RESUMEN
Bordetella pertussis is a Gram-negative bacterium that is the causative agent of the respiratory disease known as pertussis. Since the switch to the acellular vaccines of DTaP and Tap, pertussis cases in the US have risen and cyclically fallen. We have observed that mRNA pertussis vaccines are immunogenic and protective in mice. Here, we further evaluated the pertussis toxoid mRNA antigen and refined the formulation based on optimal pertussis toxin neutralization in vivo. We next evaluated the mRNA pertussis vaccine in Sprague-Dawley rats using an aerosol B. pertussis challenge model paired with whole-body plethysmography to monitor coughing and respiratory function. Female Sprague-Dawley rats were primed and boosted with either commercially available vaccines (DTaP or wP-DTP), an mRNA-DTP vaccine, or mock-vaccinated. The mRNA-DTP vaccine was immunogenic in rats and induced antigen-specific IgG antibodies comparable to DTaP. Rats were then aerosol challenged with a streptomycin-resistant emerging clinical isolate D420Sm1. Bacterial burden was assessed at days 1 and 9 post-challenge, and the mRNA vaccine reduced burden equal to both DTaP and wP-DTP. Whole-body plethysmography revealed that mRNA-DTP vaccinated rats were well protected against coughing which was comparable to the non-challenged group. These data suggest that an mRNA-DTP vaccine is immunogenic in rats and provides protection against aerosolized B. pertussis challenge in Sprague-Dawley rats.
Asunto(s)
Bordetella pertussis , Ratas Sprague-Dawley , Tos Ferina , Animales , Tos Ferina/prevención & control , Tos Ferina/inmunología , Femenino , Ratas , Bordetella pertussis/inmunología , Bordetella pertussis/genética , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Vacuna contra Difteria, Tétanos y Tos Ferina/inmunología , Vacuna contra Difteria, Tétanos y Tos Ferina/administración & dosificación , Inmunoglobulina G/sangre , Vacunas de ARNm , InmunizaciónRESUMEN
The protection afforded by acellular pertussis vaccines wanes over time, and there is a need to develop improved vaccine formulations. Options to improve the vaccines involve the utilization of different adjuvants and administration via different routes. While intramuscular (IM) vaccination provides a robust systemic immune response, intranasal (IN) vaccination theoretically induces a localized immune response within the nasal cavity. In the case of a Bordetella pertussis infection, IN vaccination results in an immune response that is similar to natural infection, which provides the longest duration of protection. Current acellular formulations utilize an alum adjuvant, and antibody levels wane over time. To overcome the current limitations with the acellular vaccine, we incorporated a novel TLR4 agonist, BECC438b, into both IM and IN acellular formulations to determine its ability to protect against infection in a murine airway challenge model. Following immunization and challenge, we observed that DTaP + BECC438b reduced bacterial burden within the lung and trachea for both administration routes when compared with mock-vaccinated and challenged (MVC) mice. Interestingly, IN administration of DTaP + BECC438b induced a Th1-polarized immune response, while IM vaccination polarized toward a Th2 immune response. RNA sequencing analysis of the lung demonstrated that DTaP + BECC438b activates biological pathways similar to natural infection. Additionally, IN administration of DTaP + BECC438b activated the expression of genes involved in a multitude of pathways associated with the immune system. Overall, these data suggest that BECC438b adjuvant and the IN vaccination route can impact efficacy and responses of pertussis vaccines in pre-clinical mouse models.
Asunto(s)
Vacunas contra Difteria, Tétanos y Tos Ferina Acelular , Tos Ferina , Animales , Ratones , Tos Ferina/prevención & control , Receptor Toll-Like 4 , Vacuna contra la Tos Ferina , Vacuna contra Difteria, Tétanos y Tos Ferina , Bordetella pertussis , Adyuvantes Inmunológicos , Inmunidad , Anticuerpos AntibacterianosRESUMEN
Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.
Asunto(s)
Adhesinas Bacterianas , Anticuerpos Antibacterianos , Bordetella pertussis , Vacuna contra la Tos Ferina , Vacunas de Partículas Similares a Virus , Tos Ferina , Animales , Bordetella pertussis/inmunología , Ratones , Tos Ferina/prevención & control , Tos Ferina/inmunología , Vacuna contra la Tos Ferina/inmunología , Vacuna contra la Tos Ferina/administración & dosificación , Anticuerpos Antibacterianos/inmunología , Adhesinas Bacterianas/inmunología , Adhesinas Bacterianas/genética , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Femenino , Ratones Endogámicos BALB C , Factores de Virulencia de Bordetella/inmunología , Infecciones del Sistema Respiratorio/prevención & control , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/microbiologíaRESUMEN
SARS-CoV-2 variants of concern (VoC) are impacting responses to the COVID-19 pandemic. Here, we utilized passive immunization using human convalescent plasma (HCP) obtained from a critically ill COVID-19 patient in the early pandemic to study the efficacy of polyclonal antibodies generated to ancestral SARS-CoV-2 against the Alpha, Beta, and Delta VoC in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model. HCP protected mice from challenge with the original WA-1 SARS-CoV-2 strain; however, only partially protected mice challenged with the Alpha VoC (60% survival) and failed to save Beta challenged mice from succumbing to disease. HCP treatment groups had elevated receptor binding domain (RBD) and nucleocapsid IgG titers in the serum; however, Beta VoC viral RNA burden in the lung and brain was not decreased due to HCP treatment. While mice could be protected from WA-1 or Alpha challenge with a single dose of HCP, six doses of HCP could not decrease mortality of Delta challenged mice. Overall, these data demonstrate that VoC have enhanced immune evasion and this work underscores the need for in vivo models to evaluate future emerging strains. IMPORTANCE Emerging SARS-CoV-2 VoC are posing new problems regarding vaccine and monoclonal antibody efficacy. To better understand immune evasion tactics of the VoC, we utilized passive immunization to study the effect of early-pandemic SARS-CoV-2 HCP against, Alpha, Beta, and Delta VoC. We observed that HCP from a human infected with the original SARS-CoV-2 was unable to control lethality of Alpha, Beta, or Delta VoC in the K18-hACE2 transgenic mouse model of SARS-CoV-2 infection. Our findings demonstrate that passive immunization can be used as a model to evaluate immune evasion of emerging VoC strains.
Asunto(s)
COVID-19/terapia , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/genética , Animales , Anticuerpos Neutralizantes/inmunología , COVID-19/prevención & control , Modelos Animales de Enfermedad , Humanos , Inmunización Pasiva , Melfalán , Ratones , Ratones Transgénicos , SARS-CoV-2/genética , SARS-CoV-2/inmunología , gammaglobulinas , Sueroterapia para COVID-19RESUMEN
BACKGROUND: Lung airway epithelial cells are part of innate immunity and the frontline of defense against bacterial infections. During infection, airway epithelial cells secrete proinflammatory mediators that participate in the recruitment of immune cells. Virulence factors expressed by bacterial pathogens can alter epithelial cell gene expression and modulate this response. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, expresses numerous virulence factors to facilitate establishment of infection and evade the host immune response. This study focused on identifying the role of two major P. aeruginosa virulence factors, type III (T3SS) and type VI (T6SS) secretion systems, on the early transcriptome response of airway epithelial cells in vitro. RESULTS: We performed RNA-seq analysis of the transcriptome response of type II pneumocytes during infection with P. aeruginosa in vitro. We observed that P. aeruginosa differentially upregulates immediate-early response genes and transcription factors that induce proinflammatory responses in type II pneumocytes. P. aeruginosa infection of type II pneumocytes was characterized by up-regulation of proinflammatory networks, including MAPK, TNF, and IL-17 signaling pathways. We also identified early response genes and proinflammatory signaling pathways whose expression change in response to infection with P. aeruginosa T3SS and T6SS mutants in type II pneumocytes. We determined that T3SS and T6SS modulate the expression of EGR1, FOS, and numerous genes that are involved in proinflammatory responses in epithelial cells during infection. T3SS and T6SS were associated with two distinct transcriptomic signatures related to the activation of transcription factors such as AP1, STAT1, and SP1, and the secretion of pro-inflammatory cytokines such as IL-6 and IL-8. CONCLUSIONS: Taken together, transcriptomic analysis of epithelial cells indicates that the expression of immediate-early response genes quickly changes upon infection with P. aeruginosa and this response varies depending on bacterial viability and injectosomes. These data shed light on how P. aeruginosa modulates host epithelial transcriptome response during infection using T3SS and T6SS.
Asunto(s)
Pseudomonas aeruginosa , Sistemas de Secreción Tipo VI , Células Epiteliales Alveolares/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/genética , Factores de Transcripción/metabolismo , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo VI/genética , Factores de Virulencia/genéticaRESUMEN
Despite high vaccine coverage in many parts of the world, pertussis is resurging in a number of areas in which acellular vaccines are the primary vaccine administered to infants and young children. This is attributed in part to the suboptimal and short-lived immunity elicited by acellular pertussis vaccines and to their inability to prevent nasal colonization and transmission of the etiologic agent Bordetella pertussis In response to this escalating public health concern, the National Institute of Allergy and Infectious Diseases held the workshop "Overcoming Waning Immunity in Pertussis Vaccines" in September 2019 to identify issues and possible solutions for the defects in immunity stimulated by acellular pertussis vaccines. Discussions covered aspects of the current problem, gaps in knowledge and possible paths forward. This review summarizes presentations and discussions of some of the key points that were raised by the workshop.
Asunto(s)
Vacuna contra la Tos Ferina/inmunología , Tos Ferina/inmunología , Animales , Bordetella pertussis/inmunología , Humanos , National Institute of Allergy and Infectious Diseases (U.S.) , Estados Unidos , Vacunas Acelulares/inmunologíaRESUMEN
Pertussis is a respiratory disease caused by the Gram-negative pathogen, Bordetella pertussis. The transition from a whole-cell pertussis vaccine (wP and DTP) to an acellular pertussis vaccine (aP, DTaP, and Tdap) correlates with an increase in pertussis cases, despite widespread vaccine implementation and coverage, and it is now appreciated that the protection provided by aP rapidly wanes. To recapitulate the localized immunity observed from natural infection, mucosal vaccination with aP was explored using the coughing rat model of pertussis. Overall, our goal was to evaluate the route of vaccination in the coughing rat model of pertussis. Immunity induced by both oral gavage and intranasal vaccination of aP in B. pertussis challenged rats over a 9-day infection was compared to intramuscular wP (IM-wP)- and IM-aP-immunized rats that were used as positive controls. Our data demonstrate that mucosal immunization of aP resulted in the production of anti-B. pertussis IgG antibody titers similar to IM-wP- and IM-aP-vaccinated controls postchallenge. IN-aP also induced anti-B. pertussis IgA antibodies in the nasal cavity. Immunization with IM-wP, IM-aP, IN-aP, and OG-aP immunization protected against B. pertussis-induced cough, whereas OG-aP immunization did not protect against respiratory distress. Mucosal immunization by both intranasal and oral gavage administration protected against acute inflammation and decreased bacterial burden in the lung compared to mock-vaccinated challenge rats. The data presented in this study suggest that mucosal vaccination with aP can induce a mucosal immune response and provide protection against B. pertussis challenge. This study highlights the potential benefits and uses of the coughing rat model of pertussis; however, further questions regarding waning immunity still require additional investigation.
Asunto(s)
Bordetella pertussis/inmunología , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/inmunología , Inmunidad Mucosa , Tos Ferina/prevención & control , Animales , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/administración & dosificación , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno/inmunología , Inmunización , Ratas , Ratas Sprague-Dawley , Tos Ferina/inmunologíaRESUMEN
Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe pulmonary infections associated with high morbidity and mortality in immunocompromised patients. The development of a vaccine against P. aeruginosa could help prevent infections caused by this highly antibiotic-resistant microorganism. We propose that identifying the vaccine-induced correlates of protection against P. aeruginosa will facilitate the development of a vaccine against this pathogen. In this study, we investigated the mechanistic correlates of protection of a curdlan-adjuvanted P. aeruginosa whole-cell vaccine (WCV) delivered intranasally. The WCV significantly decreased bacterial loads in the respiratory tract after intranasal P. aeruginosa challenge and raised antigen-specific antibody titers. To study the role of B and T cells during vaccination, anti-CD4, -CD8, and -CD20 depletions were performed prior to WCV vaccination and boosting. The depletion of CD4+, CD8+, or CD20+ cells had no impact on the bacterial burden in mock-vaccinated animals. However, depletion of CD20+ B cells, but not CD8+ or CD4+ T cells, led to the loss of vaccine-mediated bacterial clearance. Also, passive immunization with serum from WCV group mice alone protected naive mice against P. aeruginosa, supporting the role of antibodies in clearing P. aeruginosa We observed that in the absence of T cell-dependent antibody production, mice vaccinated with the WCV were still able to reduce bacterial loads. Our results collectively highlight the importance of the humoral immune response for protection against P. aeruginosa and suggest that the production of T cell-independent antibodies may be sufficient for bacterial clearance induced by whole-cell P. aeruginosa vaccination.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Antibacterianos/inmunología , Neumonía Bacteriana/prevención & control , Infecciones por Pseudomonas/prevención & control , Vacunas contra la Infección por Pseudomonas/administración & dosificación , Vacunas contra la Infección por Pseudomonas/inmunología , Animales , Humanos , Inmunización , Ratones , Modelos Animales , Neumonía Bacteriana/fisiopatología , Infecciones por Pseudomonas/fisiopatología , VacunaciónRESUMEN
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.
Asunto(s)
Bordetella pertussis/fisiología , Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno , Tos Ferina/etiología , Animales , Biomarcadores , Bordetella pertussis/patogenicidad , Modelos Animales de Enfermedad , Ratas , Tos Ferina/metabolismo , Tos Ferina/patologíaRESUMEN
Bordetella pertussis colonizes the respiratory mucosa of humans, inducing an immune response seeded in the respiratory tract. An individual, once convalescent, exhibits long-term immunity to the pathogen. Current acellular pertussis (aP) vaccines do not induce the long-term immune response observed after natural infection in humans. In this study, we evaluated the durability of protection from intranasal (i.n.) pertussis vaccines in mice. Mice that convalesced from B. pertussis infection served as a control group. Mice were immunized with a mock vaccine (phosphate-buffered saline [PBS]), aP only, or an aP base vaccine combined with one of the following adjuvants: alum, curdlan, or purified whole glucan particles (IRI-1501). We utilized two study designs: short term (challenged 35 days after priming vaccination) and long term (challenged 6 months after boost). The short-term study demonstrated that immunization with i.n. vaccine candidates decreased the bacterial burden in the respiratory tract, reduced markers of inflammation, and induced significant serum and lung antibody titers. In the long-term study, protection from bacterial challenge mirrored the results observed in the short-term challenge study. Immunization with pertussis antigens alone was surprisingly protective in both models; however, the alum and IRI-1501 adjuvants induced significant B. pertussis-specific IgG antibodies in both the serum and lung and increased numbers of anti-B. pertussis IgG-secreting plasma cells in the bone marrow. Our data indicate that humoral responses induced by the i.n. vaccines correlated with protection, suggesting that long-term antibody responses can be protective.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Bordetella pertussis/inmunología , Vacuna contra la Tos Ferina/administración & dosificación , Vacuna contra la Tos Ferina/inmunología , Tos Ferina/inmunología , Tos Ferina/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Administración Intranasal , Animales , Modelos Animales de Enfermedad , Humanos , Inmunización , Ratones , Factores de Tiempo , VacunaciónRESUMEN
To commemorate the 100th anniversary of the Nobel prize being awarded to Jules Bordet, the discoverer of Bordetella pertussis, the 12th International Bordetella Symposium was held from 9 to 12 April 2019 at the Université Libre de Bruxelles, where Jules Bordet studied and was Professor of Microbiology. The symposium attracted more than 300 Bordetella experts from 34 countries. They discussed the latest epidemiologic data and clinical aspects of pertussis, Bordetella biology and pathogenesis, immunology and vaccine development, and genomics and evolution. Advanced technological and methodological tools provided novel insights into the genomic diversity of Bordetella and a better understanding of pertussis disease and vaccine performance. New molecular approaches revealed previously unrecognized complexity of virulence gene regulation. Innovative insights into the immune responses to infection by Bordetella resulted in the development of new vaccine candidates. Such discoveries will aid in the design of more effective approaches to control pertussis and other Bordetella-related diseases.
Asunto(s)
Bordetella pertussis , Tos Ferina , Bordetella pertussis/genética , Genómica , Humanos , Vacuna contra la Tos Ferina , Virulencia , Tos Ferina/epidemiologíaRESUMEN
Bordetella pertussis is the primary causative agent of pertussis (whooping cough), which is a respiratory infection that leads to a violent cough and can be fatal in infants. There is a need to develop more effective vaccines because of the resurgence of cases of pertussis in the United States since the switch from the whole-cell pertussis vaccines (wP) to the acellular pertussis vaccines (aP; diphtheria-tetanus-acellular-pertussis vaccine/tetanus-diphtheria-pertussis vaccine). Adenylate cyclase toxin (ACT) is a major virulence factor of B. pertussis that is (i) required for establishment of infection, (ii) an effective immunogen, and (iii) a protective antigen. The C-terminal repeats-in-toxin domain (RTX) of ACT is sufficient to induce production of toxin-neutralizing antibodies. In this study, we characterized the effectiveness of vaccines containing the RTX antigen against experimental murine infection with B. pertussis RTX was not protective as a single-antigen vaccine against B. pertussis challenge, and adding RTX to 1/5 human dose of aP did not enhance protection. Since the doses of aP used in murine studies are not proportionate to mouse/human body masses, we titrated the aP from 1/20 to 1/160 of the human dose. Mice receiving 1/80 human aP dose had bacterial burden comparable to those of naive controls. Adding RTX antigen to the 1/80 aP base resulted in enhanced bacterial clearance. Inclusion of RTX induced production of antibodies recognizing RTX, enhanced production of anti-pertussis toxin, decreased secretion of proinflammatory cytokines, such as interleukin-6, and decreased recruitment of total macrophages in the lung. This study shows that adding RTX antigen to an appropriate dose of aP can enhance protection against B. pertussis challenge in mice.
Asunto(s)
Adenilil Ciclasas/inmunología , Bordetella pertussis/inmunología , Vacuna contra la Tos Ferina/inmunología , Toxoides/inmunología , Tos Ferina/inmunología , Adenilil Ciclasas/administración & dosificación , Adenilil Ciclasas/genética , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Bordetella pertussis/genética , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Vacuna contra la Tos Ferina/administración & dosificación , Vacuna contra la Tos Ferina/genética , Toxoides/administración & dosificación , Toxoides/genética , Tos Ferina/microbiologíaRESUMEN
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that requires iron for virulence. Iron homeostasis is maintained in part by the PrrF1 and PrrF2 small RNAs (sRNAs), which block the expression of iron-containing proteins under iron-depleted conditions. The PrrF sRNAs also promote the production of the Pseudomonas quinolone signal (PQS), a quorum sensing molecule that activates the expression of several virulence genes. The tandem arrangement of the prrF genes allows for expression of a third sRNA, PrrH, which is predicted to regulate gene expression through its unique sequence derived from the prrF1-prrF2 intergenic (IG) sequence (the PrrHIG sequence). Previous studies showed that the prrF locus is required for acute lung infection. However, the individual functions of the PrrF and PrrH sRNAs were not determined. Here, we describe a system for differentiating PrrF and PrrH functions by deleting the PrrHIG sequence [prrF(ΔHIG)]. Our analyses of this construct indicate that the PrrF sRNAs, but not PrrH, are required for acute lung infection by P. aeruginosa Moreover, we show that the virulence defect of the ΔprrF1-prrF2 mutant is due to decreased bacterial burden during acute lung infection. In vivo analysis of gene expression in lung homogenates shows that PrrF-mediated regulation of genes for iron-containing proteins is disrupted in the ΔprrF1-prrF2 mutant during infection, while the expression of genes that mediate PrrF-regulated PQS production are not affected by prrF deletion in vivo Combined, these studies demonstrate that regulation of iron utilization plays a critical role in P. aeruginosa's ability to survive during infection.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Genes Bacterianos , Homeostasis , Pulmón/microbiología , Ratones , Viabilidad Microbiana , ARN Interferente Pequeño/genética , Eliminación de Secuencia , VirulenciaRESUMEN
Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions.
Asunto(s)
Toxina de Adenilato Ciclasa/genética , Bordetella pertussis/genética , Bordetella pertussis/patogenicidad , Toxina del Pertussis/genética , Factor sigma/genética , Factores de Virulencia de Bordetella/genética , Virulencia/genética , Animales , Femenino , Expresión Génica/genética , Ratones , Proteómica/métodos , Factores de Transcripción/genética , Tos Ferina/microbiologíaRESUMEN
BACKGROUND: Pseudomonas aeruginosa is an environmentally ubiquitous Gram-negative bacterium and important opportunistic human pathogen, causing severe chronic respiratory infections in patients with underlying conditions such as cystic fibrosis (CF) or bronchiectasis. In order to identify mechanisms responsible for adaptation during bronchiectasis infections, a bronchiectasis isolate, PAHM4, was phenotypically and genotypically characterized. RESULTS: This strain displays phenotypes that have been associated with chronic respiratory infections in CF including alginate over-production, rough lipopolysaccharide, quorum-sensing deficiency, loss of motility, decreased protease secretion, and hypermutation. Hypermutation is a key adaptation of this bacterium during the course of chronic respiratory infections and analysis indicates that PAHM4 encodes a mutated mutS gene responsible for a ~1,000-fold increase in mutation rate compared to wild-type laboratory strain P. aeruginosa PAO1. Antibiotic resistance profiles and sequence data indicate that this strain acquired numerous mutations associated with increased resistance levels to ß-lactams, aminoglycosides, and fluoroquinolones when compared to PAO1. Sequencing of PAHM4 revealed a 6.38 Mbp genome, 5.9 % of which were unrecognized in previously reported P. aeruginosa genome sequences. Transcriptome analysis suggests a general down-regulation of virulence factors, while metabolism of amino acids and lipids is up-regulated when compared to PAO1 and metabolic modeling identified further potential differences between PAO1 and PAHM4. CONCLUSIONS: This work provides insights into the potential differential adaptation of this bacterium to the lung of patients with bronchiectasis compared to other clinical settings such as cystic fibrosis, findings that should aid the development of disease-appropriate treatment strategies for P. aeruginosa infections.
Asunto(s)
Bronquiectasia/microbiología , Fibrosis Quística/complicaciones , Genotipo , Fenotipo , Infecciones por Pseudomonas/etiología , Pseudomonas aeruginosa/fisiología , Adaptación Biológica/genética , Alelos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Enfermedad Crónica , Biología Computacional , Farmacorresistencia Bacteriana , Perfilación de la Expresión Génica , Orden Génico , Genoma Bacteriano , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación , Tasa de Mutación , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/patogenicidad , Percepción de Quorum/genética , Metabolismo Secundario , Transcriptoma , Virulencia/genéticaRESUMEN
Acellular multivalent vaccines for pertussis (DTaP and Tdap) prevent symptomatic disease and infant mortality, but immunity to Bordetella pertussis infection wanes significantly over time resulting in cyclic epidemics of pertussis. The messenger RNA (mRNA) vaccine platform provides an opportunity to address complex bacterial infections with an adaptable approach providing Th1-biased responses. In this study, immunogenicity and challenge models were used to evaluate the mRNA platform with multivalent vaccine formulations targeting both B. pertussis antigens and diphtheria and tetanus toxoids. Immunization with mRNA formulations were immunogenetic, induced antigen specific antibodies, as well as Th1 T cell responses. Upon challenge with either historical or contemporary B. pertussis strains, 6 and 10 valent mRNA DTP vaccine provided protection equal to that of 1/20th human doses of either DTaP or whole cell pertussis vaccines. mRNA DTP immunized mice were also protected from pertussis toxin challenge as measured by prevention of lymphocytosis and leukocytosis. Collectively these pre-clinical mouse studies illustrate the potential of the mRNA platform for multivalent bacterial pathogen vaccines.
RESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that can adapt to changing environments and can secrete an exopolysaccharide known as alginate as a protection response, resulting in a colony morphology and phenotype referred to as mucoid. However, how P. aeruginosa senses its environment and activates alginate overproduction is not fully understood. Previously, we showed that Pseudomonas isolation agar supplemented with ammonium metavanadate (PIAAMV) induces P. aeruginosa to overproduce alginate. Vanadate is a phosphate mimic and causes protein misfolding by disruption of disulfide bonds. Here we used PIAAMV to characterize the pathways involved in inducible alginate production and tested the global effects of P. aeruginosa growth on PIAAMV by a mutant library screen, by transcriptomics, and in a murine acute virulence model. The PA14 nonredundant mutant library was screened on PIAAMV to identify new genes that are required for the inducible alginate stress response. A functionally diverse set of genes encoding products involved in cell envelope biogenesis, peptidoglycan remodeling, uptake of phosphate and iron, phenazine biosynthesis, and other processes were identified as positive regulators of the mucoid phenotype on PIAAMV. Transcriptome analysis of P. aeruginosa cultures growing in the presence of vanadate showed differential expression of genes involved in virulence, envelope biogenesis, and cell stress pathways. In this study, it was observed that growth on PIAAMV attenuates P. aeruginosa in a mouse pneumonia model. Induction of alginate overproduction occurs as a stress response to protect P. aeruginosa, but it may be possible to modulate and inhibit these pathways based on the new genes identified in this study.
Asunto(s)
Alginatos/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Respuesta al Choque Térmico , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Vanadatos/farmacología , Enfermedad Aguda , Agar , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Medios de Cultivo/química , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Vanadatos/metabolismo , Virulencia/genéticaRESUMEN
Pseudomonas aeruginosa, a Gram-negative bacterium, is a significant opportunistic pathogen associated with skin and soft tissue infections, nosocomial pneumonia and sepsis. In addition, it can chronically colonize the lungs of cystic fibrosis (CF) patients. Overproduction of the exopolysaccharide called alginate provides P. aeruginosa with a selective advantage and facilitates survival in the CF lung. The in vitro phenotype of alginate overproduction observed on solid culture media is referred to as mucoid. Expression of the alginate machinery and biosynthetic enzymes are controlled by the extracytoplasmic sigma factor, σ(22) (AlgU/T). The key negative regulator of both σ(22) activity and the mucoid phenotype is the cognate anti-sigma factor MucA. MucA sequesters σ(22) to the inner membrane inhibiting the sigma factor's transcriptional activity. The well-studied mechanism for transition to the mucoid phenotype is mutation of mucA, leading to loss of MucA function and therefore activation of σ(22) . Recently, regulated intramembrane proteolysis (RIP) has been recognized as a mechanism whereby proteolysis of the anti-sigma factor MucA leads to active σ(22) allowing P. aeruginosa to respond to environmental stress conditions by overproduction of alginate. The goal of this review is to illuminate the pathways leading to RIP that have been identified and proposed.
Asunto(s)
Alginatos/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Pseudomonas aeruginosa/metabolismo , Factor sigma/metabolismo , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/metabolismo , Humanos , Proteínas Mutantes/metabolismo , Unión Proteica , ProteolisisRESUMEN
We describe a mini-Tn7-based broad-host-range expression cassette for arabinose-inducible gene expression from the P(BAD) promoter. This delivery vector, pTJ1, can integrate a single copy of a gene into the chromosome of Gram-negative bacteria for diverse genetic applications, of which several are discussed, using Pseudomonas aeruginosa as the model host.