Tratamiento ayurveda (Virechana y Basti) y cambios de la microbiota intestinal a nivel de Phylum y especie / Ayurveda Treatment (Virechana and Basti) and Changes of Intestinal Microbiota at Phyla and Species Level

Introducción: Se han analizado terapias ayurvédicas y prácticas médicas para un grupo de pacientes en Japón. La característica del tratamiento ayurvédico es una desintoxicación con una gran cantidad de tratamiento con aceite mediante un masaje con aceite en la superficie del cuerpo y una terapia de purificación con ghee o aceite de hierbas especialmente preparado. Los cambios de la microbiota intestinal durante estos tratamientos no han sido bien estudiados. Mé- LA PRENSA MÉDICA ARGENTINA Ayurveda Treatment (Virechana and Basti) and Changes of Intestinal Microbiota at Phyla and Species Level 79 V.107/Nº 2 todo: Los participantes fueron reclutados de la Clínica Hatai Ayurveda en Tokio. La terapia de Virechana, una terapia de purificación o la terapia de Basti (decocción y enema de aceite) se llevó a cabo en 13 pacientes con diversas manifestaciones. Todos los participantes proporcionaron el detalle de su estilo de vida, hábitos dietéticos, enfermedades pasadas y presentes mediante el cuestionario, y se registró la condición precisa durante la admisión al final del campamento. Se tomaron muestras fecales a la entrada, durante el tratamiento, al alta y tres semanas después para analizar la microbiota intestinal por el gen seqyebcubg 16srRNA. Resultados: el peso corporal disminuyó aproximadamente un 5% con la terapia de Virechana, mientras que no ocurrió con Basti, pero la grasa corporal aumentó un 4% (2,2 kg) en promedio en ambos grupos. Varias manifestaciones clínicas de los participantes mejoraron, especialmente en una erupción cutánea y un cambio atópico. El paciente deprimido también remitió mejoras en sus ganas de vivir. En su mayoría son vegetarianos y tenían más Bacteroides (48.09 ± 7.51%), Firmicutes (38.27 ± 10.82%) y Actinobacteria (3.30 ± 3.58%) que los omnívoros que tenían más Proteobacteria (10.73 ± 4.75%), Fusobacteria (2.40 ± 6.25%) y cianobacterias (0,09 ± 0,24%). Cuando los grupos se dividieron por el consumo de aceite, los usuarios de ghee mostraron más Fusobacterium y menos Firmicutes y Actinobacteria. La terapia con Virechana provocó cambios notables en la microbiota después del pretratamiento, como la disminución de Firmicutes y el aumento de Proteobacterias. A nivel género-especie, destacan el aumento de Enterobacteriaceae y la pérdida de Akkermansia municiphila. Niruha Basti y Matra Basti disminuyeron Firmicutes y aumentaron Proteobacteria (p = 0.096). Fusobacterium también aumentó. Después del alta, la Proteobateria se mantuvo alta, pero Firmicutes regresó al 30% en promedio, oscilando entre el 25% y el 50%. Tres semanas después, la variedad aumentó con Fusobacterium, Verrucomicrobia, Tenericutes y Lentisphaerae. La variedad de especies también aumentó tres semanas después. Conclusión: Varias quejas de los participantes mejoraron por el tratamiento ayurvédico con una gran cantidad de tratamiento de aceite por masaje de aceite de superficie corporal y terapia de purga. Causó cambios en la microbiota intestinal y los metabolitos bacterianos pueden afectar las lesiones cutáneas y la salud mental como la sensación depresiva

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

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

The Eukaryotic Host Factor 14-3-3 Inactivates Adenylate Cyclase Toxins of Bordetella bronchiseptica and B. parapertussis, but Not B. pertussis.

Bordetella pertussis, Bordetella bronchiseptica, and Bordetella parapertussis share highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles in Bordetella infections. We herein demonstrate that CyaAs function as virulence factors differently between B. bronchiseptica/B. parapertussis and B. pertussisBbronchiseptica CyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly to Bpertussis CyaA. The hemolytic activity of Bbronchiseptica CyaA on sheep erythrocytes was also preserved. However, in nucleated target cells, Bbronchiseptica CyaA was phosphorylated at Ser375, which constitutes a motif (RSXpSXP [pS is phosphoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects of Bbronchiseptica CyaA based on its enzyme activity were markedly attenuated. Bparapertussis CyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly to Bbronchiseptica CyaA; however, Bpertussis CyaA has Phe375 instead of Ser, and thus, was not affected by 14-3-3. In addition, Bpertussis CyaA impaired the barrier function of epithelial cells, whereas Bbronchiseptica CyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity between B. bronchiseptica/Bparapertussis and B. pertussisIMPORTANCEBordetella pertussis, B. bronchiseptica, and B. parapertussis are bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by each Bordetella species. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor of Bordetella The toxins of B. bronchiseptica and presumably B. parapertussis were inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas the B. pertussis toxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences in Bordetella are attributed to the different activities of adenylate cyclase toxins.

Ectopic Expression of O Antigen in Bordetella pertussis by a Novel Genomic Integration System.

We describe a novel genome integration system that enables the introduction of DNA fragments as large as 50 kbp into the chromosomes of recipient bacteria. This system, named BPI, comprises a bacterial artificial chromosome vector and phage-derived gene integration machinery. We introduced the wbm locus of Bordetella bronchiseptica, which is required for O antigen biosynthesis, into the chromosome of B. pertussis, which intrinsically lacks O antigen, using the BPI system. After the introduction of the wbm locus, B. pertussis presented an additional substance in the lipooligosaccharide fraction that was specifically recognized by the anti-B. bronchiseptica antibody but not the anti-B. pertussis antibody, indicating that B. pertussis expressed O antigen corresponding to that of B. bronchiseptica. O antigen-expressing B. pertussis was less sensitive to the bactericidal effects of serum and polymyxin B than the isogenic parental strain. In addition, an in vivo competitive infection assay showed that O antigen-expressing B. pertussis dominantly colonized the mouse respiratory tract over the parental strain. These results indicate that the BPI system provides a means to alter the phenotypes of bacteria by introducing large exogenous DNA fragments. IMPORTANCE Some bacterial phenotypes emerge through the cooperative functions of a number of genes residing within a large genetic locus. To transfer the phenotype of one bacterium to another, a means to introduce the large genetic locus into the recipient bacterium is needed. Therefore, we developed a novel system by combining the advantages of a bacterial artificial chromosome vector and phage-derived gene integration machinery. In this study, we succeeded for the first time in introducing a gene locus involved in O antigen biosynthesis of Bordetella bronchiseptica into the chromosome of B. pertussis, which intrinsically lacks O antigen, and using this system we analyzed phenotypic alterations in the resultant mutant strain of B. pertussis. The present results demonstrate that this system successfully accomplished the above-described purpose. We consider this system to be applicable to a number of bacteria other than Bordetella.

Protective effects of in vivo-expressed autotransporters against Bordetella pertussis infection.

Bordetella pertussis causes whooping cough, a severe and prolonged respiratory disease that results inhas high morbidity and mortality rates, particularly in developing countries. The number incidence of whooping cough cases is increasing in many countries despite high vaccine coverage. Causes for the re-emergence of the disease include the limited duration of protection conferred by the acellular pertussis vaccines (aP)s and pathogenic adaptations that involve antigenic divergence from vaccine strains. Therefore, current vaccines therefore need to be improved. In the present study, we focused on five autotransporters: namely SphB1, BatB, SphB2, Phg, and Vag8, which were previously found to be expressed by B. bronchiseptica during the course of infection in rats and examined their protective efficiencies as vaccine antigens. The passenger domains of these proteins were produced in recombinant forms and used as antigens. An intranasal murine challenge assay showed that immunization with a mixture of SphB1 and Vag8 (SV) significantly reduced bacterial load in the lower respiratory tract and a combination of aP and SV acts synergistically in effects of conferring protection against B. pertussis infection, implying that these antigens have potential as components to for improvinge th the currently available acellular pertussis vaccine.

The bvg-repressed gene brtA, encoding biofilm-associated surface adhesin, is expressed during host infection by Bordetella bronchiseptica.

Bordetella species display phase modulation between Bvg(+) and Bvg(-) phases. Because expression of known virulence factors is up-regulated in the Bvg(+) phase, bacteria in this phase are considered competent for infection. However, the Bvg(-) phase is of negligible importance for infection. No studies have shown that bacterial factors specific to the Bvg(-) phase (bvg-repressed factors) are expressed in the course of Bordetella infection. In the present study, the gene brtA (Bordetella RTX-family Adhesin), which is a typical bvg-repressed gene but is expressed in B. bronchiseptica infecting hosts, was characterized. BrtA is composed of repeated pairs of the VCBS unit and dystroglycan-type cadherin-like unit, the von Willebrand Factor A domain, RTX motif and type I secretion target signal. It is herein demonstrated that BrtA is secreted by the type I secretion system and is essential for Ca(2+) -dependent bacteria-to-substrate adherence, followed by biofilm formation. Although the contribution of BrtA to bacterial colonization of the rat trachea currently remains unclear, this is the first study to present concrete evidence for the expression of a bvg-repressed gene during infection, which may provide a novel aspect for analyses of Bordetella pathogenesis.