Inhibition of pneumococcal choline‐binding proteins and cell growth by esters of bicyclic amines

Abstract

Streptococcus pneumoniae is one of the major pathogens worldwide. The use of currently available antibiotics to treat pneumococcal diseases is hampered by increasing resistance levels; also, capsular polysaccharide‐based vaccination is of limited efficacy. Therefore, it is desirable to find targets for the development of new antimicrobial drugs specifically designed to fight pneumococcal infections. Choline‐binding proteins are a family of polypeptides, found in all S. pneumoniae strains, that take part in important physiologic processes of this bacterium. Among them are several murein hydrolases whose enzymatic activity is usually inhibited by an excess of choline. Using a simple chromatographic procedure, we have identified several choline analogs able to strongly interact with the choline‐binding module (C‐LytA) of the major autolysin of S. pneumoniae. Two of these compounds (atropine and ipratropium) display a higher binding affinity to C‐LytA than choline, and also increase the stability of the protein. CD and fluorescence spectroscopy analyses revealed that the conformational changes of C‐LytA upon binding of these alkaloids are different to those induced by choline, suggesting a different mode of binding. In vitro inhibition assays of three pneumococcal, choline‐dependent cell wall lytic enzymes also demonstrated a greater inhibitory efficiency of those molecules. Moreover, atropine and ipratropium strongly inhibited in vitro pneumococcal growth, altering cell morphology and reducing cell viability, a very different response than that observed upon addition of an excess of choline. These results may open up the possibility of the development of bicyclic amines as new antimicrobials for use against pneumococcal pathologies.

Multivalent Choline Dendrimers Increase Phagocytosis of <b><i>Streptococcus pneumoniae </i></b>R6 by Microglial Cells

“…Phagocytosis of bacteria by microglial cells was substantially increased in both cases, although to a different extent, depending on the interval of dendrimer-bacterial co-incubation, probably reflecting the different chain lengths induced in each condition. This is in accordance with previous results showing that the potency of the effect exerted by choline and its analogs depends on the metabolic state of the bacterium [5]. Addition of the g2-cho dendrimer at the beginning of the pneumococcal growth probably reflects its use as a prophylactic agent to prevent colonization while its addition at the end of the logarithmic phase reflects the conditions of manifest infection.…”

“…Therefore, CBPs might constitute a new target for the development of novel antimicrobials. Inhibition of CBPs by the addition of choline and choline analogs (atropine and ipratropium) blocked cell separation and the characteristic autolysis of Spn, thereby inducing the formation of long chains or even preventing bacterial growth [5,15]. However, this interaction was weak, requiring high concentrations (in the millimolar range) to achieve a therapeutic effect.…”

“…Choline-binding proteins (CBPs) are cell surface virulence factors [for a review, see ref. [4]] common to all pneumococcal serotypes, so they are potential drug development targets [5]. Blocking pneumococcal CBPs would indeed reduce bacterial dissemination and prevent the release of bacterial products responsible for the inflammatory response which in pneumonia and bacterial meningitis contribute to the development of tissue damage [6].…”

Multivalent Choline Dendrimers as Potent Inhibitors of Pneumococcal Cell‐Wall Hydrolysis

“…The weak interaction between choline and a single choline-binding site [10,12] explains the need for tandem chol- Figure 1. a) Schematic representation of the S. pneumoniae cell-wall structure showing the multivalent arrangement of phosphocholine groups.…”

“…Exogenously added choline and choline analogues competitively inhibit the binding of CBPs to the cell wall, blocking cell separation and the characteristic autolysis of S. pneumoniae at the end of the stationary phase of growth, inducing instead the formation of long chains [9] or even preventing growth. [10] These effects are thought to reduce bacterial virulence by preventing the release of toxins upon cell autolysis and limiting the dissemination of the bacteria on the host tissue during infection.…”