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

ABSTRACT The Streptococcus pneumoniae clone Hungary19A-6 expresses unusually high levels of β-lactam resistance, which is in part due to mutations in the MurM gene, encoding a transferase involved in the synthesis of branched peptidoglycan. Moreover, it contains the allele ciaH232, encoding the histidine kinase CiaH (M. Müller, P. Marx, R. Hakenbeck, and R. Brückner, Microbiology 157:3104–3112, 2011, https://doi.org/10.1099/mic.0.053157-0 ). High-level penicillin resistance primarily requires the presence of low-affinity (mosaic) penicillin binding protein (PBP) genes, as, for example, in strain Hu17, a closely related member of the Hungary19A-6 lineage. Interestingly, strain Hu15 is β-lactam sensitive due to the absence of mosaic PBPs. This unique situation prompted us to investigate the development of cefotaxime resistance in transformation experiments with genes known to play a role in this phenotype, pbp2x, pbp1a, murM, and ciaH, and penicillin-sensitive recipient strains R6 and Hu15. Characterization of phenotypes, peptidoglycan composition, and CiaR-mediated gene expression revealed several novel aspects of penicillin resistance. The murM gene of strain Hu17 (murMHu17), which is highly similar to murM of Streptococcus mitis, induced morphological changes which were partly reversed by ciaH232. murMHu17 conferred cefotaxime resistance only in the presence of the pbp2x of strain Hu17 (pbp2xHu17). The ciaH232 allele contributed to a remarkable increase in cefotaxime resistance in combination with pbp2xHu17 and pbp1a of strain Hu17 (pbp1aHu17), accompanied by higher levels of expression of CiaR-regulated genes, documenting that ciaH232 responds to PBP1aHu17-mediated changes in cell wall synthesis. Most importantly, the proportion of branched peptides relative to the proportion of linear muropeptides increased in cells containing mosaic PBPs, suggesting an altered enzymatic activity of these proteins.

[1]  M. Rieger,et al.  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 , 2017, Genome Announcements.

[2]  M. P. van der Linden,et al.  Insight into the Diversity of Penicillin-Binding Protein 2x Alleles and Mutations in Viridans Streptococci , 2017, Antimicrobial Agents and Chemotherapy.

[3]  P. Kappeler,et al.  Highly Variable Streptococcus oralis Strains Are Common among Viridans Streptococci Isolated from Primates , 2016, mSphere.

[4]  M. Rieger,et al.  Transfer of penicillin resistance from Streptococcus oralis to Streptococcus pneumoniae identifies murE as resistance determinant , 2015, Molecular microbiology.

[5]  A. Kaysen,et al.  Control of competence by related non-coding csRNAs in Streptococcus pneumoniae R6 , 2015, Front. Genet..

[6]  Stephen J. Salipante,et al.  A Year of Infection in the Intensive Care Unit: Prospective Whole Genome Sequencing of Bacterial Clinical Isolates Reveals Cryptic Transmissions and Novel Microbiota , 2015, PLoS genetics.

[7]  M. Kilian,et al.  Commensal Streptococci Serve as a Reservoir for β-Lactam Resistance Genes in Streptococcus pneumoniae , 2015, Antimicrobial Agents and Chemotherapy.

[8]  R. Brückner,et al.  Activity of the response regulator CiaR in mutants of Streptococcus pneumoniae R6 altered in acetyl phosphate production , 2015, Front. Microbiol..

[9]  K. R. Short,et al.  Nasopharyngeal Colonization with Streptococcus pneumoniae , 2015 .

[10]  T. Welte,et al.  Pneumococcal vaccination: what have we learnt so far and what can we expect in the future? , 2014, European Journal of Clinical Microbiology & Infectious Diseases.

[11]  W. Hanage,et al.  Comprehensive Identification of Single Nucleotide Polymorphisms Associated with Beta-lactam Resistance within Pneumococcal Mosaic Genes , 2014, PLoS genetics.

[12]  David R. Riley,et al.  Parallel Evolution of Streptococcus pneumoniae and Streptococcus mitis to Pathogenic and Mutualistic Lifestyles , 2014, mBio.

[13]  G. Minasov,et al.  Structure of the LdcB LD-Carboxypeptidase Reveals the Molecular Basis of Peptidoglycan Recognition , 2014, Structure.

[14]  W. Hanage,et al.  Evidence for Soft Selective Sweeps in the Evolution of Pneumococcal Multidrug Resistance and Vaccine Escape , 2014, Genome biology and evolution.

[15]  J. Veening,et al.  Streptococcus pneumoniae PBP2x mid‐cell localization requires the C‐terminal PASTA domains and is essential for cell shape maintenance , 2014, Molecular microbiology.

[16]  R. Brückner,et al.  Target evaluation of the non‐coding csRNAs reveals a link of the two‐component regulatory system CiaRH to competence control in Streptococcus pneumoniae R6 , 2013, Molecular microbiology.

[17]  E. Tuomanen,et al.  The pneumococcus: epidemiology, microbiology, and pathogenesis. , 2013, Cold Spring Harbor perspectives in medicine.

[18]  T. Kochan,et al.  The HtrA Protease of Streptococcus pneumoniae Controls Density-Dependent Stimulation of the Bacteriocin blp Locus via Disruption of Pheromone Secretion , 2013, Journal of bacteriology.

[19]  M. Rieger,et al.  Streptococcus pneumoniae R6 interspecies transformation: genetic analysis of penicillin resistance determinants and genome‐wide recombination events , 2012, Molecular microbiology.

[20]  M. Sebert,et al.  The HtrA Protease from Streptococcus pneumoniae Digests Both Denatured Proteins and the Competence-stimulating Peptide* , 2012, The Journal of Biological Chemistry.

[21]  W. Vollmer,et al.  Biosynthesis of teichoic acids in Streptococcus pneumoniae and closely related species: lessons from genomes. , 2012, Microbial drug resistance.

[22]  R. Hakenbeck,et al.  Molecular mechanisms of β-lactam resistance in Streptococcus pneumoniae. , 2012, Future microbiology.

[23]  A. Tomasz,et al.  Isolation and analysis of cell wall components from Streptococcus pneumoniae. , 2012, Analytical biochemistry.

[24]  W. Schaffner,et al.  Prevention of antibiotic-nonsusceptible Streptococcus pneumoniae with conjugate vaccines. , 2012, The Journal of infectious diseases.

[25]  R. Hakenbeck,et al.  Mechanisms of Penicillin Resistance in Streptococcus pneumoniae: Targets, Gene Transfer and Mutations , 2012 .

[26]  R. Hakenbeck,et al.  Effect of new alleles of the histidine kinase gene ciaH on the activity of the response regulator CiaR in Streptococcus pneumoniae R6. , 2011, Microbiology.

[27]  M. Winkler,et al.  The Requirement for Pneumococcal MreC and MreD Is Relieved by Inactivation of the Gene Encoding PBP1a , 2011, Journal of bacteriology.

[28]  R. Hakenbeck,et al.  Activity of the Two-Component Regulatory System CiaRH in Streptococcus pneumoniae R6 , 2011, Journal of Molecular Microbiology and Biotechnology.

[29]  M. Winkler,et al.  Characterization of Mutants Deficient in the l,d-Carboxypeptidase (DacB) and WalRK (VicRK) Regulon, Involved in Peptidoglycan Maturation of Streptococcus pneumoniae Serotype 2 Strain D39 , 2011, Journal of bacteriology.

[30]  M. Nuhn,et al.  Identification of genes for small non-coding RNAs that belong to the regulon of the two-component regulatory system CiaRH in Streptococcus , 2010, BMC Genomics.

[31]  Ernesto García,et al.  Vancomycin tolerance in clinical and laboratory Streptococcus pneumoniae isolates depends on reduced enzyme activity of the major LytA autolysin or cooperation between CiaH histidine kinase and capsular polysaccharide , 2010, Molecular microbiology.

[32]  T. Cherian,et al.  Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates , 2009, The Lancet.

[33]  M. Winkler,et al.  Influences of Capsule on Cell Shape and Chain Formation of Wild-Type and pcsB Mutants of Serotype 2 Streptococcus pneumoniae , 2009, Journal of bacteriology.

[34]  M. Sebert,et al.  Bacteriocin Activity of Streptococcus pneumoniae Is Controlled by the Serine Protease HtrA via Posttranscriptional Regulation , 2008, Journal of bacteriology.

[35]  R. Hakenbeck,et al.  An Important Site in PBP2x of Penicillin-Resistant Clinical Isolates of Streptococcus pneumoniae: Mutational Analysis of Thr338 , 2008, Antimicrobial Agents and Chemotherapy.

[36]  A. Tomasz,et al.  Characterization of tRNA-dependent Peptide Bond Formation by MurM in the Synthesis of Streptococcus pneumoniae Peptidoglycan* , 2008, Journal of Biological Chemistry.

[37]  T. Vernet,et al.  Penicillin-binding proteins and beta-lactam resistance. , 2008, FEMS microbiology reviews.

[38]  M. de Pedro,et al.  Peptidoglycan structure and architecture. , 2008, FEMS microbiology reviews.

[39]  R. Hakenbeck,et al.  Identification of the genes directly controlled by the response regulator CiaR in Streptococcus pneumoniae: five out of 15 promoters drive expression of small non‐coding RNAs , 2007, Molecular microbiology.

[40]  R. Hakenbeck,et al.  A new integrative reporter plasmid for Streptococcus pneumoniae. , 2007, FEMS microbiology letters.

[41]  M. Lipsitch,et al.  Age- and Serogroup-Related Differences in Observed Durations of Nasopharyngeal Carriage of Penicillin-Resistant Pneumococci , 2007, Journal of Clinical Microbiology.

[42]  J. Glass,et al.  Genome Sequence of Avery's Virulent Serotype 2 Strain D39 of Streptococcus pneumoniae and Comparison with That of Unencapsulated Laboratory Strain R6 , 2006, Journal of bacteriology.

[43]  W. Schaffner,et al.  Effect of introduction of the pneumococcal conjugate vaccine on drug-resistant Streptococcus pneumoniae. , 2006, The New England journal of medicine.

[44]  T. Mascher,et al.  The CiaRH System of Streptococcus pneumoniae Prevents Lysis during Stress Induced by Treatment with Cell Wall Inhibitors and by Mutations in pbp2x Involved in β-Lactam Resistance , 2006, Journal of bacteriology.

[45]  M. Sebert,et al.  Pneumococcal HtrA Protease Mediates Inhibition of Competence by the CiaRH Two-Component Signaling System , 2005, Journal of bacteriology.

[46]  T. Mitchell,et al.  Control of Virulence by the Two-Component System CiaR/H Is Mediated via HtrA, a Major Virulence Factor of Streptococcus pneumoniae , 2004, Journal of bacteriology.

[47]  Sébastien Guiral,et al.  Interconnection of competence, stress and CiaR regulons in Streptococcus pneumoniae: competence triggers stationary phase autolysis of ciaR mutant cells , 2004, Molecular microbiology.

[48]  K. Klugman,et al.  Site-Specific Mutagenesis Analysis of PBP 1A from a Penicillin-Cephalosporin-Resistant Pneumococcal Isolate , 2003, Antimicrobial Agents and Chemotherapy.

[49]  T. Mascher,et al.  The Streptococcus pneumoniae cia Regulon: CiaR Target Sites and Transcription Profile Analysis , 2003, Journal of bacteriology.

[50]  M. Sebert,et al.  Microarray-Based Identification of htrA, a Streptococcus pneumoniae Gene That Is Regulated by the CiaRH Two-Component System and Contributes to Nasopharyngeal Colonization , 2002, Infection and Immunity.

[51]  T. Mascher,et al.  The ciaR/ciaH regulatory network of Streptococcus pneumoniae. , 2002, Journal of molecular microbiology and biotechnology.

[52]  W. P. Schneider,et al.  Differential Fluorescence Induction Analysis of Streptococcus pneumoniae Identifies Genes Involved in Pathogenesis , 2002, Infection and Immunity.

[53]  J. Claverys,et al.  An rpsL Cassette, Janus, for Gene Replacement through Negative Selection in Streptococcus pneumoniae , 2001, Applied and Environmental Microbiology.

[54]  A. Tomasz,et al.  Functional Analysis of Streptococcus pneumoniae MurM Reveals the Region Responsible for Its Specificity in the Synthesis of Branched Cell Wall Peptides* , 2001, The Journal of Biological Chemistry.

[55]  K. Klugman,et al.  Alterations in MurM, a Cell Wall Muropeptide Branching Enzyme, Increase High-Level Penicillin and Cephalosporin Resistance in Streptococcus pneumoniae , 2001, Antimicrobial Agents and Chemotherapy.

[56]  A. Tomasz,et al.  Nomenclature of Major Antimicrobial-Resistant Clones of Streptococcus pneumoniae Defined by the Pneumococcal Molecular Epidemiology Network , 2001, Journal of Clinical Microbiology.

[57]  R. Hakenbeck,et al.  Mosaic Genes and Mosaic Chromosomes: Intra- and Interspecies Genomic Variation of Streptococcus pneumoniae , 2001, Infection and Immunity.

[58]  L. Harrison,et al.  Active bacterial core surveillance of the emerging infections program network. , 2001, Emerging infectious diseases.

[59]  A. Tomasz,et al.  Distribution of the Mosaic StructuredmurM Genes among Natural Populations ofStreptococcus pneumoniae , 2000, Journal of bacteriology.

[60]  R. Hakenbeck,et al.  The fib locus in Streptococcus pneumoniae is required for peptidoglycan crosslinking and PBP-mediated beta-lactam resistance. , 2000, FEMS microbiology letters.

[61]  A. Tomasz,et al.  Inhibition of the expression of penicillin resistance in Streptococcus pneumoniae by inactivation of cell wall muropeptide branching genes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[62]  A. Tomasz,et al.  The pgdA Gene Encodes for a PeptidoglycanN-Acetylglucosamine Deacetylase in Streptococcus pneumoniae * , 2000, The Journal of Biological Chemistry.

[63]  James R. Brown,et al.  A genomic analysis of two‐component signal transduction in Streptococcus pneumoniae , 2000, Molecular microbiology.

[64]  Z. Mészner,et al.  Epidemiological studies on drug resistance in Streptococcus pneumoniae in Hungary: an update for the 1990s. , 1999, Microbial drug resistance.

[65]  L. Gutmann,et al.  Acquisition of Five High-MrPenicillin-Binding Protein Variants during Transfer of High-Level β-Lactam Resistance from Streptococcus mitis toStreptococcus pneumoniae , 1998, Journal of bacteriology.

[66]  J. Claverys,et al.  Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae , 1996, Molecular microbiology.

[67]  R. Hakenbeck,et al.  Penicillin-binding proteins 2b and 2x of Streptococcus pneumoniae are primary resistance determinants for different classes of beta-lactam antibiotics , 1996, Antimicrobial agents and chemotherapy.

[68]  A. Tomasz,et al.  Separation of abnormal cell wall composition from penicillin resistance through genetic transformation of Streptococcus pneumoniae , 1996, Journal of bacteriology.

[69]  L. Gutmann,et al.  In vitro selection of one-step mutants of Streptococcus pneumoniae resistant to different oral beta-lactam antibiotics is associated with alterations of PBP2x , 1996, Antimicrobial agents and chemotherapy.

[70]  A. Tomasz,et al.  Naturally occurring peptidoglycan variants of Streptococcus pneumoniae , 1996, Journal of bacteriology.

[71]  R. Hakenbeck,et al.  Penicillin-binding proteins as resistance determinants in clinical isolates of Streptococcus pneumoniae. , 1996, Microbial drug resistance.

[72]  R. Lüttiken,et al.  Penicillin-resistant Streptococcus pneumoniae in Germany: genetic relationship to clones from other European countries. , 1995, Journal of medical microbiology.

[73]  B. Spratt,et al.  Genetics and Molecular Biology of β-Lactam-Resistant Pneumococci , 1995 .

[74]  B. Spratt,et al.  Genetics and molecular biology of beta-lactam-resistant pneumococci. , 1995, Microbial drug resistance.

[75]  A. Tomasz,et al.  Novel penicillin-resistant clones of Streptococcus pneumoniae in the Czech Republic and in Slovakia. , 1995, Microbial drug resistance.

[76]  R. Hakenbeck,et al.  Mosaic pbpX genes of major clones of penicillin‐resistant Streptococcus pneumoniae have evolved from pbpX genes of a penicillin‐sensitive Streptococcus oralis , 1994, Molecular microbiology.

[77]  R. Hakenbeck,et al.  A two‐component signal‐transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae , 1994, Molecular microbiology.

[78]  C. Dowson,et al.  Evolution of penicillin resistance in Streptococcus pneumoniae; the role of Streptococcus mitis in the formation of a low affinity PBP2B in S. pneumoniae , 1993, Molecular microbiology.

[79]  L. Créancier,et al.  The high level streptomycin resistance gene from Streptococcus pneumoniae is a homologue of the ribosomal protein S12 gene from Escherichia coli. , 1992, Nucleic acids research.

[80]  A. Tomasz,et al.  Altered murein composition in a DD-carboxypeptidase mutant of Streptococcus pneumoniae , 1992, Journal of bacteriology.

[81]  B. Spratt,et al.  Interspecies recombinational events during the evolution of altered PBP 2x genes in penicillin‐resistant clinical isolates of Streptococcus pneumoniae , 1991, Molecular microbiology.

[82]  A. Tomasz,et al.  Extremely high incidence of antibiotic resistance in clinical isolates of Streptococcus pneumoniae in Hungary. , 1991, The Journal of infectious diseases.

[83]  A. Tomasz,et al.  A biological price of antibiotic resistance: major changes in the peptidoglycan structure of penicillin-resistant pneumococci. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[84]  B. Chait,et al.  Structure of the peptide network of pneumococcal peptidoglycan. , 1987, The Journal of biological chemistry.

[85]  R. Hakenbeck,et al.  Interaction of non-lytic beta-lactams with penicillin-binding proteins in Streptococcus pneumoniae. , 1987, Journal of general microbiology.

[86]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[87]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[88]  R. Hotchkiss,et al.  RELEASE OF GENETIC TRANSFORMING AGENT FROM PNEUMOCOCCAL CULTURES DURING GROWTH AND DISINTEGRATION , 1962, The Journal of experimental medicine.

[89]  S. Lacks,et al.  A study of the genetic material determining an enzyme activity in Pneumococcus , 1960 .