Searching for Antipneumococcal Targets: Choline-Binding Modules as Phagocytosis Enhancers.

Choline-binding proteins (CBPs) from Streptococcus pneumoniae comprise a family of modular polypeptides involved in essential events of this pathogen. They recognize the choline residues present in the teichoic and lipoteichoic acids of the cell wall using the so-called choline-binding modules (CBMs). The importance of CBPs in pneumococcal physiology points to them as novel targets to combat antimicrobial resistances shown by this organism. In this work we have tested the ability of exogenously added CBMs to act as CBP inhibitors by competing with the latter for the binding to the choline molecules in the bacterial surface. First, we carried out a thorough physicochemical characterization of three native CBMs, namely C-LytA, C-Cpl1, and C-CbpD, and assessed their affinity for choline and macromolecular, pneumococcal cell-wall mimics. The interaction with these substrates was evaluated by molecular modeling, analytical ultracentrifugation, surface plasmon resonance, and fluorescence and circular dichroism spectroscopies. Van't Hoff thermal analyses unveiled the existence of one noncanonical choline binding site in each of the C-Cpl1 and C-CbpD proteins, leading in total to 5 ligand-binding sites per dimer and 4 sites per monomer, respectively. Remarkably, the binding affinities of the CBMs do not directly correlate with their native oligomeric state or with the number of choline-binding sites, suggesting that choline recognition by these modules is a complex phenomenon. On the other hand, the exogenous addition of CBMs to pneumococcal planktonic cultures caused extensive cell-chaining probably as a consequence of the inhibition of CBP attachment to the cell wall. This was accompanied by bacterial aggregation and sedimentation, causing an enhancement of bacterial phagocytosis by peritoneal macrophages. In addition, the rational design of an oligomeric variant of a native CBM led to a substantial increase in its antibacterial activity by multivalency effects. These results suggest that CBMs might constitute promising nonlytic antimicrobial candidates based on the natural induction of the host defense system.

Widening the antimicrobial spectrum of esters of bicyclic amines: In vitro effect on gram-positive Streptococcus pneumoniae and gram-negative non-typeable Haemophilus influenzae biofilms.

Antibiotic resistance is a global current threat of increasing importance. Moreover, biofilms represent a medical challenge since the inherent antibiotic resistance of their producers demands the use of high doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence, therefore demanding the development of novel antimicrobials. Esters of bicyclic amines (EBAs), which are strong inhibitors of Streptococcus pneumoniae growth, were initially designed as inhibitors of pneumococcal choline-binding proteins on the basis of their structural analogy to the choline residues in the cell wall. However, instead of mimicking the characteristic cell chaining phenotype caused by exogenously added choline on planktonic cultures of pneumococcal cells, EBAs showed an unexpected lytic activity. In this work we demonstrate that EBAs display a second, and even more important, function as cell membrane destabilizers. We then assayed the inhibitory and disintegrating activity of these molecules on pneumococcal biofilms. The selected compound (EBA 31) produced the highest effect on S. pneumoniae (encapsulated and non-encapsulated) biofilms at very low concentrations. EBA 31 was also effective on mixed biofilms of non-encapsulated S. pneumoniae plus non-typeable Haemophilus influenzae, two pathogens frequently forming a self-produced biofilm in the human nasopharynx. These results support the role of EBAs as a promising alternative for the development of novel, broad-range antimicrobial drugs encompassing both Gram-positive and Gram-negative pathogens.

An enzymatic system for decolorization of wastewater dyes using immobilized CueO laccase-like multicopper oxidase on poly-3-hydroxybutyrate.

The presence of synthetic dyes in wastewaters generated by the textile industry constitutes a serious environmental and health problem that urges the scientific community on an appropriate action. As a proof-of-concept, we have developed a novel approach to design enzymatic bioreactors with the ability to decolorize dye solutions through the immobilization of the bacterial CueO laccase-like multicopper oxidase from Escherichia coli on polyhydroxybutyrate (PHB) beads by making use of the BioF affinity tag. The decolorization efficiency of the system was characterized by a series of parameters, namely maximum enzyme adsorption capacity, pH profile, kinetic constants, substrate range, temperature and bioreactor recycling. Depending on the tested dye, immobilization increased the catalytic activity of CueO by up to 40-fold with respect to the soluble enzyme, reaching decolorization efficiencies of 45-90%. Our results indicate that oxidase bioreactors based on polyhydroxyalkanoates are a promising alternative for the treatment of coloured industrial wastewaters.