Background and Objectives: Pertussis is an infectious disease caused by the Gram-negative bacterium Bordetella pertussis. In Peru, actual public health programs indicate that vaccination against B. pertussis must be mandatory and generalized, be-sides all detected cases must be reported. The objective of this study was to determine the prevalence of B. pertussis among children under five years of age with a presumptive diagnosis of whopping cough in Cajamarca, a region located in northern Peru. Materials and Methods: The population of this cross-sectional study were children under 5 years old hospitalized as presumptive cases of pertussis during December 2017 to December 2018. The nasopharyngeal samples were analyzed by real-time PCR for the detection of B. pertussis. Results: B. pertussis was identified as PCR + in 42.3% of our sample (33/78). The clinical presentation that was observed most frequently includes paroxysmal coughing (97%), difficulty breathing (69.7%), cyanosis (72.7%) and post-tussive em-esis (60.6%). Additionally, pneumonia was the most observed complication (33.3%). Four of the patients with PCR+ for B. pertussis presented only lymphocytosis, five only leukocytosis, two patients with decreased leukocytosis and lymphocytes and only one patient with leukopenia and relative lymphocytosis. There was a percentage of 84.8% of unvaccinated children in the PCR+ group. Finally, the mother was the most frequent symptom carrier (18.2%). Conclusion: In conclusion, in the studied population there is a high rate of PCR+ cases for B. pertussis. Laboratory values may show leukopenia or lymphopenia in patients with pertussis. It is necessary to use appropriate laboratory diagnostic tests in all infants with respiratory symptoms for B. pertussis. Since, the clinical diagnosis overestimates the diagnosis of pertussis. / Revisión por pares

Mittels Tissue Engineering hergestellte humane 3D in vitro-Testsysteme sind ein neuer Ansatz, um u.a. Erkrankungen der Atemwege zu simulieren und zu untersuchen. Obwohl gegen B. pertussis, den Erreger des Keuchhustens, Impfstoffe zur Verfügung stehen, nimmt die Erkrankungs-Inzidenz in den letzten Jahren deutlich zu. Da B. pertussis zu den obligat humanpathogenen Erregern zählt, sind die aus Tierversuchen stammenden Daten nur unzureichend auf den Menschen übertragbar. Die genauen Pathomechanismen der Infektion sind bisher nicht geklärt. Auf einer biologischen Kollagenmatrix wurde eine Ko-Kultur aus humanen tracheobronchialen Fibroblasten und humanen tracheobronchialen Epithelzellen (hTEC) angesiedelt und 3 Wochen unter apikaler Belüftung kultiviert. Die ausdifferenzierten 3D Testsysteme wurden mit Überständen von Bordetella pertussis-Kulturen inkubiert und auf licht- und elektronenmikroskopischer Ebene analysiert. Weiterhin wurden 2D Kulturen der hTEC mit Hilfe der Ramanspektroskopie nicht-invasiv auf intrazelluläre Veränderungen nach der Inkubation mit den bakteriellen Überständen untersucht. Das 3D Testsystem der humanen Atemwegschleimhaut zeigte auf lichtmikroskopischer und ultrastruktureller Ebene eine hohe in vitro – in vivo-Korrelation. Die elektronenmikroskopische Analyse zeigte morphologische Veränderungen nach der Inkubation mit den B. pertussis Überständen, die mit vorbeschrieben Effekten einer B. pertussis Infektion korrelieren. Mittels der Ramanspektroskopie ließen sich Gruppen von unbehandelten Zellen von Gruppen, die zuvor mit Bakterienüberständen inkubiert wurden, trennen. Somit zeigte sich die Ramanspektroskopie sensitiv für intrazelluläre Infektionsfolgen. Zusammenfassend wurde belegt, dass das 3D-Modell der humanen Atemwegschleimhaut zur Untersuchung obligat humanpathogener Infektionserreger geeignet ist und dass die Ramanspektroskopie eine nicht-invasive Methode ist, um durch Infektionen hervorgerufene intrazellulären Pathologien zu analysieren. / Three dimensional (3D) tissue-engineered human tissue models are of high relevance, e.g. to investigate virulence mechanisms of human obligate pathogens in vitro. One major obligate agent causing acute respiratory diseases is Bordetella pertussis (Bp), the agent of whooping cough. The progress towards elimination Bp has stalled which is mainly caused due to an absence of suitable models to gain more knowledge about its pathomechanism. On a biological collagen matrix (SISser) a co-culture of human fibroblasts and human airway epithelial cells (hTEC) was seeded and cultured under airlift conditions. The completely differentiated test systems were treated with sterile-filtrated supernatants of Bp and afterwards analyzed with light and transmission electron microscopy. 2D cultures of hTEC were also infected and analyzed with Raman spectroscopy. The 3D test system of the human airway mucosa shows high in vitro - in vivo - correlation on both structural and ultrastructural level. Preliminary morphological analysis after infection with Bp supernatant reveals considerable ultrastructural changes which were not observed in control samples and correlate to effects knows from Bp infections in vivo. In 2D cultivation conditions the Raman spectra of infected hTEC clearly differ from spectra of the control. It is shown that the 3D airway mucosa model represents pathological effects of Bp toxins and offers an opportunity to further examine their pathomechanisms. Raman spectroscopy appears to be a practical method to show intracellular changes on living cells non-invasively.

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Bordetella pertussis is a strictly human pathogen colonizing the upper respiratory tract, causing a respiratory disease known as whooping cough or pertussis. The introduction of whole-cell vaccines and acellular vaccines, resulted in a significant reduction in the incidence of disease and reduce the fatalities associated with infection. However, epidemiological data show a significant increase in the incidence of the disease in recent decades. The increasing incidence is mainly attributed to the transition from the whole- cell vaccine to an acellular vaccine. Based on research from recent years has shown that acellular vaccines have many drawbacks, and it is therefore necessary to change the vaccination strategy. One possible solution to the situation is the development of a new generation of whole-cell vaccines with reduced reactogenicity. The new whole-cell vaccine was prepared by a genetically modified B. pertussis strain. B. pertussis was modified using allelic exchange to develop strain encoding enzymatically inactive pertussis toxin, modified lipid A and lacking dermonecrotic toxin. This combination of genetic modifications in mice led to a decrease in reactogenicity test vaccine in vivo. In case of intranasal infection whole-cell vaccine containing genetically modified strain is providing...

Em trabalhos anteriores o nosso grupo mostrou que a vacina celular pertussis (wP) apresenta atividade adjuvante quando combinada a proteína A da superfície de Streptococcus pneumoniae (pneumococo), PspA, A formulação PspA5-wP induz altos níveis de anticorpos e proteção em camundongos após desafio com pneumococo. Neste trabalho foram avaliados os mecanismos da ação adjuvante e os componentes de B. pertussis responsáveis por este efeito. A imunização de camundongos com PspA em combinação a mutantes de B. pertussis ou componentes desta bactéria purificados mostrou que a presença da toxina pertussis (PT) capaz é essencial para induzir altos níveis de anticorpos anti-PspA e proteção significativa contra o desafio letal com pneumococo. Este efeito não foi dependente da atividade enzimática. Anticorpos anti-PspA e as proteínas do sistema complemento foram essenciais para a proteção conferida por PspA-wP. As vacinas celulares BCG e DTP combinadas a PspA, também apresentaram atividade adjuvante, induzindo anticorpos anti-PspA e proteção contra o desafio invasivo por pneumococo. / Previously our group showed that the pneumococcal surface protein A from Streptococcus pneumoniae (pneumococcus), PspA, when combined with cellular pertussis vaccine (wP) as adjuvant, induce high levels of antibodies and mice protection on pneumococcal challenge. In this work were used mutants and purified components derived from B. pertussis for investigate the components responsible for this adjuvant effect, through of mice immunizations in combination with the PspA5 (Clade 5 PspA) antigen. Pertussis toxin (PT) was able to inducing the higher levels of PspA5 specific antibodies and significative protection against the pneumococcal lethal challenge, independently of its enzymatic effect. Was observed that in the protection conferred by the PspA5-wP formulation thus as the anti-PspA5 antibodies, the complements proteins are essentials. Lastly, prime-boost experiments showed that the cellular vaccines BCG and DTP when combined to PspA5, induced high levels of anti-PspA5 antibodies and protection against invasive challenge with pneumococcus.

La tos convulsa es una enfermedad aguda, respiratoria y altamente contagiosa que a pesar de ser inmunoprevenible, hoy en día es considerada una enfermedad resurgente. Surge entonces la necesidad de aplicar refuerzos de inmunización de forma de prolongar la duración de la inmunidad contra la enfermedad, así como también de mejorar las vacunas actuales de manera tal que no solo ofrezcan una protección duradera en el tiempo sino que también protejan contra cepas circulantes antigénicamente distintas. Es por esto que la inclusión de antígenos expresados por cepas circulantes en las formulaciones vacunales podría ofrecer una mejora en el control de esta enfermedad Por otra parte, ante las vacunas parenterales convencionales, una vacuna mucosal podría ser una alternativa válida para mejorar las vacunas existentes, ya que imita a la infección natural y puede prevenir la colonización de B. pertussis mediante la generación de anticuerpos en la mucosa. Sin embargo, la inducción de una respuesta inmune protectora es difícil de lograr mediante inmunización por vía mucosal con antígenos recombinantes solubles. Es por ésto que el uso correcto de adyuvantes es fundamental a la hora de diseñar vacunas recombinantes administradas por vía de mucosas. En este sentido, se ha demostrado que la subunidad B de la toxina colérica es un potente inmunoestimulador que puede actuar como adyuvante estimulando la respuesta inmune sistémica y de mucosas contra antígenos administrados por vía mucosal, además de ser seguro para su uso en humanos. Con el objetivo de contribuir al desarrollo de una vacuna recombinante acelular para la mejora de la prevención de la infección por B. pertussis, el objetivo general de este trabajo fue generar herramientas moleculares que resulten útiles en el control de la infección causada por B. pertussis. Nuestro trabajo contempló la identificación, caracterización, y la validación de proteínas de B. pertussis como candidatos vacunales así como el estudio de la respuesta del huésped a dichas proteínas. Nuestra hipótesis es que proteínas recombinantes de B. pertussis fusionadas con CTB son buenos candidatos vacunales para ser utilizados por vía mucosal.

Bordetella pertussis est l’agent responsable de la coqueluche. Pour coloniser le tractus respiratoire humain, cette bactérie à Gram négatif, aérobie stricte, produit de nombreux facteurs de virulence dont l’expression est sous la dépendance du système à deux composants BvgAS. BvgS est un senseur-kinase dimérique. Chaque monomère est constitué de trois domaines putatifs de perception - deux domaines Venus flytrap périplasmiques et un domaine PAS cytoplasmique -, suivis du domaine enzymatique et deux autres domaines, de phospho-transfert et receveur, impliqués dans la cascade de phosphorylation. L’expression du régulon de virulence est activée suite à la phosphorylation par BvgS du régulateur de réponse BvgA. BvgS est en mode kinase à l’état basal, et la perception de basses températures ou de signaux chimiques comme les ions sulfate ou nicotinate cause son passage en mode phosphatase. L’étude présentée dans ce manuscrit vise à caractériser le senseur-kinase BvgS en analysant les domaines putatifs de perception ainsi que la transduction de signal qui s’effectue au sein de la molécule. L’étude de la portion périplasmique a permis de mettre en évidence, à l’état basal, un gradient de dynamique décroissant. En se fixant au domaine VFT2 proximal à la membrane, le nicotinate induirait une diminution de la dynamique du second lobe du VFT1, causée par la formation d’un bloc compact entre le domaine VFT2 et le deuxième lobe du domaine VFT1. Cette rigidification exercerait une tension sur les hélices α qui précèdent les segments transmembranaires, provoquant une transition de la portion cytoplasmique vers l’état phosphatase. La perception de modulateurs par le domaine VFT2 - ou possiblement la fixation d’un ligand dans la cavité du VFT1- modifierait cette dynamique et causerait le changement d’activité de BvgS. Ainsi, nous proposons un modèle dans lequel le VFT1 est considéré comme le moteur du système, lui impulsant une dynamique qui serait relayée ou atténuée par le domaine VFT2. Une recherche de ligands antagonistes pour le domaine VFT1 a été entreprise, selon l’idée que la fixation d’un ligand réduirait la dynamique de ce dernier. Au sein du dimère, des connecteurs prédits pour former des enroulements d’hélices α (‘coiled coil’) relient entre eux les domaines VFT et PAS, et les domaines PAS et kinase de BvgS. La transduction d’information entre les domaines périplasmiques et le site enzymatique de BvgS a été analysée par mutagénèse dirigée et ‘cysteine scanning’. Des contacts proches sont observés entre les hélices constituant le segment transmembranaire, qui ne semblent pas être modifiés après perception de modulateur. Nous suggérons donc un modèle de piston symétrique pour la transmission d’information au travers de la membrane. Le coiled coil putatif précédant le domaine PAS présente une certaine dynamique rotationnelle à l’état basal. La perception de modulateurs semble induire l’écartement de ces hélices, ce qui pourrait permettre un changement de l’interface des domaines PAS. L’étude de la topologie du domaine PAS confirme une modification de cette interface entre les modes kinase et phosphatase de BvgS. Enfin, le coiled coil reliant les domaines PAS et kinase est sujet à une forte dynamique rotationnelle à l’état basal, en accord avec un modèle de régulation de l’activité kinase proposé dans d’autres systèmes. Suite à la perception de modulateur, une rigidification marquée de ce coiled coil est observée, permettant le passage en mode phosphatase. L’existence de deux états dynamiques différents de ce coiled coil a également été mise en évidence en absence du domaine PAS.Ces études ont permis d’avancer dans la compréhension de BvgS et de proposer un modèle de la signalisation au sein de ce senseur-kinase, qui pourrait s’appliquer aux autres membres de la famille de BvgS. / Bordetella pertussis is the agent of an acute and highly contagious respiratory disease, whooping cough. In order to colonize the human respiratory tract, this strictly aerobic Gram negative bacterium produces many virulence factors, the expression of which is regulated by the BvgAS two-component system. BvgS is a sensor-kinase composed of three putative domains of perception –two periplasmic Venus flytrap domains and a cytoplasmic PAS domain -, followed by the enzymatic domain and two other domains called phosphotransfert and receiver involved in the phophorelay. The expression of the virulence regulon is activated after the phosphorylation by BvgS of the response regulator BvgA. BvgS is in a kinase mode at the basal state, and the perception of low temperatures or chemical signals like sulfate ions or nicotinate causes a shift to the phosphatase state. The study presented in this manuscript has focused on the characterization of the BvgS sensor-kinase. We have analyzed its putative domains of perception and the mechanisms of signal transduction.Investigations into the dynamics of the periplasmic moiety has provided evidence for a decreasing gradient of dynamics from N to C-terminus at the basal state. Nicotinate binding to the membrane-proximal VFT2 domains decreases the dynamics of the second lobe of VFT1. Tighter interactions between the latter and the VFT2 domain cause a tension on the α helices that precede the transmembrane segments, triggering the transition to the phosphatase state of the enzymatic portion. Perception of modulator by the VFT2 domains –or possibly binding of a ligand in the VFT1 cavity- thus appears to modify periplasmic dynamics, which shifts BvgS activity. We propose that the VFT1 domains are the motor for BvgS activity, and their dynamics are relayed or attenuated by the VFT2 domains. A search for antagonistic VFT1 ligands has been undertaken, along the idea that ligand binding may reduce their dynamics.The VFT and PAS domains, and the PAS and kinase domains are joined to each other by long α helices predicted to form coiled coils. We performed directed mutagenesis and cysteine scanning analyses to decipher signal transduction between the periplasmic domains and the enzymatic moiety of BvgS. The close contacts between the helices of the transmembrane segment are not modified after perception of the modulator, suggesting that signal transduction across the membrane is mediated by symmetrical piston motions. The putative coiled coil before the PAS domain shows rotational dynamics at the basal state. Modulator perception causes the helices to splay, and this motion may modify the PAS domains interface. Our topology analyses of the PAS domain confirm that changes occur at this interface between the kinase and phosphatase states of BvgS. Finally, the coiled coil between the PAS and kinase domains presents a strong rotational dynamics at the basal state, which is consistent with the model of regulation of kinase activity proposed for other sensor-kinases. After perception of a modulator, this coiled coil becomes more rigid, allowing the shift to the phosphatase state. The occurrence of two states of dynamics for this coiled coil has also been demonstrated in the absence of the PAS domain.These studies have advanced our understanding of BvgS and allow us to propose a model of signaling by this sensor-kinase, which may apply more broadly to other family members.

Bordetella pertussis, l’agent responsable de la coqueluche, provoque près de 200000 morts par an dans le monde. Malgré une forte couverture vaccinale, une réémergence de la maladie a été observée dans les pays développés, liée en partie à une adaptation à la pression vaccinale. Les souches capables d’échapper à la réponse immunitaire montrent des réarrangements génomiques importants, provoqués par des éléments génétiques mobiles (IS) présents en plus de 230 copies, qui pourraient impacter sur la transcription des gènes et ARN régulateurs du pathogène.Pour élucider l’impact des IS sur le transcriptome global de Bordetella pertussis, nous avons d’abord déterminé le transcriptome codant et non-codant du pathogène par des approches de séquençage d’ARN couplées à des prédictions bioinformatiques. Cette étude a permis d’identifier les structures codantes mono- et polycistroniques, incluant des structures régulatrices telles que des riboswitches, un excludon et de longs 5’ et 3’UTR chevauchants. Une cartographie de candidats ARN régulateurs non-codant a été édifiée à partir de nouveaux transcrits localisés en régions intergéniques (IGR) et de transcrits orientés en antisens de séquences codantes. Des prolongements de transcriptions des IS ont été observés, prenant leur origine de promoteurs internes à l’IS ou formés par insertion de celles-ci. Ces transcrits sont spécifiques d’une souche à l’autre du pathogène, et s’orientent en sens ou en antisens des gènes environnant, ou dans des IGR. Le potentiel caractère régulateur de ces transcrits a été étudié par la caractérisation et l’étude du mode d’action d’un ARN régulateur, BPnc264, orienté en antisens du gène de virulence fim2. / Bordetella pertussis, the causative agent of whooping cough, is responsible of more than 200000 deaths worldwide. Despite a high vaccine coverage in developed countries, a reemergence of the disease has been observed, which is in part linked to vaccine-pressure. Strains able to evade vaccine-induced immunity show high genome organization rearrangements, essentially due to mobile genetic elements (IS) present in more than 230 copies, which could impact on messenger and regulatory transcription of the pathogene.To assess the impact of IS on the global transcriptome of Bordetella pertussis, we first determined the coding and non-coding primary transcriptome by a combination of differents RNA-sequencing approaches and predictive bioinformatics analysis software packages. We identify mono- and polycistronic coding structures, including regulatories structures like riboswitches, excludon, and long overlapping 5’ and 3’UTR. A list of candidates regulating transcripts (small RNA) has been mapped from new transcripts located in intergenic regions (IGR) and transcripts oriented in antisense of annotated coding sequences. Extended transcriptions emerging from IS elements have been observed, originating from internal promoters or newly formed promoters by insertion in a specific genomic region. Those transcripts can extend in sense or in antisense of the flanking gene, or in IGR. The regulatory function of those transcripts has been studied from the characterization and the mode of action of an extended regulatory RNA, BPnc264, oriented in antisense of the virulence gene fim2.

Em trabalhos anteriores o nosso grupo mostrou que a vacina celular pertussis (wP) apresenta atividade adjuvante quando combinada a proteína A da superfície de Streptococcus pneumoniae (pneumococo), PspA, A formulação PspA5-wP induz altos níveis de anticorpos e proteção em camundongos após desafio com pneumococo. Neste trabalho foram avaliados os mecanismos da ação adjuvante e os componentes de B. pertussis responsáveis por este efeito. A imunização de camundongos com PspA em combinação a mutantes de B. pertussis ou componentes desta bactéria purificados mostrou que a presença da toxina pertussis (PT) capaz é essencial para induzir altos níveis de anticorpos anti-PspA e proteção significativa contra o desafio letal com pneumococo. Este efeito não foi dependente da atividade enzimática. Anticorpos anti-PspA e as proteínas do sistema complemento foram essenciais para a proteção conferida por PspA-wP. As vacinas celulares BCG e DTP combinadas a PspA, também apresentaram atividade adjuvante, induzindo anticorpos anti-PspA e proteção contra o desafio invasivo por pneumococo. / Previously our group showed that the pneumococcal surface protein A from Streptococcus pneumoniae (pneumococcus), PspA, when combined with cellular pertussis vaccine (wP) as adjuvant, induce high levels of antibodies and mice protection on pneumococcal challenge. In this work were used mutants and purified components derived from B. pertussis for investigate the components responsible for this adjuvant effect, through of mice immunizations in combination with the PspA5 (Clade 5 PspA) antigen. Pertussis toxin (PT) was able to inducing the higher levels of PspA5 specific antibodies and significative protection against the pneumococcal lethal challenge, independently of its enzymatic effect. Was observed that in the protection conferred by the PspA5-wP formulation thus as the anti-PspA5 antibodies, the complements proteins are essentials. Lastly, prime-boost experiments showed that the cellular vaccines BCG and DTP when combined to PspA5, induced high levels of anti-PspA5 antibodies and protection against invasive challenge with pneumococcus.