RESUMEN
Alpha-hemolysin (HlyA) of uropathogenic strains of Escherichia coli irreversibly binds to human erythrocytes (RBCs) and triggers activation of ATP release and metabolic changes ultimately leading to hemolysis. We studied the regulation of extracellular ATP (ATPe) of RBCs exposed to HlyA. Luminometry was used to assess ATP release and ATPe hydrolysis, whereas changes in cell volume and morphology were determined by electrical impedance, ektacytometry and aggregometry. Exposure of RBCs to HlyA induced a strong increase of [ATPe] (3-36-fold) and hemolysis (1-44-fold), partially compensated by [ATPe] hydrolysis by ectoATPases and intracellular ATPases released by dead cells. Carbenoxolone, a pannexin 1 inhibitor, partially inhibited ATP release (43-67%). The un-acylated toxin ProHlyA and the deletion analog HlyA∆914-936 were unable to induce ATP release or hemolysis. For HlyA treated RBCs, a data driven mathematical model showed that simultaneous lytic and non-lytic release mainly governed ATPe kinetics, while ATPe hydrolysis became important after prolonged toxin exposure. HlyA induced a 1.5-fold swelling, while blocking this swelling reduced ATP release by 77%. Blocking ATPe activation of purinergic P2X receptors reduced swelling by 60-80%. HlyA-RBCs showed an acute 1.3-2.2-fold increase of Ca2+i, increased crenation and externalization of phosphatidylserine. Perfusion of HlyA-RBCs through adhesion platforms showed strong adhesion to activated HMEC cells, followed by rapid detachment. HlyA exposed RBCs exhibited increased sphericity under osmotic stress, reduced elongation under shear stress, and very low aggregation in viscous media. Overall results showed that HlyA-RBCs displayed activated ATP release, high but weak adhesivity, low deformability and aggregability and high sphericity.
Asunto(s)
Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Deformación Eritrocítica/efectos de los fármacos , Proteínas de Escherichia coli/farmacología , Proteínas Hemolisinas/farmacología , Hemólisis/efectos de los fármacos , Presión Osmótica/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , HumanosRESUMEN
After invading red blood cells (RBCs), Plasmodium falciparum (Pf) can export its own proteins to the host membrane and activate endogenous channels that are present in the membrane of RBCs. This transport pathway involves the Voltage Dependent Anion Channel (VDAC). Moreover, ligands of the VDAC partner TranSlocator PrOtein (TSPO) were demonstrated to inhibit the growth of the parasite. We studied the expression of TSPO and VDAC isoforms in late erythroid precursors, examined the presence of these proteins in membranes of non-infected and infected human RBCs, and evaluated the efficiency of TSPO ligands in inhibiting plasmodium growth, transporting the haem analogue Zn-protoporphyrin-IX (ZnPPIX) and enhancing the accumulation of reactive oxygen species (ROS). TSPO and VDAC isoforms are differentially expressed on erythroid cells in late differentiation states. TSPO2 and VDAC are present in the membranes of mature RBCs in a unique protein complex that changes the affinity of TSPO ligands after Pf infection. TSPO ligands dose-dependently inhibited parasite growth, and this inhibition was correlated to ZnPPIX uptake and ROS accumulation in the infected RBCs. Our results demonstrate that TSPO ligands can induce Pf death by increasing the uptake of porphyrins through a TSPO2-VDAC complex, which leads to an accumulation of ROS.
Asunto(s)
Plasmodium falciparum/crecimiento & desarrollo , Protoporfirinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de GABA/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD34/metabolismo , Transporte Biológico , Diferenciación Celular , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Eritrocitos/parasitología , Células Eritroides/citología , Células Eritroides/metabolismo , Perfilación de la Expresión Génica , Glutatión/metabolismo , Humanos , Ligandos , Espectrometría de Masas , Parásitos/crecimiento & desarrollo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de GABA/química , Receptores de GABA/genética , Canales Aniónicos Dependientes del Voltaje/química , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
Staphylococcus aureus and various coagulase-negative staphylococci were isolated from turkeys with staphylococcosis. Virulent S. aureus adhered well (averaged more than 100 bacteria per tissue cell) in vitro to cells from tissues of the respiratory tract but did not adhere well (averaged fewer than 12 bacteria per tissue cell) to cells from tissues of the alimentary tract. Some avirulent coagulase-negative staphylococci also adhered well to cells from the respiratory tissues. Lungs and livers of turkeys became colonized with virulent S. aureus following experimental aerosol exposure. Tracheas, livers, and hock joints of some market-age turkeys were naturally colonized with S. aureus and various species of coagulase-negative staphylococci.
Asunto(s)
Enfermedades de las Aves de Corral/microbiología , Infecciones Estafilocócicas/veterinaria , Pavos/microbiología , Aerosoles , Animales , Adhesión Bacteriana , Hígado/microbiología , Pulmón/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/aislamiento & purificación , Tráquea/microbiologíaRESUMEN
Ninety-eight Staphylococcus aureus isolates and 227 coagulase-negative staphylococcal isolates from turkeys were assayed for protein A content. The amount of protein A of each isolate was quantitated using a direct enzyme-linked immunosorbent assay. Of the S. aureus isolates, 83% possessed some protein A, whereas only 13% of the coagulase-negative staphylococci contained some protein A. No correlation was seen between protein A content and ability to adhere to turkey cells. No differences in virulence were seen between isolates of S. aureus possessing high or low levels of protein A; however, an isolate with no protein A was avirulent.