Gene transfer and the evolution of penicillin resistance: PBPs and non-PBP genes in laboratory mutants and clinical isolates in Streptococcus pneumoniae
Final Report Abstract
Intra- and inter-species gene transfer events that include commensal streptococci are a driving force for the emergence and spread of penicillin resistant Streptococcus pneumoniae (PRSP). This results in mosaic genes involved in the resistance phenotype. In contrast, selection with β-lactams in the laboratory leads to point mutations in relevant genes. Using a unique collection of PRSP, laboratory mutants, and transformants obtained with DNA of high-level β-lactam resistant strains, several new aspects on the mechanism and dynamics of penicillin resistance could be revealed. All high molecular weight PBPs including PBP1b, and MurM, contributed to resistance in transformants obtained with S. mitis B6 DNA. In contrast, PBP2x, 2b and 1a, and surprisingly MurE, a UDP-N-acetylmuramyl tripeptide synthetase, were identified in transformants with S. oralis Uo5 DNA, but PBP2a, PBP1b and MurM were not involved. In piperacillin resistant mutants, the α-GalGlcDAG synthase CpoA is mutated resulting in an altered glycolipid profile; in addition, a two component system and ImdH (inosin-monophosphate dehydrogenase) play a role in some laboratory mutants. PBP2a which is rarely involved in resistance displays a mosaic structure in clinical isolates, whereas in cefotaxime resistant laboratory mutants disruption of pbp2a occurred due to point mutations or internal duplications, confirming fundamental differences between these two settings. The histidine kinase CiaH plays a role in resistance in cefotaxime resistant laboratory mutants and in one high-level PRSP Hu17. Localization studies of the histidine kinase CiaH using gfp-ciaH derivatives revealed that also this protein is at least temporarily located at septal sites. CiaH localization was strongly affected by ciaH232 of S. pneumoniae Hu17 containing a mutation in the external sensor domain, adding another complexity to the process of cell division. Alterations in PBP2x are a prerequisite for the development of high-level PRSP. Comparative analysis of pbp2x from PRSP and commensal streptococci revealed that four pbp2x alleles of penicillin sensitive S. mitis account for most of the diverse sequence blocks in resistant S. pneumoniae, S. pseudopneumoniae, and S. mitis; moreover, S. infantis and S. oralis sequences were observed in rare PRSP. Specific mutational patterns were detected in PBP2x, depending on the parental sequence blocks. Cells containing mosaic PBP genes of the high-level PRSP Hu17 have an altered muropeptide composition in their cell wall, strongly suggesting an altered enzymatic activity of these proteins. High-level penicillin resistance involves a low affinity PBP2b in addition to PBP2x and PBP1a, whereas by itself it confers only small decrease in penicillin susceptibility. One phenotype associated with a low affinity PBP2b, a tolerant response and reduced lysis upon treatment with -lactam antibiotics, was now shown to be not only beneficial during growth in the laboratory, but also in in vivo mouse models, implicating that this resistance determinant is advantageous for the survival in vivo. Mutations distinct from those of clinical isolates occur in PBP2x of two laboratory mutants, C606 and C405, which apparently affect the amount of the protein. The amount of PBP2xC405 could be restored to wild type levels upon deletion of the chaperone/serine protease HtrA which is part of the regulon controlled by the two component system CiaRH. Thus, HtrA targets PBP2xC405 similar to GFP-PBP2x derivatives, but not native PBP2x. Interestingly, mutations observed in two laboratory mutants are located at the surface of the transpeptidase domain and face the C-terminal PASTA domains. Deletions of 3'-terminal sequences affecting the C-terminal PASTA domains of PBP2x documented for the first time that this region is important for β-lactam binding and thus for the enzymatic activity of PBP2x. Moreover, the PASTA domains are crucial for PBP2x localization at the division septum. Positioning of PBP2x at the division site is similar to PBP1a and the serinethreonine kinase StkP, confirming that PBP2x is a key element of the divisome complex.
Publications
- 2012. Molecular mechanism of beta-lactam resistance in Streptococcus pneumoniae. Future Microbiology 7:395-410
Hakenbeck R, Brückner R, Denapaite D, and Maurer P
(See online at https://doi.org/10.2217/FMB.12.2) - 2012. Streptococcus pneumoniae R6 interspecies transformation: genetic analysis of penicillin resistance determinants and genome-wide recombination events. Mol Microbiol 86:692-706
Sauerbier J, Maurer P, Rieger M, and Hakenbeck R
(See online at https://doi.org/10.1111/mmi.12009) - 2012. The C-terminal PASTA-domains of Streptococcus pneumoniae PBP2x are important for beta-lactam binding. Microb Drug Resist 18:314- 321
Maurer P, Todorova K, Sauerbier J, and Hakenbeck R
(See online at https://doi.org/10.1089/mdr.2012.0022) - 2014. A low-affinity penicillin-binding protein 2x variant is required for heteroresistance in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 58:3934-3941
Engel, H., M. Mika, D. Denapaite, R. Hakenbeck, K. Mühlemann, M. Heller, L. J. Hathaway, and M. Hilty
(See online at https://doi.org/10.1128/AAC.02547-14) - 2014. Altered lipid composition in Streptococcus pneumoniae cpoA mutants. BMC. Microbiol. 14:12
Meiers, M., C. Volz, J. Eisel, P. Maurer, B. Henrich, and R. Hakenbeck
(See online at https://doi.org/10.1186/1471-2180-14-12) - 2014. Penicillin-binding protein 2x of Streptococcus pneumoniae: the mutation Ala707Asp within the C-terminal PASTA2 domain leads to destabilization. Microb. Drug Resist. 20:250-257
Schweizer, I., K. Peters, C. Stahlmann, R. Hakenbeck, and D. Denapaite
(See online at https://doi.org/10.1089/mdr.2014.0082) - 2014. Streptococcus pneumoniae PBP2x mid-cell localization requires the C-terminal PASTA domains and is essential for cell shape maintenance. Mol. Microbiol. 92:733-755
Peters, K., I. Schweizer, K. Beilharz, C. Stahlmann, J. W. Veening, R. Hakenbeck, and D. Denapaite
(See online at https://doi.org/10.1111/mmi.12588) - 2015. Genomics, genetic variation, and regions of differences, p. 81-107. In: J. Brown, S. Hammerschmidt, and C. Orihuela (eds.), Streptococcus pneumoniae Molecular mechanisms of host-pathogen interaction. Academic Press, London
Tettelin, H., S. Chancey, T. Mitchell, D. Denapaite, Y. Schähle, M. Rieger, and R. Hakenbeck
(See online at https://doi.org/10.1016/B978-0-12-410530-0.00005-3) - 2015. Lipoteichoic acid of Streptococcus oralis Uo5: a novel biochemical structure comprising an unusual phosphorylcholine substitution pattern compared to Streptococcus pneumoniae. Sci Rep 5:16718
Gisch N, Schwudke D, Thomsen S, Hess N, Hakenbeck R, and Denapaite D
(See online at https://doi.org/10.1038/srep16718) - 2015. Mechanism of beta-lactam action in Streptococcus pneumoniae: the piperacillin paradox. Antimicrob.Agents Chemother. 59:609-621
Philippe, J., B. Gallet, C. Morlot, D. Denapaite, R. Hakenbeck, Y. Chen, T. Vernet, and A. Zapun
(See online at https://doi.org/10.1128/AAC.04283-14) - 2015. Transfer of penicillin resistance from Streptococcus oralis to Streptococcus pneumoniae identifies murE as resistance determinant. Mol. Microbiol. 97:866-880
Todorova, K., P. Maurer, M. Rieger, T. Becker, N. K. Bui, J. Gray, W. Vollmer, and R. Hakenbeck
(See online at https://doi.org/10.1111/mmi.13070) - 2016. Promoter identification and transcription analysis of penicillin-binding protein genes in Streptococcus pneumoniae R6. Microb. Drug Resist. 22:487-498
Peters, K., J. Pipo, I. Schweizer, R. Hakenbeck, and D. Denapaite
(See online at https://doi.org/10.1089/mdr.2016.0084) - 2017. Diversity of mosaic pbp2x families of penicillin-resistant Streptococcus pneumoniae from Iran and Romania. Antimicrob. Agents Chemother.
Mousavi, S. F., M. Pana, M. Feizabadi, P. Jalali, M. Ghita, D. Denapaite, and R. Hakenbeck
(See online at https://doi.org/10.1128/AAC.01535-17) - 2017. Draft genome sequences of two Streptococcus pneumoniae serotype 19A sequence type 226 clinical isolates from Hungary, Hu17 with high-level beta-lactam resistance and Hu15 of a penicillin-sensitive phenotype. Genome Announc. 5
Rieger, M., D. Denapaite, R. Brückner, P. Maurer, and R. Hakenbeck
(See online at https://dx.doi.org/10.1128/genomeA.00401-17) - 2017. Insight into the diversity of penicillin-binding protein 2x alleles and mutations in viridans streptococci. Antimicrob. Agents Chemother. 61
van der Linden, M., J. Otten, C. Bergmann, C. Latorre, J. Linares, and R. Hakenbeck
(See online at https://doi.org/10.1128/AAC.02646-16) - 2017. Long persistence of a Streptococcus pneumoniae 23F clone in a cystic fibrosis patient. mSphere 2
Rieger, M., H. Mauch, and R. Hakenbeck
(See online at https://doi.org/10.1128/mSphere.00201-17) - 2017. New aspects of the interplay between penicillin binding proteins, murM, and the two-component system CiaRH of penicillin-resistant Streptococcus pneumoniae serotype 19A isolates from Hungary. Antimicrob. Agents Chemother. 61
Schweizer, I., S. Blättner, P. Maurer, K. Peters, D. Vollmer, W. Vollmer, R. Hakenbeck, and D. Denapaite
(See online at https://doi.org/10.1128/AAC.00414-17) - (2018) PBP2a in Beta-Lactam Resistant Laboratory Mutants and Clinical Isolates: Disruption versus Reduced Penicillin-Affinity. 2017. Microbial Drug Resistance 24 (6)
van der Linden, Mark; Rutschmann, Jens; Maurer, Patrick; Hakenbeck, Regine
(See online at https://doi.org/10.1089/mdr.2017.0302)
