Role of the Putative Histidine Kinase BP1092 in Bordetella pertussis Virulence Regulation and Intracellular Survival.

Bordetella pertussis persists inside host cells, and virulence factors are crucial for intracellular adaptation. The regulation of B. pertussis virulence factor transcription primarily occurs through the modulation of the two-component system (TCS) known as BvgAS. However, additional regulatory systems have emerged as potential contributors to virulence regulation. Here, we investigate the impact of BP1092, a putative TCS histidine kinase that shows increased levels after bacterial internalization by macrophages, on B. pertussis proteome adaptation under nonmodulating (Bvg+) and modulating (Bvg-) conditions. Using mass spectrometry, we compare B. pertussis wild-type (wt), a BP1092-deficient mutant (ΔBP1092), and a ΔBP1092 trans-complemented strain under both conditions. We find an altered abundance of 10 proteins, including five virulence factors. Specifically, under nonmodulating conditions, the mutant strain showed decreased levels of FhaB, FhaS, and Cya compared to the wt. Conversely, under modulating conditions, the mutant strain exhibited reduced levels of BvgA and BvgS compared to those of the wt. Functional assays further revealed that the deletion of BP1092 gene impaired B. pertussis ability to survive within human macrophage THP-1 cells. Taken together, our findings allow us to propose BP1092 as a novel player involved in the intricate regulation of B. pertussis virulence factors and thus in adaptation to the intracellular environment. The data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD041940.

Bordetella filamentous hemagglutinin and adenylate cyclase toxin interactions on the bacterial surface are consistent with FhaB-mediated delivery of ACT to phagocytic cells.

Bordetella species that cause respiratory infections in mammals include B. pertussis, which causes human whooping cough, and B. bronchiseptica, which infects nearly all mammals. Both bacterial species produce filamentous hemagglutinin (FhaB) and adenylate cyclase toxin (ACT), prominent surface-associated and secreted virulence factors that contribute to persistence in the lower respiratory tract by inhibiting clearance by phagocytic cells. FhaB and ACT proteins interact with themselves, each other, and host cells. Using immunoblot analyses, we showed that ACT binds to FhaB on the bacterial surface before it can be detected in culture supernatants. We determined that SphB1, a surface protease identified based on its requirement for FhaB cleavage, is also required for ACT cleavage, and we determined that the presence of ACT blocks SphB1-dependent and -independent cleavage of FhaB, but the presence of FhaB does not affect SphB1-dependent cleavage of ACT. The primary SphB1-dependent cleavage site on ACT is proximal to ACT's active site, in a region that is critical for ACT activity. We also determined that FhaB-bound ACT on the bacterial surface can intoxicate host cells producing CR3, the receptor for ACT. In addition to increasing our understanding of FhaB, ACT, and FhaB-ACT interactions on the Bordetella surface, our data are consistent with a model in which FhaB functions as a novel toxin delivery system by binding to ACT and allowing its release upon binding of ACT to its receptor, CR3, on phagocytic cells.IMPORTANCEBacteria need to control the variety, abundance, and conformation of proteins on their surface to survive. Members of the Gram-negative bacterial genus Bordetella include B. pertussis, which causes whooping cough in humans, and B. bronchiseptica, which causes respiratory infections in a broad range of mammals. These species produce two prominent virulence factors, the two-partner secretion (TPS) effector FhaB and adenylate cyclase toxin (ACT), that interact with themselves, each other, and host cells. Here, we determined that ACT binds FhaB on the bacterial surface before being detected in culture supernatants and that ACT bound to FhaB can be delivered to eukaryotic cells. Our data are consistent with a model in which FhaB delivers ACT specifically to phagocytic cells. This is the first report of a TPS system facilitating the delivery of a separate polypeptide toxin to target cells and expands our understanding of how TPS systems contribute to bacterial pathogenesis.

Bordetella pertussis targets the basolateral membrane of polarized respiratory epithelial cells, gets internalized, and survives in intracellular locations.

The airway epithelial barrier is a continuous highly organized cell layer that separates the exterior from the underlying mucosal tissue, preventing pathogen invasion. Several respiratory pathogens have evolved mechanisms to compromise this barrier, invade and even reside alive within the epithelium. Bordetella pertussis is a persistent pathogen that infects the human airway epithelium, causing whooping cough. Previous studies have shown that B. pertussis survives inside phagocytic and nonphagocytic cells, suggesting that there might be an intracellular stage involved in the bacterial infectious process and/or in the pathogen persistence inside the host. In this study we found evidence that B. pertussis is able to survive inside respiratory epithelial cells. According to our results, this pathogen preferentially attaches near or on top of the tight junctions in polarized human bronchial epithelial cells and disrupts these structures in an adenylate cyclase-dependent manner, exposing their basolateral membrane. We further found that the bacterial internalization is significantly higher in cells exposing this membrane compared with cells only exposing the apical membrane. Once internalized, B. pertussis mainly remains in nondegradative phagosomes with access to nutrients. Taken together, these results point at the respiratory epithelial cells as a potential niche of persistence.

Delivery of Mycobacterium tuberculosis epitopes by Bordetella pertussis adenylate cyclase toxoid expands HLA-E-restricted cytotoxic CD8+ T cells.

Introduction: Tuberculosis (TB) remains the first cause of death from infection caused by a bacterial pathogen. Chemotherapy does not eradicate Mycobacterium tuberculosis (Mtb) from human lungs, and the pathogen causes a latent tuberculosis infection that cannot be prevented by the currently available Bacille Calmette Guerin (BCG) vaccine, which is ineffective in the prevention of pulmonary TB in adults. HLA-E-restricted CD8+ T lymphocytes are essential players in protective immune responses against Mtb. Hence, expanding this population in vivo or ex vivo may be crucial for vaccination or immunotherapy against TB. Methods: The enzymatically inactive Bordetella pertussis adenylate cyclase (CyaA) toxoid is an effective tool for delivering peptide epitopes into the cytosol of antigen-presenting cells (APC) for presentation and stimulation of specific CD8+ T-cell responses. In this study, we have investigated the capacity of the CyaA toxoid to deliver Mtb epitopes known to bind HLA-E for the expansion of human CD8+ T cells in vitro. Results: Our results show that the CyaA-toxoid containing five HLA-E-restricted Mtb epitopes causes significant expansion of HLA-E-restricted antigen-specific CD8+ T cells, which produce IFN-γ and exert significant cytotoxic activity towards peptide-pulsed macrophages. Discussion: HLA-E represents a promising platform for the development of new vaccines; our study indicates that the CyaA construct represents a suitable delivery system of the HLA-E-binding Mtb epitopes for ex vivo and in vitro expansion of HLA-E-restricted CD8+ T cells inducing a predominant Tc1 cytokine profile with a significant increase of IFN-γ production, for prophylactic and immunotherapeutic applications against Mtb.

Adenylate cyclase toxin of Bordetella parapertussis disrupts the epithelial barrier granting the bacterial access to the intracellular space of epithelial cells.

B. parapertussis is one of the etiological agents of whooping cough. Once inhaled, the bacteria bind to the respiratory epithelium and start the infection. Little is known about this first step of host colonization and the role of the human airway epithelial barrier on B. parapertussis infection. We here investigated the outcome of the interaction of B. parapertussis with a polarized monolayer of respiratory epithelial cells. Our results show that B. parapertussis preferentially attaches to the intercellular boundaries, and causes the disruption of the tight junction integrity through the action of adenylate cyclase toxin (CyaA). We further found evidence indicating that this disruption enables the bacterial access to components of the basolateral membrane of epithelial cells to which B. parapertussis efficiently attaches and gains access to the intracellular location, where it can survive and eventually spread back into the extracellular environment. Altogether, these results suggest that the adenylate cyclase toxin enables B. parapertussis to overcome the epithelial barrier and eventually establish a niche of persistence within the respiratory epithelial cells.

Bordetella spp. utilize the type 3 secretion system to manipulate the VIP/VPAC2 signaling and promote colonization and persistence of the three classical Bordetella in the lower respiratory tract.

Introduction: Bordetella are respiratory pathogens comprised of three classical Bordetella species: B. pertussis, B. parapertussis, and B. bronchiseptica. With recent surges in Bordetella spp. cases and antibiotics becoming less effective to combat infectious diseases, there is an imperative need for novel antimicrobial therapies. Our goal is to investigate the possible targets of host immunomodulatory mechanisms that can be exploited to promote clearance of Bordetella spp. infections. Vasoactive intestinal peptide (VIP) is a neuropeptide that promotes Th2 anti-inflammatory responses through VPAC1 and VPAC2 receptor binding and activation of downstream signaling cascades. Methods: We used classical growth in vitro assays to evaluate the effects of VIP on Bordetella spp. growth and survival. Using the three classical Bordetella spp. in combination with different mouse strains we were able to evaluate the role of VIP/VPAC2 signaling in the infectious dose 50 and infection dynamics. Finally using the B. bronchiseptica murine model we determine the suitability of VPAC2 antagonists as possible therapy for Bordetella spp. infections. Results: Under the hypothesis that inhibition of VIP/VPAC2 signaling would promote clearance, we found that VPAC2-/- mice, lacking a functional VIP/VPAC2 axis, hinder the ability of the bacteria to colonize the lungs, resulting in decreased bacterial burden by all three classical Bordetella species. Moreover, treatment with VPAC2 antagonists decrease lung pathology, suggesting its potential use to prevent lung damage and dysfunction caused by infection. Our results indicate that the ability of Bordetella spp. to manipulate VIP/VPAC signaling pathway appears to be mediated by the type 3 secretion system (T3SS), suggesting that this might serve as a therapeutical target for other gram-negative bacteria. Conclusion: Taken together, our findings uncover a novel mechanism of bacteria-host crosstalk that could provide a target for the future treatment for whooping cough as well as other infectious diseases caused primarily by persistent mucosal infections.

Multiple rib and vertebral fractures associated with Bordetella pertussis infection: a case report.

BACKGROUND: Pertussis is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis, characterized by paroxysms of severe coughing, and predominantly affects children. We report the first case of multiple fractures in the ribs, lumbar spine, and sacrum associated with severe coughing caused by Bordetella pertussis infection in an adult. CASE PRESENTATION: A 49-year-old female presented with acute-onset chest wall pain for 3 weeks. Imaging results revealed multiple fractures in the ribs and vertebrae, as well as bilateral pleural effusion, pericardial effusion, right pneumothorax, and enlargement of the left parapharyngeal and subclavian lymph nodes. The patient's bone density scan, autoimmune antibodies, bone marrow biopsy, and sacral bone biopsy all came back normal. Imaging test results found no evidence of solid tumors or active TB infection. The patient later recalled having violent coughing prior to the onset of chest pain and several family members having similar symptoms. Her blood sample was sent to the CDC, revealing Bordetella pertussis toxin (PT) IgG titer of 110.68 IU/mL. The patient was diagnosed with pertussis and multiple stress fractures from violent coughing. Symptomatic treatments were administered, and the patient's symptoms improved. The patient was followed up 8 weeks later, she reported no more coughing or chest pain. CONCLUSIONS: Pertussis is not just a pediatric disease, but diagnosis in adults is challenging as patients may present with a myriad of confusing symptoms, such as multiple stress fractures due to violent coughing. Medical and epidemiological histories are key to reaching the correct diagnosis, which is essential for appropriate treatments to avoid further complications. Adult immunization should be suggested both for the protection of the adult population and to prevent transmission to children.

Pasteurian Contributions to the Study of Bordetella pertussis Toxins.

As a tribute to Louis Pasteur on the occasion of the 200th anniversary of his birth, this article summarizes the main contributions of scientists from Pasteur Institutes to the current knowledge of toxins produced by Bordetella pertussis. The article therefore focuses on publications authored by researchers from Pasteur Institutes and is not intended as a systematic review of B. pertussis toxins. Besides identifying B. pertussis as the causative agent of whooping cough, Pasteurians have made several major contributions with respect to the structure-function relationship of the Bordetella lipo-oligosaccharide, adenylyl cyclase toxin and pertussis toxin. In addition to contributing to the understanding of these toxins' mechanisms at the molecular and cellular levels and their role in pathogenesis, scientists at Pasteur Institutes have also exploited potential applications of the gathered knowledge of these toxins. These applications range from the development of novel tools to study protein-protein interactions over the design of novel antigen delivery tools, such as prophylactic or therapeutic vaccine candidates against cancer and viral infection, to the development of a live attenuated nasal pertussis vaccine. This scientific journey from basic science to applications in the field of human health matches perfectly with the overall scientific objectives outlined by Louis Pasteur himself.

Bordetella parapertussis adenylate cyclase toxin promotes the bacterial survival to the encounter with macrophages.

B. parapertussis is a whooping cough etiological agent, whose incidence in the population has increased remarkably. Virulence factors involved in the bacterial infection, however, remain poorly investigated. We here studied the role of adenylate cyclase (CyaA), the main toxin of B. parapertussis, in the outcome of the bacterial interaction with macrophages. Our results showed that B. parapertussis CyaA intoxicates human macrophages, prevents bacterial phagocytosis and precludes phago-lysosomal fusion eventually promoting the bacterial survival to the encounter with these immune cells. Accordingly, we found that B. parapertussis CyaA induces the transcriptional downregulation of host genes encoding for antimicrobial peptides, proteins involved in bacterial intracellular killing, and the pro-inflammatory cytokine TNF-α, while induces the upregulation of the anti-inflammatory cytokine IL-10. Together with previous reports suggesting a protective role of B. parapertussis CyaA against neutrophils bactericidal activity, the results of this study suggest a central role of CyaA in B. parapertussis immune evasion and persistence.

Avirulent phenotype promotes Bordetella pertussis adaptation to the intramacrophage environment.

Bordetella pertussis, the causative agent of whooping cough, is an extracellular, strictly human pathogen. However, it has been shown that B. pertussis cells can escape phagocytic killing and survive in macrophages upon internalization. Our time-resolved RNA-seq data suggest that B. pertussis efficiently adapts to the intramacrophage environment and responds to host bactericidal activities. We show that this adaptive response is multifaceted and, surprisingly, related to the BvgAS two-component system, a master regulator of virulence. Our results show that the expression of this regulatory circuit is downregulated upon internalization. Moreover, we demonstrate that the switch to the avirulent Bvg- phase augments a very complex process based on the adjustment of central and energy metabolism, cell wall reinforcement, maintenance of appropriate redox and metal homeostasis, and repair of damaged macromolecules. Nevertheless, not all observed effects could be simply attributed to the transition to Bvg- phase, suggesting that additional regulators are involved in the adaptation to the intramacrophage environment. Interestingly, a large number of genes required for the metabolism of sulphur were strongly modulated within macrophages. In particular, the mutant lacking two genes encoding cysteine dioxygenases displayed strongly attenuated cytotoxicity toward THP-1 cells. Collectively, our results suggest that intracellular B. pertussis cells have adopted the Bvg- mode to acclimate to the intramacrophage environment and respond to antimicrobial activities elicited by THP-1 cells. Therefore, we hypothesize that the avirulent phase represents an authentic phenotype of internalized B. pertussis cells.

Estandarización de un ELISA para la cuantificación de anticuerpos IgG humanos contra células enteras de Bordetella pertussis / Standardization of an ELISA for the quantification of human IgG antibodies against whole cells of Bordetella pertussis

Bordetella pertussis es un patógeno exclusivo de humanos que causa la tos ferina, enfermedad respiratoria aguda que afecta principalmente a la población pediátrica. Existen dos tipos de vacunas comercializadas contra este patógeno: celulares y acelulares. Las vacunas celulares han sido extensamente utilizadas y siguen teniendo gran relevancia. El presente trabajo tuvo como objetivo la estandarización de un ELISA para la cuantificación de anticuerpos IgG contra células enteras de Bordetella pertussis. Para ello se determinó la concentración de recubrimiento, el rango lineal de la curva, los parámetros de precisión intra e interensayo, la especificidad, el valor de corte y el límite de detección. Se determinó como concentración de recubrimiento 0,5 UO/mL de células enteras. La curva estándar utilizando un suero de referencia internacional presentó un buen ajuste a una función polinómica en un intervalo entre las diluciones 1/100 y 1/24.300 con un coeficiente de correlación R2≥0,98. Los coeficientes de variación en los ensayos de precisión intra e interensayo estuvieron en los intervalos establecidos para cada uno (≤10 por ciento, ≤20 por ciento respectivamente). Los resultados obtenidos avalan el empleo de este ELISA cuantitativo para la evaluación de la respuesta a células enteras de Bordetella pertussis en ensayos clínicos(AU)

Bordetella pertussis is a pathogen exclusive to humans that causes pertussis, an acute respiratory disease that mainly affects the pediatric population. There are two types of vaccines commercially available against this pathogen: cellular and acellular. Cellular vaccines have been widely used and continue to be of great relevance. The aim of the present work was to standardize an ELISA for the quantification of IgG antibodies against whole cells of Bordetella pertussis. For this purpose, the coating concentration, the linear range of the curve, the intra- and inter-assay precision parameters, the specificity, the cut-off value and the detection limit were determined. The coating concentration was determined as 0.5 UO/mL of whole cells. The standard curve using an international reference serum presented a good fit to a polynomial function in a range between dilutions 1/100 and 1/24,300 with a correlation coefficient R2≥0.98. The coefficients of variation in the intra- and inter-assay precision tests were in the intervals established for each (≤10percent, ≤20percent respectively). The results obtained support the use of this quantitative ELISA for the evaluation of whole-cell response to Bordetella pertussis in clinical trials(AU)

A Robust and Sensitive Spectrophotometric Assay for the Enzymatic Activity of Bacterial Adenylate Cyclase Toxins.

Various bacterial pathogens are producing toxins that target the cyclic Nucleotide Monophosphate (cNMPs) signaling pathways in order to facilitate host colonization. Among them, several are exhibiting potent nucleotidyl cyclase activities that are activated by eukaryotic factors, such as the adenylate cyclase (AC) toxin, CyaA, from Bordetella pertussis or the edema factor, EF, from Bacillus anthracis. The characterization of these toxins frequently requires accurate measurements of their enzymatic activity in vitro, in particular for deciphering their structure-to-function relationships by protein engineering and site-directed mutagenesis. Here we describe a simple and robust in vitro assay for AC activity based on the spectrophotometric detection of cyclic AMP (cAMP) after chromatographic separation on aluminum oxide. This assay can accurately detect down to fmol amounts of B. pertussis CyaA and can even be used in complex media, such as cell extracts. The relative advantages and disadvantages of this assay in comparison with other currently available methods are briefly discussed.

A Bacterial Two-Hybrid System for In Vivo Assays of Protein-Protein Interactions and Drug Discovery.

The bacterial adenylate cyclase-based two-hybrid (BACTH) system is a robust and simple genetic assay used to monitor protein-protein interactions in vivo. This system is based on functional complementation between two fragments from the catalytic domain of Bordetella pertussis adenylate cyclase (AC) to reconstitute a cyclic AMP (cAMP)-signaling cascade in Escherichia coli. Interactions between two chimeric proteins result in the synthesis of cAMP, which activates the transcription of various catabolite operons, leading to selectable phenotypes. One advantageous feature of this signaling cascade is that the physical association between the two interacting hybrid proteins is spatially separated from the transcriptional activation readout. Consequently, the BACTH system can detect protein-protein interactions occurring at various subcellular localizations. The system has been used to characterize interactions between soluble or membrane proteins of prokaryotic, eukaryotic, or viral origin. The BACTH assay can be used to uncover the region(s), domain(s), or amino acid residue(s) of a protein involved in an interaction with a specific partner. The BACTH system can also be adapted for the high-throughput screening of small molecules able to interfere with protein-protein interactions.

The whole-cell pertussis vaccine imposes a broad effector B cell response in mouse heterologous prime-boost settings.

ÍSince the introduction of new generation pertussis vaccines, resurgence of pertussis has been observed in many developed countries. Former whole-cell pertussis (wP) vaccines are able to protect against disease and transmission but have been replaced in several industrialized countries because of their reactogenicity and adverse effects. Current acellular pertussis (aP) vaccines, made of purified proteins of Bordetella pertussis, are efficient at preventing disease but fail to induce long-term protection from infection. While the systemic and mucosal T cell immunity induced by the 2 types of vaccines has been well described, much less is known concerning B cell responses. Taking advantage of an inducible activation-induced cytidine deaminase fate-mapping mouse model, we compared effector and memory B cells induced by the 2 classes of vaccines and showed that a stronger and broader memory B cell and plasma cell response was achieved by a wP prime. We also observed that homologous or heterologous vaccine combinations that include at least 1 wP administration, even as a booster dose, were sufficient to induce this broad effector response, thus highlighting its dominant imprint on the B cell profile. Finally, we describe the settlement of memory B cell populations in the lung following subcutaneous wP prime vaccination.

Amplificación directa de ADN de Bordetella pertussis purificado de hisopados nasofaríngeos por un método de bajo costo, rápido (60-segundos) y libre de equipos / Direct amplification of Bordetella pertussis DNA purified from nasopharyngeal swabs by a low-cost, fast (60-second), and equipment-free method

RESUMEN Objetivo. Desarrollar y evaluar un método de bajo costo basado en celulosa para la purificación rápida y amplificación directa de ADN de Bordetella pertussis de hisopados nasofaríngeos. Materiales y métodos. Se prepararon discos de celulosa y se evaluaron diferentes parámetros (buffers de lisis/lavado, número de discos y elución de ADN). El método se acopló a una amplificación directa por PCR en tiempo real (qPCR) y se estimó el rendimiento utilizando hisopados nasofaríngeos que fueron positivos (n=100) y negativos (n=50) para ADN B. pertussis por qPCR, comparado con el método basado en columnas de sílice. Se calculó el grado de concordancia, sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). Se evaluó la factibilidad del método rápido para ser acoplado a un ensayo colorimétrico de amplificación isotérmica mediada por lazo (LAMP). Resultados. El método rápido con un disco de celulosa y buffer de lisis y lavado conteniendo PVP-40 y Tween 20, respectivamente, mostró una mayor capacidad para purificar ADN amplificable de B. pertussis. El método tuvo una sensibilidad de 89,0% (IC95%, 80,2%-94,9%) y una especificidad de 98,5% (IC95%, 92,1%-100,0%), con un buen grado de concordancia (Kappa=0,867; IC95% 0,788 - 0,946), respecto al método referencial. Los VPP y VPN fueron 98,6% (IC95%, 92,7,2%-100,0%) y 88,2% (IC95%, 78,7%-94,4%), respectivamente. Se evidenció una amplificación exitosa por LAMP, y se obtuvieron resultados comparables con el método por columnas de sílice. Conclusión. El método desarrollado es simple, de bajo costo y libre de equipos para la obtención rápida (60 segundos) de ADN en el punto de atención, y puede ser implementado en diversas técnicas moleculares orientados al diagnóstico oportuno y al estudio epidemiológico de tos ferina.

ABSTRACT Objective. To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. Materials and methods. We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated. Results. The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had a sensitivity of 89.0% (95%CI: 80.2%-94.9%) and a specificity of 98.5% (95%CI: 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95%CI: 0.788 - 0.946), compared to the reference method. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method. Conclusion. The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.

Bordetella pertussis outer membrane vesicles as virulence factor vehicles that influence bacterial interaction with macrophages.

Gram-negative pathogenic bacteria constitutively shed outer membrane vesicles (OMVs) which play a significant role in the host-pathogen interaction, eventually determining the outcome of the infection. We previously found that Bordetella pertussis, the etiological agent of whooping cough, survives the innate interaction with human macrophages remaining alive inside these immune cells. Adenylate cyclase (CyaA), one of the main toxins of this pathogen, was found involved in the modulation of the macrophage defense response, eventually promoting bacterial survival within the cells. We here investigated whether B. pertussis OMVs, loaded with most of the bacterial toxins and CyaA among them, modulate the macrophage response to the bacterial infection. We observed that the pre-incubation of macrophages with OMVs led to a decreased macrophage defense response to the encounter with the bacteria, in a CyaA dependent way. Our results suggest that CyaA delivered by B. pertussis OMVs dampens macrophages protective function by decreasing phagocytosis and the bactericidal capability of these host cells. By increasing the chances of bacterial survival to the innate encounter with the macrophages, B. pertussis OMVs might play a relevant role in the course of infection, promoting bacterial persistence within the host and eventually, shaping the whole infection process.

Novel 2-arylthiazolidin-4-one-thiazole hybrids with potent activity against Mycobacterium tuberculosis.

Substituted aldehydes, ethyl 2-(2-amino-thiazol-4-yl)acetate), and 2-mercaptoacetic acid, in a three-component one-pot green synthetic approach afforded 2-arylthiazolidin-4-one- thiazole hybrids(T1-T13). Compounds showed good anti-tubercular activity towards sensitive M. tuberculosis strain. Compound T8 was as potent as isoniazide (INH) with MIC = 0.12 µg/ml. Compounds T2 and T13 showed potent activity with MIC = 0.48 µg/ml. Other compounds showed moderate to good anti-tubercular activity towards MDR M. tuberculosis strain with MIC range 1.95-125 µg/ml. Compounds T2, T8, T9, and T13 showed anti-tubercular activity towards XDR M. tuberculosis strain with MIC range 7.81-125 µg/ml. Compounds T2 and T8 were capable of inhibiting M. tuberculosis InhA enzyme in-vitro with IC50 = 1.3 ± 0.61 µM and 1.06 ± 0.97 µM, respectively. Molecular docking simulation showed that T2 and T8 were also capable of interacting at the catalytic site of InhA enzyme in a similar mode to the native ligand through binding with NAD+ and Tyr158. The 3D- QSAR study highlighted the relevance of substitution of phenyl group at position-2 of thiazolidin-4-one where bulky electronegative substitution at position-4 of the phenyl ring favored the activity against M. tuberculosis H37R. Additionally, compounds showed good antibacterial activity against bronchitis causing bacteria M. pneumoniae, S. pneumonia, K. pneumonia, and B. pertussis compared to Azithromycin. In-silico studies of ADMET descriptors and drug-likeness were conducted for all synthesized compounds. Compounds showed good oral bioavailability, good gastrointestinal absorption and showed no signs of adverse effects to the liver or CNS. Compounds showed no potential carcinogenicity as well.

Genotypes of Bordetella pertussis isolated from infants in Xi'an and Shanghai.

OBJECTIVE: To compare the genotypes of Bordetella pertussis isolated from infants in Xi'an and Shanghai. METHODS: Samples were collected by nasopharyngeal swab from infants aged <1 year hospitalized with suspected pertussis in Xi'an and Shanghai during 2018 and 2019. Bordetella pertussis was isolated, and multilocus antigen sequence typing (MAST) and multilocus variable-number tandem repeat analysis (MLVA) were used to analyse the genotypes. RESULTS: A total of 1200 samples were collected from infants suspected of pertussis and 60 strains of Bordetella pertussis were isolated, including 34 strains in Xi'an and 26 strains in Shanghai. There were significant differences in the MAST types between Xi'an and Shanghai ( χ 2=18.642, P<0.01); the prn1/ ptxP1/ ptxA1/ fim3-1/ fim2-1 strains dominated in Xi'an (32/34, 94.12%), while the dominated MAST types in Shanghai were prn1/ ptxP1/ ptxA1/ fim3-1/ fim2-1 (13/26, 50.00%) and prn2/ ptxP3/ ptxA1/ fim3-1/ fim2-1 (11/26, 42.31%). The composition of MLVA type of pertussis strains was also significantly different between Xi'an and Shanghai ( χ 2=15.866, P<0.01); the MT195 (13/34, 38.24%), MT55 (10/34, 29.41%) and MT104 (9/34, 26.47%) strains dominated in Xi'an, while the MT27 (12/26, 46.15%) strain was most common in Shanghai. CONCLUSION: There are differences in molecular types of Bordetella pertussis isolated from infants with suspected persussis in Xi'an and Shanghai, indicating that further monitoring of Bordetella pertussis is necessary for better understanding the pathogen evolution in China.

Crystal structures of pertussis toxin with NAD+ and analogs provide structural insights into the mechanism of its cytosolic ADP-ribosylation activity.

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum, where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the endoplasmic reticulum into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (Gαi) of heterotrimeric G proteins, thus promoting dysregulation of G protein-coupled receptor signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide and NAD+, which we name benzamide amino adenine dinucleotide. These crystal structures provide unprecedented insights into pre- and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small-molecule inhibitors.