DETECTION AND ISOLATION OF THE BACTERIAL SECOND MESSENGER (P)PPGPP

This work has been carried out within the framework of the ERC funded project ERACHRON (Eradicating chronic infection), directed to the study of the chemical biology of bacterial persisters. Persisters are dormant phenotypic variants that are present in small amount in microbial populations. This phenotype shows high tolerance to antibiotics and for this reason it plays a key role in chronic infections. Indeed, after antibiotic treatment kills most of the bacterial population, persisters surviving the treatment can revert to the active phenotype, resuming growth and restoring the original, antibiotic sensitive population. One of the hypotheses for the formation of persisters is the activation of the stringent response, a signaling cascade that starts with the accumulation of guanosine tetra- or penta-phosphate, collectively called (p)ppGpp (or simply alarmone). The accumulation of the latter is promoted by a superfamily of enzymes called RSH (RelA/SpoT Homolog) that catalyze the bis-phosphorylation of either GDP or GTP at the level of their 3’ position. The aim of the ERACHRON project is to design and synthesize inhibitors of RSH proteins. The goal of my thesis work has been to make available to the laboratory a reliable and safe assay for real-time detection of the enzymatic activity of RSH enzymes, to synthesize the assay components, and to establish a source for pure (p)ppGpp, required for enzymatic inhibition studies. So far, RSHs enzymatic activity has been quantitatively monitored only using radiolabeled substrates such as 32P-GDP, entailing not only obvious safety concerns but also preventing real time reaction monitoring. In this context, we significantly optimized the synthesis of the known (p)ppGpp selective chemosensor PyPDA. The reported procedure consisted of 6 steps and 9% overall yield, while our sequence afforded PyDPA in 7 steps and a 67% overall yield (M. Minneci et al. ChemBioChem, 2019, 20(18), 1717-21). On the other hand, we synthesized ppGpp on a 30mg scale exploiting the enzymatic activity of an RSH protein. Isolation of the pure product from the reaction mixture was successfully achieved in 60-80% yield using a scavenging resin followed by a precipitation step. After performing the PyDPA-based fluorescent assay, we observed that the results were not reproducible in our hands, probably due to the non-linearity of the response with respect to the analyte concentration and to the limited operative concentration range. While searching for alternative quantitative detection methods, inspired by the working mechanism of the chemosensor, we set out to build a scavenging system able to selectively extract (p)ppGpp from the reaction mixture. As a proof of concept, a simplified binding unit capable of a specifically retaining nucleotides was synthesized and, in collaboration with Dr. Marcella Chiari (CNR, Milan), a linear polymer was chosen to immobilize it. The functionalized polymer was used to cover an inert solid support. The scavenging system obtained in this way was tested using several nucleotides solutions, showing a loading ability of ca. 10 µmol/g (similar or even higher than other commercially available solid-supported scavengers of nucleotides). Notably, the system is potentially reusable but still needs further optimization. A binding unit potentially selective for (p)ppGpp was also synthesized, showing a preferential interaction with the alarmone in solution (by STD-NMR), and will be used in the near future for the construction of a selective system.