RESUMO
In order to survive and cause disease, microbial pathogens must be able to proliferate at the temperature of their infected host. We identified novel microbial features associated with thermotolerance in the opportunistic fungal pathogen Cryptococcus neoformans using a random insertional mutagenesis strategy, screening for mutants with defective growth at 37°C. Among several thermosensitive mutants, we identified one bearing a disruption in a gene predicted to encode the Ape4 aspartyl aminopeptidase protein. Ape4 metalloproteases in other fungi, including Saccharomyces cerevisiae, are activated by nitrogen starvation, and they are required for autophagy and the cytoplasm-to-vacuole targeting (Cvt) pathway. However, none have been previously associated with altered growth at elevated temperatures. We demonstrated that the C. neoformans ape4 mutant does not grow at 37°C, and it also has defects in the expression of important virulence factors such as phospholipase production and capsule formation. C. neoformans Ape4 activity was required for this facultative intracellular pathogen to survive within macrophages, as well as for virulence in an animal model of cryptococcal infection. Similar to S. cerevisiae Ape4, the C. neoformans GFP-Ape4 fusion protein co-localized with intracytoplasmic vesicles during nitrogen depletion. APE4 expression was also induced by the combination of nutrient and thermal stress. Together these results suggest that autophagy is an important cellular process for this microbial pathogen to survive within the environment of the infected host.
Assuntos
Autofagia/fisiologia , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidade , Glutamil Aminopeptidase/metabolismo , Fatores de Virulência/metabolismo , Virulência/fisiologia , Animais , Autofagia/genética , Linhagem Celular , Cryptococcus neoformans/genética , Citoplasma/genética , Citoplasma/metabolismo , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutamil Aminopeptidase/genética , Macrófagos/metabolismo , Camundongos , Mutagênese Insercional/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Obesity is assumed to be a major cause of human essential hypertension; however, the mechanisms responsible for weight-related increase in blood pressure (BP) are not fully understood. The prevalence of hypertension induced by obesity has grown over the years, and the role of the renin-angiotensin-aldosterone system (RAAS) in this process continues to be elucidated. In this scenario, the ob/ob mice are a genetic obesity model generally used for metabolic disorder studies. These mice are normotensive even though they present several metabolic conditions that predispose them to hypertension. Although the normotensive trait in these mice is associated with the poor activation of sympathetic nervous system by the lack of leptin, we demonstrated that ob/ob mice present massively increased aminopeptidase A (APA) activity in the circulation. APA enzyme metabolizes angiotensin (ANG) II into ANG III, a peptide associated with intrarenal angiotensin type 2 (AT2) receptor activation and induction of natriuresis. In these mice, we found increased ANG-III levels in the circulation, high AT2 receptor expression in the kidney, and enhanced natriuresis. AT2 receptor blocking and APA inhibition increased BP, suggesting the ANG III-AT2 receptor axis as a complementary BP control mechanism. Circulating APA activity was significantly reduced by weight loss independently of leptin, indicating the role of fat tissue in APA production. Therefore, in this study we provide new data supporting the role of APA in BP control in ob/ob mouse strain. These findings improve our comprehension about obesity-related hypertension and suggest new tools for its treatment.NEW & NOTEWORTHY In this study, we reported an increased angiotensin III generation in the circulation of ob/ob mice caused by a high aminopeptidase A activity. These findings are associated with an increased natriuresis found in these mice and support the role of renin-angiotensin-aldosterone system as additional mechanism regulating blood pressure in this genetic obese strain.
Assuntos
Pressão Sanguínea , Glutamil Aminopeptidase/metabolismo , Obesidade/fisiopatologia , Receptor Tipo 2 de Angiotensina/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Angiotensinas/sangue , Animais , Restrição Calórica , GMP Cíclico/metabolismo , Dieta Hiperlipídica , Inibidores Enzimáticos/farmacologia , Glutamil Aminopeptidase/antagonistas & inibidores , Glutamil Aminopeptidase/sangue , Rim/enzimologia , Leptina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sódio/urinaRESUMO
Acid (aspartyl), basic (arginyl) and neutral (alanyl) aminopeptidases degrade angiotensins, vasopressin, oxytocin, bradykinin and enkephalins. These peptides regulate memory, energy homeostasis, water-salt balance and blood pressure, functions that are mainly exerted in the hippocampus and hypothalamus, and that can be affected by diabetes mellitus. To evaluate the relationship between the diabetes mellitus and processing and inactivation roles of these representative aminopeptidases, we measured their activities in both brain structures of control and streptozotocin-diabetic rats. Hypothalamic soluble aspartyl and arginyl aminopeptidases presented significant decreased activity levels in diabetic rats, which were mitigated by insulin therapy. In addition to membrane-bound puromycin sensitive and insensitive alanyl aminopeptidases, its soluble puromycin sensitive form did not differ between diabetic and control rats in both brain structures. Glucose and/or insulin did not seem to alter in vitro the hypothalamic activities of soluble aspartyl and arginyl aminopeptidases. The implied hypothalamic control of regulatory peptide activity by aspartyl and arginyl aminopeptidases supports the hypothesis that the hydrolytic ability of these enzyme types could be a common link for the disruptions of water-salt balance, blood pressure and energy homeostasis in diabetes mellitus.