Making and Breaking Streptococcal Rhamnose Polysaccharides

Abstract

Already for more than half a century, scientists have made efforts to develop vaccines that prevent streptococcal infections in humans and animals. Antibiotics that target these bacteria are readily available and still active, but are not sufficient to prevent the millions of deaths that result from streptococcal infections each year worldwide. Especially vaccines targeting Streptococcus pyogenes and Streptococcus mutans have received special attention. S. pyogenes ranks in the top ten of infection associated mortality worldwide, with a disproportionate disease burden in low and middle-income countries. S. mutans is considered the main etiological agent of dental caries, causing a less severe, but widespread global burden for human health. Current vaccine strategies mainly focus on protein antigens on the bacterial cell walls of these bacteria. However, the use of Streptococcal Rhamnose Polysaccharides (SRPs) as vaccine antigens is of considerable interest. SRPs are glycopolymers that are covalently attached to peptidoglycan and contain substantial amounts of the sugar rhamnose. The majority of the SRPs characterized to date are composed of a polyrhamnose backbone and differ in the identity or linkage of the glycan side chains that are attached to this backbone. In contrast to cell wall-anchored proteins, SRPs are more abundant representing up to 60% of the cell wall mass. In addition, SRPs have essential roles in bacterial physiology, host colonization, pathogenesis and immune evasion. However, knowledge regarding SRP biosynthesis or structure is limited. In this thesis, I studied the function of three enzymes involved in dTDP-L-rhamnose biosynthesis at a biochemical, structural and genetic level, in both S. pyogenes and S. mutans. Even though currently available antibiotics are still effective against most streptococcal species, this is not always sufficient in cases of severe fast-developing invasive disease. In addition, the rapid increase of antibiotic resistance observed in other bacteria is a big concern. With this knowledge, we aimed to identify small chemical compounds to prevent streptococcal infection by targeting dTDP-L-rhamnose production, which is not only essential for streptococci, but for many other medically relevant human pathogens like Mycobacterium tuberculosis. One of the identified compounds is of particular interest to develop further due to its ability to prevent streptococcal growth with low toxic effects on human cells and bacteria that do not produce rhamnose. Besides the rhamnose biosynthesis pathway, I also studied how SRPs are transported across the cell membrane of S. mutans. This process involves two proteins, which generate a pore in the membrane and provide the energy for transport, respectively. I also presented evidence for the involvement of a third protein. Based on currently available knowledge on polysaccharide translocation, we hypothesize that this protein may modify SRPs with -as yet- unidentified modifications to determine SRP chain length and trigger transport. Moreover, SRP transport would provide an interesting new target for the development of antibiotics as well. Finally, I hope that my research creates awareness of the therapeutic potential of SRPs, not only as vaccine candidates, but also as targets for antibiotic development.