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.

A gene regulatory network to control EMT programs in development and disease.

The Epithelial to Mesenchymal Transition (EMT) regulates cell plasticity during embryonic development and in disease. It is dynamically orchestrated by transcription factors (EMT-TFs), including Snail, Zeb, Twist and Prrx, all activated by TGF-ß among other signals. Here we find that Snail1 and Prrx1, which respectively associate with gain or loss of stem-like properties and with bad or good prognosis in cancer patients, are expressed in complementary patterns during vertebrate development and in cancer. We show that this complementarity is established through a feedback loop in which Snail1 directly represses Prrx1, and Prrx1, through direct activation of the miR-15 family, attenuates the expression of Snail1. We also describe how this gene regulatory network can establish a hierarchical temporal expression of Snail1 and Prrx1 during EMT and validate its existence in vitro and in vivo, providing a mechanism to switch and select different EMT programs with important implications in development and disease.

MicroRNAs Establish the Right-Handed Dominance of the Heart Laterality Pathway in Vertebrates.

Despite their external bilateral symmetry, vertebrates have internal left/right (L/R) asymmetries required for optimal organ function. BMP-induced epithelial to mesenchymal transition (EMT) in the lateral plate mesoderm (LPM) triggers L/R asymmetric cell movements toward the midline, higher from the right, which are crucial for heart laterality in vertebrates. However, how the L/R asymmetric levels of EMT factors are achieved is not known. Here, we show that the posterior-to-anterior Nodal wave upregulates several microRNAs (miRNAs) to transiently attenuate the levels of EMT factors (Prrx1a and Snail1) on the left LPM in a Pitx2-independent manner in the fish and mouse. These data clarify the role of Nodal in heart laterality and explain how Nodal and BMP exert their respective dominance on the left and right sides through the mutual inhibition of their respective targets, ensuring the proper balance of L/R information required for heart laterality and morphogenesis.