RESUMEN
The extracytoplasmic function sigma factor σVreI of the human pathogen Pseudomonas aeruginosa promotes transcription of potential virulence determinants, including secretion systems and secreted proteins. Its activity is modulated by the VreR anti-σ factor that inhibits the binding of σVreI to the RNA polymerase in the absence of a (still unknown) inducing signal. The vreI-vreR genes are expressed under inorganic phosphate (Pi) starvation, a physiological condition often encountered in the host that increases P. aeruginosa pathogenicity. However, whether or not σVreI is active in vivo during infection and contributes to the Pi starvation-induced virulence of this pathogen has not been analyzed yet. Using zebrafish embryos and a human alveolar basal epithelial cell line as P. aeruginosa hosts, we demonstrate in this work that σVreI is active during infection and that lack of σVreI considerably reduces the Pi starvation-induced virulence of this pathogen. Surprisingly, lack of the σVreI inhibitor, the VreR anti-σ factor, also diminishes the virulence of P. aeruginosa. By transcriptomic analyses we show that VreR modulates gene expression not only in a σVreI-dependent but also in a σVreI-independent manner. This includes potential virulence determinants and transcriptional regulators that could be responsible for the reduced virulence of the ΔvreR mutant.
Asunto(s)
Citoplasma/metabolismo , Células Epiteliales/microbiología , Fosfatos/metabolismo , Pseudomonas aeruginosa/metabolismo , Factor sigma/metabolismo , Virulencia , Células A549 , Animales , Proteínas Bacterianas/metabolismo , Análisis por Conglomerados , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Pulmón/metabolismo , Modelos Genéticos , Mutación , Filogenia , Alveolos Pulmonares/citología , Transducción de Señal , Transcriptoma , Factores de Virulencia/metabolismo , Pez CebraRESUMEN
RhoGTPases regulate cytoskeletal dynamics, migration and cell-cell adhesion in endothelial cells. Besides regulation at the level of guanine nucleotide binding, they also undergo post-translational modifications, for example ubiquitination. RhoGTPases are ubiquitinated by Cullin RING ligases which are in turn regulated by neddylation. Previously we showed that inhibition of Cullin RING ligase activity by the neddylation inhibitor MLN4924 is detrimental for endothelial barrier function, due to accumulation of RhoB and the consequent induction of contractility. Here we analyzed the effect of pharmacological activation of Cullin RING ligases on endothelial barrier integrity in vitro and in vivo. CSN5i-3 induced endothelial barrier disruption and increased macromolecule leakage in vitro and in vivo. Mechanistically, CSN5i-3 strongly induced the expression and activation of RhoB and to lesser extent of RhoA in endothelial cells, which enhanced cell contraction. Elevated expression of RhoGTPases was a consequence of activation of the NF-κB pathway. In line with this notion, CSN5i-3 treatment decreased IκBα expression and increased NF-κB-mediated ICAM-1 expression and consequent adhesion of neutrophils to endothelial cells. This study shows that sustained neddylation of Cullin RING-ligases leads to activation the NF-κB pathway in endothelial cells, elevated expression of RhoGTPases, Rho/ROCK-dependent activation of MLC and disruption of the endothelial barrier.
Asunto(s)
Complejo del Señalosoma COP9/metabolismo , Endotelio Vascular/metabolismo , Inflamación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptido Hidrolasas/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Animales , Ciclopentanos/farmacología , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Neutrófilos/metabolismo , Pirimidinas/farmacología , Ubiquitina/química , Regulación hacia Arriba , Pez CebraAsunto(s)
Coinfección/líquido cefalorraquídeo , Meningitis Bacterianas/complicaciones , Meningitis Viral/complicaciones , Coinfección/microbiología , Coinfección/virología , Infecciones Comunitarias Adquiridas/líquido cefalorraquídeo , Infecciones Comunitarias Adquiridas/complicaciones , Infecciones Comunitarias Adquiridas/microbiología , Infecciones Comunitarias Adquiridas/virología , Humanos , Meningitis Bacterianas/líquido cefalorraquídeo , Meningitis Bacterianas/virología , Meningitis Viral/líquido cefalorraquídeo , Países Bajos/epidemiologíaRESUMEN
OBJECTIVE: To evaluate the occurrence, treatment, and outcome of subdural empyema complicating community-acquired bacterial meningitis in adults. METHODS: Case series from a prospective nationwide cohort study from Dutch hospitals from 2006 to 2011. RESULTS: Subdural empyema was diagnosed in 28 of 1,034 episodes (2.7%), and was present on admission in 10 episodes and diagnosed during admission in 18. Predisposing conditions were present in 26 patients (93%), and consisted of otitis or sinusitis in 21 patients (75%). In all these patients the otitis or sinusitis spread to the subdural space. Twenty-three patients (82%) presented with neurologic symptoms (paresis, focal seizures, dysesthesia contralateral to the empyema). Streptococcus pneumoniae was identified in 26 patients (93%) and Streptococcus pyogenes in 1 (3%); 1 patient had negative CSF cultures. Clinical course was frequently complicated with seizures (50%), focal neurologic abnormalities (54%), and hearing impairment (39%), causing an unfavorable outcome in 19 episodes (68%). Neurosurgical evacuation of the empyema was performed in 5 patients, all with considerable midline shift. CONCLUSIONS: Although rare, subdural empyema must be considered in patients with community-acquired bacterial meningitis and otitis or sinusitis, focal neurologic deficits, or epileptic seizures. S pneumoniae is the predominant causative organism and neurosurgical intervention should be regarded as first-choice therapy in patients with empyema causing midline shift and focal neurologic abnormalities or a decreased level of consciousness.