RESUMO
Even though several in vitro studies have focused on bacterial biology, the extent of such knowledge is not complete when considering an actual infection. As culture-independent microbiology methods such as high-throughput sequencing became available, important aspects of host-bacterium interactions will be elucidated. Based on microbiological relevance, we considered Bacteroides fragilis in a murine experimental infection as a model system to evaluate the in vivo bacterial transcriptome in host exudates. A disproportionate number of reads belonging to the host genome were retrieved in the first round of pyrosequencing, even after depletion of ribosomal RNA; the average number of reads related to the eukaryotic genome was 71.924-67.7%, whereas prokaryotic reads represented 34.338-32.3% in host exudates. Thus, different treatments were used to improve the prokaryotic RNA yield: i) centrifugation; ii) ultrasonic treatment; and iii) ultrasonic treatment followed by centrifugation. The latter treatment was found to be the most efficient in generating bacterial yields, as it resulted in a higher number of Bacteroides cells. However, the RNA extracted after this treatment was not of sufficient quality to be used in cDNA synthesis. Our results suggest that the methodology routinely used for RNA extraction in transcriptional analysis is not appropriate for in vivo studies in complex samples. Furthermore, the most efficient treatment for generating good bacterial cell yields was not suitable to retrieve high-quality RNA. Therefore, as an alternative methodological approach to enable in vivo studies on host-bacterium interactions, we advise increasing the sequencing depth despite the high costs.
Assuntos
Bacteroides fragilis/genética , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , Transcriptoma/genética , Animais , Bacteroides fragilis/patogenicidade , Regulação Bacteriana da Expressão Gênica/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , RNA Mensageiro/isolamento & purificação , Análise de Sequência de RNARESUMO
Splenectomized mice control Listeria monocytogenes infection better than non-splenectomized mice. Here, BALB/c mice subjected to splenectomy and autogenous grafting of spleen were evaluated after 3 and 7 days of intravenous L. monocytogenes infection. The group of splenectomized animals (SP) presented a lower number of bacteria in the liver in comparison with both the sham-operated control group (CT) and the group that received splenic autotransplantation (AT) in the retroperitoneal site. The AT group presented bacterial counts in the liver similar to the CT group. SP animals showed larger production of interferon-gamma (IFN-γ) and nitric oxide (NO) in the liver in comparison with CT and AT, this being associated with greater accumulation of mononuclear cells. IFN-γ production by spleen cells after stimulation with heat-killed Listeria was similar between the AT and CT groups, suggesting that the implanted fragments behaved like the original organ. The autogenous grafting of spleen fragments reverses the resistance to L. monocytogenes infection found in splenectomized mice, associated with a reduced IFN-γ and NO production in the liver. The present study shows that splenic autotransplantation restores the function of the spleen in splenectomized mice, even though in this case it does favor the susceptibility to L. monocytogenes infection.
Assuntos
Interferon gama/metabolismo , Listeria monocytogenes , Listeriose/imunologia , Óxido Nítrico/metabolismo , Baço/transplante , Esplenectomia , Animais , Interferon gama/genética , Listeriose/microbiologia , Listeriose/patologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The high incidence of overwhelming postsplenectomy infection caused by Streptococcus pneumoniae can be reduced by splenic autotransplantation. In this study the effect of splenectomy and splenic autotransplantation on the immune response to S. pneumoniae infection was investigated. Balb/c mice were divided into three groups: splenectomized (SP), splenectomized and autotransplanted (AT), and sham operated control (CT). Five days post-infection the serum antibody levels were measured and the number of S. pneumoniae CFU, neutrophil accumulation and IL-17 production in the liver and lungs were investigated. SP mice showed greater number of bacteria in both organs and lower serum levels of S. pneumoniae-specific IgM, IgG1 and IgG2a antibodies. IL-17 production and neutrophil recruitment to the liver and lungs were lower in SP mice, in comparison with both the CT and the AT groups. Levels of S. pneumoniae-specific IgM, CFU counts, neutrophil accumulation and IL-17 production did not differ significantly between the CT and AT groups. These results suggest that splenic autotransplantation restores the capacity of splenectomized mice to fight S. pneumoniae infection.
Assuntos
Fígado/imunologia , Pulmão/imunologia , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Antibacterianos/sangue , Movimento Celular , Contagem de Colônia Microbiana , Imunidade , Interleucina-17/imunologia , Interleucina-17/metabolismo , Fígado/metabolismo , Fígado/microbiologia , Fígado/patologia , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/patologia , Infecções Pneumocócicas/sangue , Infecções Pneumocócicas/cirurgia , Infecções Pneumocócicas/terapia , Esplenectomia/efeitos adversos , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/patogenicidade , Transplante Autólogo , VirulênciaRESUMO
Splenectomy results in an increased risk of sepsis. The autogenous transplant of the spleen is an option for preserving splenic functions after total splenectomy. In this study, the capacity of animals undergoing autogenous spleen transplantation to respond to Staphylococcus aureus infection was investigated. BALB/c mice were divided into three groups: splenectomy followed by autotransplantation in the retroperitonium (AT), splenectomized only (SP) and operated non-splenectomized sham control (CT). Thirty days after surgery the mice were infected intravenously with S. aureus. Splenectomized mice had a higher number of colony-forming units (CFU) of S. aureus in liver and lungs in comparison with either AT or with CT mice (P < 0.05). Higher CFU numbers in lung of SP mice correlated with elevated production of interleukin-10 associated with a lower production of interferon-gamma and tumour necrosis factor-alpha. However, systemically, the level of tumour necrosis factor-alpha was higher in the SP group than in CT or AT. Lower titres of specific anti-S. aureus immunoglobulin (Ig)M and IgG1 were observed 6 days after infection in SP mice in comparison either with the AT or CT groups. Thus, splenectomy is detrimental to the immune response of BALB/c mice against infection by S. aureus which can be re-established by autogenous implantation of the spleen.