chain reaction and leukocytosis with lymphocytosis. Treatment depends on macrolide antibiotic therapy and intubation, with assisted ventilation and oxygen. Prevention is based on prophylactic macrolide treatment, immunization starting at 6 weeks of age, and immunization of all pregnant women in the second or third trimester.

Did a then unknown virus, HHV-6/7, give rise to the whooping cough vaccine controversy of the 1970s?

During the 1970s there was a gross loss of public confidence in infant diphtheria-tetanus-pertussis (DTP) vaccination in the UK. As well as febrile reactions and convulsions, permanent neurological damage was ascribed to the pertussis component of the vaccine, and those concerns resonated worldwide. The subsequent recognition of human herpes virus 6 (HHV-6) and 7 (HHV-7) as common sources of fever in infancy suggests that they were the main underlying cause of what was reported as DTP constitutional side-effects. With more precise data on the incidence of HHV-6/7 and other virus infections in early life it would be possible to model the concurrence of viral illnesses with routine immunizations. Adventitious viral infections may be the cause of side-effects ascribed to the numerous childhood immunizations now being given.

Isolated CyaA-RTX subdomain from Bordetella pertussis: Structural and functional implications for its interaction with target erythrocyte membranes.

The 126-kDa Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was previously expressed in Escherichia coli as a soluble precursor that can be acylated to retain hemolytic activity. Here, we investigated structural and functional characteristics of a ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of CyaA-Hly. Initially, we succeeded in producing a large amount with high purity of the His-tagged CyaA-RTX fragment and in establishing the interaction of acylated CyaA-Hly with sheep red blood cell (sRBC) membranes by immuno-localization. Following pre-incubation of sRBCs with non-acylated CyaA-Hly or with the CyaA-RTX fragment that itself produces no hemolytic activity, there was a dramatic decrease in CyaA-Hly-induced hemolysis. When CyaA-RTX was pre-incubated with anti-CyaA-RTX antisera, the capability of CyaA-RTX to neutralize the hemolytic activity of CyaA-Hly was greatly decreased. A homology-based model of the 100-kDa CyaA-RTX subdomain revealed a loop structure in Linker II sharing sequence similarity to human WW domains. Sequence alignment of Linker II with the human WW-domain family revealed highly conserved aromatic residues important for protein-protein interactions. Altogether, our present study demonstrates that the recombinant CyaA-RTX subdomain retains its functionality with respect to binding to target erythrocyte membranes and the WW-homologous region in Linker II conceivably serves as a functional segment required for receptor-binding activity.