Endotoxin-dependent release of IL-1β from mouse microglial cells is a very inefficient process, as it is slow and leads to accumulation of a modest amount of extracellular cytokine. Furthermore, secreted IL-1β is mostly in the procytokine unprocessed form. Addition of extracellular ATP to LPS-primed microglia caused a burst of release of a large amount of processed IL-1β. ATP had no effect on the accumulation of intracellular pro-IL-1β in the absence of LPS. In LPS-treated cells, ATP slightly increased the synthesis of pro-IL-1β. Optimal ATP concentration for IL-1β secretion was between 3 and 5 mM, but significant release could be observed at concentrations as low as 1 mM. At all ATP concentrations IL-1β release could be inhibited by increasing the extracellular K+ concentration. ATP-dependent IL-1β release was also inhibited by 90 and 60% by the caspase inhibitors YVAD and DEVD, respectively. Accordingly, in ATP-stimulated microglia, the p20 proteolytic fragment derived from activation of the IL-1-β-converting enzyme could be detected by immunoblot analysis. These experiments show that in mouse microglial cells extracellular ATP triggers fast maturation and release of intracellularly accumulated IL-β by activating the IL-1-β-converting enzyme/caspase 1.

SummaryThe PhaF is a nucleoid-associated like protein of Pseudomonas putida KT2442 involved in the polyhydroxyalkanoate (

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Phasins are intracellular polyhydroxyalkanoat4e (PHA)-associated proteins involved in the stabilization of these bacterial carbon storage granules. Despite its importance in PHA metabolism and regulation, only few reports have focused so far on the structure of these proteins. In this work we have investigated the structure and stability of the PhaF phasin from Pseudomonas putida KT2440, a protein that is involved in PHA granule stabilization and distribution to daughter cells upon cell division. A structural, three-dimensional model of the protein was built from homology modeling procedures and consensus secondary structure predictions. The model predicts that PhaF is an elongated protein, with a long, amphipathic N-terminal helix with PHA binding capacity, followed by a short leucine zipper involved in protein oligomerization and a superhelical C-terminal domain wrapped around the chromosomal DNA. Hydrodynamic, spectroscopical and thermodynamic experiments validated the model and confirmed both that free PhaF is a tetramer in solution and that most part of the protein is intrinsically disordered in the absence of its ligands. The results lay a molecular basis for the explanation of the biological role of PhaF and, along with an exhaustive analysis of phasin sequence databases, suggest that intrinsic disorder and oligomerization through coiled-coils may be a widespread mechanism among these proteins.

Streptococcus pneumoniae (pneumococcus) is an important pathogen responsible for acute invasive and non-invasive infections such as meningitis, sepsis and otitis media, being the major cause of community-acquired pneumonia. The fight against pneumococcus is currently hampered both by insufficient vaccine coverage and by rising antimicrobial resistances to traditional antibiotics, making necessary the research on novel targets. Choline binding proteins (CBPs) are a family of polypeptides found in pneumococcus and related species, as well as in some of their associated bacteriophages. They are characterized by a structural organization in two modules: a functional module (FM), and a choline-binding module (CBM) that anchors the protein to the choline residues present in the cell wall through non-covalent interactions. Pneumococcal CBPs include cell wall hydrolases, adhesins and other virulence factors, all playing relevant physiological roles for bacterial viability and virulence. Moreover, many pneumococcal phages also make use of hydrolytic CBPs to fulfill their infectivity cycle. Consequently, CBPs may play a dual role for the development of novel antipneumococcal drugs, both as targets for inhibitors of their binding to the cell wall and as active cell lytic agents (enzybiotics). In this article, we review the current state of knowledge about host- and phage-encoded pneumococcal CBPs, with a special focus on structural issues, together with their perspectives for effective anti-infectious treatments.

Polyhydroxyalkanoates (PHAs) can be catabolized by many microorganisms using intra-or extracellular PHA depolymerases. Most of our current knowledge of these intracellular enzyme-coding genes comes from the analysis of short chain length PHA depolymerases, whereas medium chain length PHA (mcl-PHA) intracellular depolymerization systems still remained to be characterized. The phaZ gene of some Pseudomonas putida strains has been identified only by mutagenesis and complementation techniques as putative intracellular mcl-PHA depolymerase. However, none of their corresponding encoded PhaZ enzymes have been characterized in depth. In this study the PhaZ depolymerase from P. putida KT2442 has been purified and biochemically characterized after its overexpression in Escherichia coli. To facilitate these studies we have developed a new and very sensitive radioactive method for detecting PHA hydrolysis in vitro. We have demonstrated that PhaZ is an intracellular depolymerase that is located in PHA granules and that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. We have modeled the three-dimensional structure of PhaZ complexed with a 3-hydroxyoctanoate dimer. Using this model, we found that the enzyme appears to be built up from a core ␣/␤ hydrolase-type domain capped with a lid structure with an active site containing a catalytic triad buried near the connection between domains. All these data constitute the first biochemical characterization of PhaZ and allow us to propose this enzyme as the paradigmatic representative of intracellular endo/ exo-mcl-PHA depolymerases.

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We have developed a new single-step system, using a DEAE matrix, to immobilize and/or purify fusion proteins containing the choline-binding domain of the Streptococcus pneumoniae murein hydrolases. We have constructed a choline-binding-domain -P-galactosidase chimera, which can be purified by this procedure and shows a high P-galactosidase activity when immobilized in the column. A vector plasmid, pCUZl, containing the Ippp-5/lac promoter as well as 13 restriction sites, was constructed to facilitate the cloning and expression of gene fusions. This plasmid also allows the selection of recombinants by the well-known blue/white 5-bromo-4-chloro-3-indolyl-~-~-galactoside procedure.