RESUMEN
Mice with a null mutation in the cystic fibrosis transmembrane conductance regulator (Cftr) gene show intestinal structure alterations and bacterial overgrowth. To determine whether these changes are model-dependent and whether the intestinal microbiome is altered in cystic fibrosis (CF) mouse models, we characterized the ileal tissue and intestinal microbiome of mice with the clinically common ΔF508 Cftr mutation (FVB/N Cftr(tm1Eur)) and with Cftr null mutations (BALB/c Cftr(tm1UNC) and C57BL/6 Cftr(tm1UNC)). Intestinal disease in 12-week-old CF mice, relative to wild-type strain controls, was measured histologically. The microbiome was characterized by pyrosequencing of the V4-V6 region of the 16S rRNA gene and intestinal load was measured by RT-PCR of the 16S rRNA gene. The CF-associated increases in ileal crypt to villus axis distention, goblet cell hyperplasia, and muscularis externa thickness were more severe in the BALB/c and C57BL/6 Cftr(tm1UNC) mice than in the FVB/N Cftr(tm1Eur) mice. Intestinal bacterial load was significantly increased in all CF models, compared to levels in controls, and positively correlated with circular muscle thickness in CF, but not wild-type, mice. Microbiome profiling identified Bifidobacterium and groups of Lactobacillus to be of altered abundance in the CF mice but overall bacterial frequencies were not common to the three CF strains and were not correlative of major histological changes. In conclusion, intestinal structure alterations, bacterial overgrowth, and dysbiosis were each more severe in BALB/c and C57BL/6 Cftr(tm1UNC) mice than in the FVB/N Cftr(tm1Eur) mice. The intestinal microbiome differed among the three CF mouse models.
Asunto(s)
Fibrosis Quística/microbiología , Microbioma Gastrointestinal , Intestinos/patología , Animales , Carga Bacteriana , Peso Corporal , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , ADN Bacteriano/genética , Modelos Animales de Enfermedad , Intestinos/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fenotipo , ARN Ribosómico 16S/genéticaRESUMEN
Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin's active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H¹98A], HAXXH [E¹99A], and HEXXA [H²°²A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro-domain to attain full host component-directed proteolytic activity. Furthermore, our finding that expression of pallilysin confers upon T. phagedenis the capacity to degrade fibrin clots suggests this capability may contribute to the dissemination potential of T. pallidum.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Fibrinógeno/metabolismo , Laminina/metabolismo , Metaloproteasas/metabolismo , Treponema pallidum/enzimología , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/aislamiento & purificación , Secuencias de Aminoácidos , Animales , Dominio Catalítico , Activación Enzimática , Fibrina/metabolismo , Metaloproteasas/química , Metaloproteasas/genética , Metaloproteasas/aislamiento & purificación , Fagocitosis , Conejos , Trombina/metabolismo , Treponema pallidum/patogenicidad , Zinc/metabolismoRESUMEN
BACKGROUND: Idiopathic pulmonary fibrosis is a disease characterized by alveolar epithelial cell injury, inflammatory cell infiltration and deposition of extracellular matrix in lung tissue. As mouse models of bleomycin-induced pulmonary fibrosis display many of the same phenotypes observed in patients with idiopathic pulmonary fibrosis, they have been used to study various aspects of the disease, including altered expression of microRNAs. RESULTS: In this work, microRNA expression profiling of the lungs from treated C57BL/6J mice, relative to that of untreated controls, was undertaken to determine which alterations in microRNAs could in part regulate the fibrosis phenotype induced by bleomycin delivered through mini-osmotic pumps. We identified 11 microRNAs, including miR-21 and miR-34a, to be significantly differentially expressed (P < 0.01) in lungs of bleomycin treated mice and confirmed these data with real time PCR measurements. In situ hybridization of both miR-21 and miR-34a indicated that they were expressed in alveolar macrophages. Using a previously reported gene expression profile, we identified 195 genes to be both predicted targets of the 11 microRNAs and of altered expression in bleomycin-induced lung disease of C57BL/6J mice. Pathway analysis with these 195 genes indicated that altered microRNA expression may be associated with hepatocyte growth factor signaling, cholecystokinin/gastrin-mediated signaling, and insulin-like growth factor (IGF-1) signaling, among others, in fibrotic lung disease. The relevance of the IGF-1 pathway in this model was then demonstrated by showing lung tissue of bleomycin treated C57BL/6J mice had increased expression of Igf1 and that increased numbers of Igf-1 positive cells, predominantly in macrophages, were detected in the lungs. CONCLUSIONS: We conclude that altered microRNA expression in macrophages is a feature which putatively influences the insulin-like growth factor signaling component of bleomycin-induced pulmonary fibrosis.