ABSTRACT
Biofilms, composed of bacterial cells embedded in a secreted polysaccharide and protein matrix, often cause problems such as chronic and refractory infections. Staphylococcus pseudintermedius, which is an important pathogen in veterinary medicine, has a high rate of biofilm production. Although it is considered that S. pseudintermedius biofilms are associated with prolonged inflammatory disorders, there are no reports that S. pseudintermedius biofilm directly regulates inflammatory reactions. In this study, we focused on the metabolites derived from biofilm cultures of S. pseudintermedius and evaluated their inflammatory effects in vitro. Expression levels of interleukin-1 beta and interleukin-6 mRNA significantly increased in RAW264.7 cells that were cultured with biofilm-conditioned medium (BCM). The secreted proteins in BCM were heat resistance and activated a Toll-like receptor (TLR) signalling pathway. Moreover, based on SDS-PAGE analysis, isolates with stronger biofilm-forming capabilities induced more inflammatory reactions and had specific banding patterns compared with those of weak biofilm producers. Collectively, our results suggest that the proteins derived from S. pseudintermedius biofilm induce a host inflammatory response via a TLR pathway. Furthermore, the severity of inflammation depends on the biofilm formation capacity of the S. pseudintermedius strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus pseudintermedius is a biofilm-forming bacterium. We identified some biofilm secreted heat-resistant proteins that induce inflammatory reactions through Toll-like receptor signalling. The expression of the secreted protein varied depending on the potency of biofilm production. Our data suggest that these proteins may be the factors causing biofilm-related inflammation during S. pseudintermedius infections. Identification of these proteins may lead to the development of novel medications to prevent the exacerbation of infections caused by S. pseudintermedius.
Subject(s)
Biofilms , Staphylococcal Infections/immunology , Staphylococcus/physiology , Animals , Culture Media, Conditioned/chemistry , Culture Media, Conditioned/metabolism , Humans , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Macrophages/drug effects , Macrophages/immunology , Mice , RAW 264.7 Cells , Staphylococcal Infections/genetics , Staphylococcal Infections/microbiology , Staphylococcus/geneticsABSTRACT
The Clostridium coccoides group, including the genus Blautia and other genera, is one of the predominant bacterial groups in the human intestine. We re-examined 266 human faecal clones and 58 isolates in the C. coccoides group isolated by Hayashi et al. (2002) in order to elucidate the detailed distribution of Blautia wexlerae and Blautia luti in human faeces. Subsequently, we designed a primer pair specific for B. wexlerae and B. luti based on the 16S ribosomal RNA (16S rRNA) gene sequence. The number of B. wexlerae and B. luti in faecal samples of 12 healthy Japanese subjects was examined by real-time PCR assay. The number of the C. coccoides group in the 12 faecal samples was also determined using C. coccoides group-specific primers. Re-examination of the human faecal clones and isolates revealed that B. wexlerae and B. luti accounted for 19.5% of the clones and 25.9% of the isolates. B. wexlerae and B. luti were detected in all faecal samples with 5.3±3.2×10(9) cells/g faeces (wet weight, average ± standard deviation) as assessed by real-time PCR. Furthermore, B. wexlerae and B. luti constituted 32.3±12.7% (average ± standard deviation) of the C. coccoides group (1.7±0.8×10(10) cells/g faeces). This demonstrates that B. wexlerae and B. luti were presented in human faeces with a high frequency as the dominant bacteria.
Subject(s)
Bacterial Load/methods , Clostridiales/genetics , Clostridiales/isolation & purification , DNA Primers/genetics , Feces/microbiology , Real-Time Polymerase Chain Reaction/methods , Adult , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Healthy Volunteers , Humans , Japan , Male , RNA, Ribosomal, 16S/geneticsABSTRACT
The intestinal microbiota composition of 92 volunteers living in Japan was identified following the consumption of 'identical meals' (1,879 kcal/day) for 3 days. When faecal samples were analysed by terminal restriction fragment length polymorphism with several primer-restriction enzyme systems and then clustered, the patterns could be divided into 2 clusters. Contribution tests and partition modelling showed that OTU211 of the 35f-MspI system and OTU237 of the 35f-AluI system were key factors in the distribution of these groups. However, significant differences among these groups in terms of body mass index and age were not observed.
Subject(s)
Biodiversity , Eating , Meals , Metagenome/drug effects , Adult , Cluster Analysis , DNA Fingerprinting , Feces/microbiology , Human Experimentation , Humans , Japan , Male , Middle Aged , Polymorphism, Restriction Fragment Length , Young AdultABSTRACT
Interleukin-7 (IL-7) is an essential T-cell survival cytokine. IL-7 receptor (IL-7Rα) deficiency severely impairs T-cell development due to substantial apoptosis. We hypothesized that IL-7Rα(null)-induced apoptosis is partially contributed by an elevated p53 activity. To investigate the genetic association of IL-7/IL-7Rα signaling with the p53 pathway, we generated IL-7Rα(null)p53(null) (DKO) mice. DKO mice exhibited a marked reduction of apoptosis in developing T cells and an augmented thymic lymphomagenesis with telomere erosions and exacerbated chromosomal anomalies, including chromosome duplications, breaks, and translocations. In particular, Robertsonian translocations, in which telocentric chromosomes fuse at the centromeric region, and a complete loss of telomeres at the fusion site occurred frequently in DKO thymic lymphomas. Cellular and molecular investigations revealed that IL-7/IL-7Rα signaling withdrawal diminished the protein synthesis of protection of telomere 1 (POT1), a subunit of telomere protective complex shelterin, leading to telomere erosion and the activation of the p53 pathway. Blockade of IL-7/IL-7Rα signaling in IL-7-dependent p53(null) cells reduced POT1 expression and caused telomere and chromosome abnormalities similar to those observed in DKO lymphomas. This study underscores a novel function of IL-7/IL-7Rα during T-cell development in regulating telomere integrity via POT1 expression and provides new insights into cytokine-mediated survival signals and T-cell lymphomagenesis.