Investigating peptidoglycan synthesis as a mechanism for maintaining the fidelity of protein translation in Streptococcus pneumoniae
Peptidoglycan (PG) is an essential component of the bacterial cell wall, the precursors of which are comprised of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) with an appended pentapeptide stem (Figure 1). These are polymerised and the pentapeptide stem is cross-linked either directly or indirectly (via dipeptide bridges) to adjacent peptide stems (Figure 2). This mesh-like structure is important in providing structural strength and rigidity, flexibility during growth and division, as well as countering osmotic pressures of the cytoplasm .
S.pneumoniae is a gram-positive, facultative anaerobe that resides asymptomatically in the nasopharynx of 5-10% and 20-40% of healthy adults and children respectively . It is clinically important, as it is the leading cause of community-acquired pneumonia, and is also an etiological agent for otitis media, meningitis and septacemia .
The introduction of β-lactams, such as penicillin, to treat pneumococcal infections resulted in the emergence of resistant strains of S. pneumoniae. The first penicillin-resistant strain was identified in 1967 and subsequently numerous extensively multi-drug resistant strains began to emerge, putting understanding of the mechanisms of antibiotic resistance in pneumococci at the forefront of medical research .
Mutations inthe penicillin binding proteins (PBP’s) that confer reduced binding to β-lactams are a major resistance mechanism in S. pneumoniae. In resistant pneumococci, the peptidoglycan layer contains an increase in abnormal indirect cross-links  comprised of a dipeptide ‘bridge’ of Ala-Ala or Ser-Ala. MurM and MurN are ATP dependent aminoacyl-tRNA ligases that are responsible for the addition of the first position Serine/Alanine and the second position Alanine respectively, to the lipid linked peptidoglycan precursors lipid I and lipid II, resulting in the formation of branched peptidoglycan containing the dipeptide ‘bridge’.
MurM can utilise both seryl-tRNASer and alanyl-tRNAAla as substrates however, interestingly, the preferred substrate is the mis-aminoacylated tRNA (seryl-tRNAAla). MurM is necessary but not sufficient for penicillin resistance, since some β-lactam sensitive strains of S. pneumoniae such as R6, contain levels of indirect cross-linking comparable to that of resistant pneumococci, however deletion of the MurMN operon in resistant strains results in the disappearance of cross-bridges and a complete loss in penicillin resistance . Mutations occurring in the transpeptidase active site of PBPs that cause decreased penicillin binding may also cause changes to the substrate binding site resulting in an altered substrate specificity for peptidoglycan precursors with MurM driven incorporation of Ala-Ala or Ala-Ser branches. This presents a new paradigm for novel antibiotic development such that inhibition of cell wall branching enzymes such as MurM may restore penicillin sensitivity to previously resistant strains .
S. pneumoniae produces H2O2 as a by-product of aerobic metabolism, but unlike most aerobes it does not express catalase (the enzyme responsible for elimination of H2O2). Therefore it must possess other mechanisms for mitigating the impact of oxidative stress, particularly as the reactivity of H2O2 impairs mechanisms employed by aminoacyl-tRNA synthetases to avoid incorrect (mis)-aminoacylation of tRNAs with noncognate amino acids and consequent corruption of the fidelity of protein synthesis. Therefore, the premise of this PhD is that under oxidative stress, as the frequency of mis-aminoacylation of tRNA’s increases, MurM may act to eliminate mis-aminoacylated species by directing them into peptidoglycan synthesis, thereby contributing to maintaining the fidelity of protein synthesis.
My research aims to evaluate this premise by elucidating and comparing the effects of oxidative stress on the kinetics of MurM in resistant and susceptible strains and those aminoacyl-tRNA synthetases that supply MurM with aminoacyl-tRNA substrates. Furthermore, an array of techniques such as circular dichroism, isothermal calorimetry, surface plasmon resonance and X-ray crystallography will be used to determine the structure and potential protein-protein, protein:RNA and protein:peptidoglycan precursor interactions (and combinations thereof) of MurM.
 Longenecker, K.L. et al., 2005. Structure of MurF from Streptococcus pneumoniae co-crystallized with a small molecule inhibitor exhibits interdomain closure. Protein science : a publication of the Protein Society, 14(12), pp.3039–47.
 Shepherd, J. & Ibba, M., 2013. Lipid II-independent trans editing of mischarged tRNAs by the penicillin resistance factor MurM. The Journal of biological chemistry, 288(36), pp.25915–23.
 Philippe, J. et al., 2015. Mechanism of β-lactam action in Streptococcus pneumoniae: the piperacillin paradox. Antimicrobial agents and chemotherapy, 59(1), pp.609–21.
 Filipe, S.R., Severina, E. & Tomasz, A., 2001. The role of murMN operon in penicillin resistance and antibiotic tolerance of Streptococcus pneumoniae. Microbial drug resistance (Larchmont, N.Y.), 7(4), pp.303–16.
 Fiser, A., Filipe, S.R. & Tomasz, A., 2003. Cell wall branches, penicillin resistance and the secrets of the MurM protein. Trends in Microbiology, 11(12), pp.547–553.
Dr David Roper
University of Warwick
David dot Roper at warwick dot ac dot uk
Dr Adrian Lloyd
University of Warwick
Adrian dot Lloyd at warwick dot ac dot uk