Escaping the Phagocytic Oxidative Burst: The Role of SODB in the Survival of Pseudomonas aeruginosa Within Macrophages

Reactive oxygen species (ROS) are small oxygen-derived molecules that are used to control infections by phagocytic cells. In macrophages, the oxidative burst produced by the NOX2 NADPH-oxidase is essential to eradicate engulfed pathogens by both oxidative and non-oxidative killing. Indeed, while the superoxide anion (O2-) produced by NOX2, and the other ROS derived from its transformation, can directly target pathogens, ROS also contribute to activation of non-oxidative microbicidal effectors. The response of pathogens to the phagocytic oxidative burst includes the expression of different enzymes that target ROS to reduce their toxicity. Superoxide dismutases (SODs) are the primary scavengers of O2-, which is transformed into H2O2. In the Gram-negative Salmonella typhimurium, periplasmic SODCI has a major role in bacterial resistance to NOX-mediated oxidative stress. In Pseudomonas aeruginosa, the two periplasmic SODs, SODB, and SODM, appear to contribute to bacterial virulence in small-animal models. Furthermore, NOX2 oxidative stress is essential to restrict P. aeruginosa survival in macrophages early after infection. Here, we focused on the role of P. aeruginosa SODs in the counteracting of the lethal effects of the macrophage oxidative burst. Through this study of the survival of sod mutants in macrophages and the measurement of ROS in infected macrophages, we have identified a dual, antagonistic, role for SODB in P. aeruginosa survival. Indeed, the survival of the sodB mutants, but not of the sodM mutants, was greater than that of the wild-type (WT) bacteria early after infection, and sodB-infected macrophages showed higher levels of O2- and lower levels of H2O2. This suggests that SODB contributes to the production of lethal doses of H2O2 within the phagosome. However, later on following infection, the sodB mutants survived less that the WT bacteria, which highlights the pro-survival role of SODB. We have explained this defensive role through an investigation of the activation of autophagy, which was greater in the sodB-infected macrophages

Thyroid hormone inhibition in L6 myoblasts of IGF-I-mediated glucose uptake and proliferation: New roles for integrin αvβ3

Thyroid hormones L-thyroxine (T4) and 3,3′,5-triiodo-L-thyronine (Se

Calcium ion independent membrane leakage induced by phospholipase-like myotoxins

The two snake venom myotoxins ammodytin L and myotoxin II, purified respectively from Vipera ammodytes ammodytes and Bothrops asper, have phospholipase-like structures but lack an Asp-49 in the active site and are without normal phospholipase activity. The interaction of these proteins with different types of liposomes indicated that the myotoxins were able to provoke rapid and extensive release of the aqueous content of liposomes. Leakage was measured by two different methods: fluorescence dequenching of liposome-entrapped carboxyfluorescein and ESR measurement of intravesicular TEM-POcholine reduction by external ascorbate. The process was independent of Ca2+ and took place without any detectable phospholipid hydrolysis. Nonmyotoxic phospholipases tested under the same conditions were unable to induce liposome leakage, which could be detected only when Ca2+ was added to the medium and with the concomitant hydrolysis of phospholipids. The kinetics of Ca(2+)-dependent and Ca(2+)-independent leakage were completely different, indicating two different mechanisms of interaction with the lipid bilayer. Studies using diphenylhexatriene as a probe of lipid membrane organization indicated that the myotoxins gave rise to a profound perturbation of the arrangement of the lipid chains in the membrane interior, whereas interaction of Naja naja phospholipase A2 with the membrane surface did not affect lipid organization. On the basis of these results we suggest that a new type of cytolytic reaction mechanism is responsible for the effects of phospholipase-like myotoxins in vivo.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Isolation and characterization of a myotoxin from the venom of Macrovipera lebetina transmediterranea

The Macrovipera lebetina venom consists of a complex mixture of proteins belonging to a few main families according to their enzymatic and pharmacological activity. Given the serious pathophysiological effects caused by M. lebetina bites mainly induced by muscle degeneration, we decided to investigate the myotoxic activity of some venom fractions. In the present study we describe the purification and characterization of a 22.600 kDa protein, named in the following Mlp4.2, that shares myotoxic but not haemorrhagic activity in vivo. Herein we report that Mlp4.2 is a metalloproteinase belonging to the PI-SVMPS family able, in vitro, to proteolyse extracellular matrix proteins as laminin and fibronectin. Histological observations of mouse anterior tibialis Mlp4.2-treated muscle, demonstrate that this protein induces a massive degeneration of myofibers but not haemorrhage. The immunofluorescence analysis of protein-treated anterior tibialis, demonstrates that Mlp4.2 is able to disarray the laminin network surrounding muscle fibers. Finally Mlp4.2 did not show any direct cytolytic activity towards the myogenic cell line C2C12 in culture. The data reported herein suggest that the myotoxicity of Mlp4.2 is primarily linked to the disruption of the muscle fibers interaction with extracellular matrix proteins