Spontaneous mutations and mutational responses to penicillin treatment in the bacterial pathogen Streptococcus pneumoniae D39

Abstract

Bacteria with functional DNA repair systems are expected to have low mutation rates due to strong natural selection for genomic stability. However, our study of the wild-type Streptococcus pneumoniae D39, a pathogen responsible for many common diseases, revealed a high spontaneous mutation rate of 0.02 per genome per cell division in mutation-accumulation (MA) lines. This rate is orders of magnitude higher than that of other non-mutator bacteria and is characterized by a high mutation bias in the A/T direction. The high mutation rate may have resulted from a reduction in the overall efficiency of selection, conferred by the tiny effective population size in nature. In line with this, S. pneumoniae D39 also exhibited the lowest DNA mismatch-repair (MMR) efficiency among bacteria. Treatment with the antibiotic penicillin did not elevate the mutation rate, as penicillin did not induce DNA damage and S. pneumoniae lacks a stress response pathway. Our findings suggested that the MA results are applicable to within-host scenarios and provide insights into pathogen evolution.

Keywords

Neutral evolution / Mutation spectrum / Antibiotics / DNA mismatch repair / Oxidative damage repair

References

[] Ames BN, Gold LS. Endogenous mutagens and the causes of aging and cancer. Mutat Res, 1991, 250: 3-16.

[] Andersson DI, Hughes D. Microbiological effects of sublethal levels of antibiotics. Nat Rev Microbiol, 2014, 12: 465-478.

[] Andreani NA, Carraro L, Zhang L, Vos M, Cardazzo B. Transposon mutagenesis in Pseudomonas fluorescens reveals genes involved in blue pigment production and antioxidant protection. Food Microbiol, 2019, 82: 497-503.

[] Asokan GV, Ramadhan T, Ahmed E, Sanad H. WHO global priority pathogens list: a bibliometric analysis of medline-pubmed for knowledge mobilization to infection prevention and control practices in Bahrain. Oman Med J, 2019, 34: 184-193.

[] Baquero F, Martínez J-L, Cantón R. Antibiotics and antibiotic resistance in water environments. Curr Opin Biotechnol, 2008, 19: 260-265.

[] Blake R, Hess ST, Nicholson-Tuell J. The influence of nearest neighbors on the rate and pattern of spontaneous point mutations. J Mol Evol, 1992, 34: 189-200.

[] Bogaert D, de Groot R, Hermans PWM. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect Dis, 2004, 4: 144-154.

[] Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 2014, 30: 2114-2120.

[] Briles DE, Novak L, Hotomi M, van Ginkel FW, King J. Nasal colonization with Streptococcus pneumoniae includes subpopulations of surface and invasive pneumococci. Infect Immun, 2005, 73: 6945-6951.

[] Carmeli Y, Troillet N, Karchmer AW, Samore MH. Health and economic outcomes of antibiotic resistance in Pseudomonas aeruginosa. Arch Intern Med, 1999, 159: 1127-1132.

[] Chaguza C, Senghore M, Bojang E, Gladstone RA, Lo SW, Tientcheu P-E, Bancroft RE, Worwui A, Foster-Nyarko E, Ceesay F. Within-host microevolution of Streptococcus pneumoniae is rapid and adaptive during natural colonisation. Nat Commun, 2020, 11: 3442.

[] Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant, 2020, 13: 1194-1202.

[] Chen S, Zhou Y, Chen Y, Gu J. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 2018, 34: i884-i890.

[] Dahiya R, Speck M. Hydrogen peroxide formation by lactobacilli and its effect on Staphylococcus aureus. J Dairy Sci, 1968, 51: 1568-1572.

[] Deatherage DE, Barrick JE. Sun L, Shou W. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Engineering and analyzing multicellular systems, 2014 New York Humana Press 165-188.

[] DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, Del Angel G, Rivas MA, Hanna M. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet, 2011, 43: 491-498.

[] Dettman JR, Sztepanacz JL, Kassen R. The properties of spontaneous mutations in the opportunistic pathogen Pseudomonas aeruginosa. BMC Genom, 2016, 17: 27.

[] Dillon MM, Sung W, Lynch M, Cooper VS. The rate and molecular spectrum of spontaneous mutations in the GC-rich multichromosome genome of Burkholderia cenocepacia. Genetics, 2015, 200: 935-946.

[] Dillon MM, Sung W, Sebra R, Lynch M, Cooper VS. Genome-wide biases in the rate and molecular spectrum of spontaneous mutations in Vibrio cholerae and Vibrio fischeri. Mol Biol Evol, 2017, 34: 93-109.

[] Fineran PC, Everson L, Slater H, Salmond GPC. A GntR family transcriptional regulator (PigT) controls gluconate-mediated repression and defines a new, independent pathway for regulation of the tripyrrole antibiotic, prodigiosin, in Serratia. Microbiology, 2005, 151: 3833-3845.

[] Gasc A, Sicard N, Claverys J, Sicard A. Lack of SOS repair in Streptococcus pneumoniae. Mutat Res, 1980, 70: 157-165.

[] Green AE, Howarth D, Chaguza C, Echlin H, Langendonk RF, Munro C, Barton TE, Hinton JCD, Bentley SD, Rosch JW, Neill DR. Pneumococcal colonization and virulence factors identified via experimental evolution in infection models. Mol Biol Evol, 2021, 38: 2209-2226.

[] Gu Z, Gu L, Eils R, Schlesner M, Brors B. Circlize implements and enhances circular visualization in R. Bioinformatics, 2014, 30: 2811-2812.

[] Hess ST, Blake JD, Blake R. Wide variations in neighbor-dependent substitution rates. J Mol Biol, 1994, 236: 1022-1033.

[] Hillerich B, Westpheling J. A new GntR family transcriptional regulator in Streptomyces coelicolor is required for morphogenesis and antibiotic production and controls transcription of an ABC transporter in response to carbon source. J Bacteriol, 2006, 188: 7477-7487.

[] Hirschmann S, Gómez-Mejia A, Mäder U, Karsunke J, Driesch D, Rohde M, Häussler S, Burchhardt G, Hammerschmidt S. The two-component system 09 regulates pneumococcal carbohydrate metabolism and capsule expression. Microorganisms, 2021, 9: 468.

[] Jacobs MR. Treatment and diagnosis of infections caused by drug-resistant Streptococcus pneumoniae. Clin Infect Dis, 1992, 15: 119-127.

[] Jiang L, Hu X, Xu T, Zhang H, Sheng D, Yin D. Prevalence of antibiotic resistance genes and their relationship with antibiotics in the Huangpu River and the drinking water sources, Shanghai, China. Sci Total Environ, 2013, 458: 267-272.

[] Jones M, Wagner R, Radman M. Repair of a mismatch is influenced by the base composition of the surrounding nucleotide sequence. Genetics, 1987, 115: 605-610.

[] Jørgensen KM, Wassermann T, Jensen PØ, Hengzuang W, Molin S, Høiby N, Ciofu O. Sublethal ciprofloxacin treatment leads to rapid development of high-level ciprofloxacin resistance during long-term experimental evolution of Pseudomonas aeruginosa. Antimicrob Agents Chemother, 2013, 57: 4215-4221.

[] Jun SH, Kim TG, Ban C. DNA mismatch repair system: classical and fresh roles. FEBS J, 2006, 273: 1609-1619.

[] Kadioglu A, Weiser JN, Paton JC, Andrew PW. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol, 2008, 6: 288-301.

[] Keightley PD, Halligan DL. Analysis and implications of mutational variation. Genetica, 2009, 136: 359-369.

[] Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods, 2015, 12: 357-360.

[] Kolodner RD, Marsischky GT. Eukaryotic DNA mismatch repair. Curr Opin Genet Dev, 1999, 9: 89-96.

[] Krisko A, Radman M. Biology of extreme radiation resistance: the way of Deinococcus radiodurans. Cold Spring Harb Perspect Biol, 2013, 5.

[] Kucukyildirim S, Long H, Sung W, Miller SF, Doak TG, Lynch M. The rate and spectrum of spontaneous mutations in Mycobacterium smegmatis, a bacterium naturally devoid of the postreplicative mismatch repair pathway. G3 Genes Genom Genet, 2016, 6: 2157-2163.

[] Kunkel TA, Erie DA. DNA mismatch repair. Annu Rev Biochem, 2005, 74: 681-710.

[] Lanie JA, Ng WL, Kazmierczak KM, Andrzejewski TM, Davidsen TM, Wayne KJ. Genome sequence of Avery's virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6. J Bacteriol, 2007, 189: 38-51.

[] Lee H, Popodi E, Tang H, Foster PL. Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing. Proc Natl Acad Sci USA, 2012, 109: E2774-E2783.

[] Levinson G, Gutman GA. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol Biol Evol, 1987, 4: 203-221.

[] Li G-M. Mechanisms and functions of DNA mismatch repair. Cell Res, 2008, 18: 85-98.

[] Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 2009, 25: 1754-1760.

[] Long H, Kucukyildirim S, Sung W, Williams E, Lee H, Ackerman M, Doak TG, Tang H, Lynch M. Background mutational features of the radiation-resistant bacterium Deinococcus radiodurans. Mol Biol Evol, 2015, 32: 2383-2392.

[] Long H, Miller SF, Strauss C, Zhao C, Cheng L, Ye Z, Griffin K, Te R, Lee H, Chen CC, Lynch M. Antibiotic treatment enhances the genome-wide mutation rate of target cells. Proc Natl Acad Sci USA, 2016, 113: E2498-E2505.

[] Long H, Miller SF, Williams E, Lynch M. Specificity of the DNA mismatch repair system (MMR) and mutagenesis bias in bacteria. Mol Biol Evol, 2018, 35: 2414-2421.

[] Long H, Sung W, Kucukyildirim S, Williams E, Miller SF, Guo W, Patterson C, Gregory C, Strauss C, Stone C, Berne C, Kysela D, Shoemaker WR, Muscarella ME, Luo H, Lennon JT, Brun YV, Lynch M. Evolutionary determinants of genome-wide nucleotide composition. Nat Ecol Evol, 2018, 2: 237-240.

[] Longerich S, Galloway AM, Harris RS, Wong C, Rosenberg SM. Adaptive mutation sequences reproduced by mismatch repair deficiency. Proc Natl Acad Sci USA, 1995, 92: 12017-12020.

[] Lynch M. Evolution of the mutation rate. Trends Genet, 2010, 26: 345-352.

[] Lynch M. Evolutionary layering and the limits to cellular perfection. Proc Natl Acad Sci USA, 2012, 109: 18851-18856.

[] Lynch M, Ackerman MS, Gout JF, Long H, Sung W, Thomas WK, Foster PL. Genetic drift, selection and the evolution of the mutation rate. Nat Rev Genet, 2016, 17: 704-714.

[] Lynch M, Sung W, Morris K, Coffey N, Landry CR, Dopman EB, Dickinson WJ, Okamoto K, Kulkarni S, Hartl DL. A genome-wide view of the spectrum of spontaneous mutations in yeast. Proc Natl Acad Sci USA, 2008, 105: 9272-9277.

[] Lynch M, Walsh B. Genetics and analysis of quantitative traits, 1998 Sunderland Sinauer

[] Mandell LA, Wunderink RG, Anzueto A, Bartlett JG, Campbell GD, Dean NC, Dowell SF, File TM Jr Musher DM, Niederman MS. Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis, 2007, 44: S27-S72.

[] McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res, 2010, 20: 1297-1303.

[] Mejean V, Salles C, Bullions LC, Bessman MJ, Claverys JP. Characterization of the mutX gene of Streptococcus pneumoniae as a homologue of Escherichia coli mutT, and tentative definition of a catalytic domain of the dGTP pyrophosphohydroiases. Mol Microbiol, 1994, 11: 323-330.

[] Michaels ML, Cruz C, Grollman AP, Miller JH. Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proc Natl Acad Sci USA, 1992, 89: 7022-7025.

[] Mitchell AM, Mitchell TJ. Streptococcus pneumoniae: virulence factors and variation. Clin Microbiol Infect, 2010, 16: 411-418.

[] Modrich P, Lahue R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu Rev Biochem, 1996, 65: 101-133.

[] Morton BR. The role of context-dependent mutations in generating compositional and codon usage bias in grass chloroplast DNA. J Mol Evol, 2003, 56: 616-629.

[] Munita JM, Arias CA. Mechanisms of antibiotic resistance. Microbiol Spectrum, 2016, 4: 481-511.

[] Musher DM. Infections caused by Streptococcus pneumoniae: clinical spectrum, pathogenesis, immunity, and treatment. Clin Infect Dis, 1992, 14: 801-807.

[] Napolitano R, Janel-Bintz R, Wagner J, Fuchs R. All three SOS-inducible DNA polymerases (Pol II, Pol IV and Pol V) are involved in induced mutagenesis. EMBO J, 2000, 19: 6259-6265.

[] Nghiem Y, Cabrera M, Cupples C, Miller J. The mutY gene: a mutator locus in Escherichia coli that generates G.C→T.A transversions. Proc Natl Acad Sci USA, 1988, 85: 2709-2713.

[] Pan J, Li W, Ni J, Wu K, Konigsberg I, Rivera CE, Tincher C, Gregory C, Zhou X, Doak TG. Rates of mutations and transcript errors in the foodborne pathogen Salmonella enterica subsp. enterica. Mol Biol Evol, 2022, 39: msac081.

[] Pan J, Williams E, Sung W, Lynch M, Long H. The insect-killing bacterium Photorhabdus luminescens has the lowest mutation rate among bacteria. Mar Life Sci Technol, 2021, 3: 20-27.

[] Pericone CD, Overweg K, Hermans PW, Weiser JN. Inhibitory and bactericidal effects of hydrogen peroxide production by Streptococcus pneumoniae on other inhabitants of the upper respiratory tract. Infect Immun, 2000, 68: 3990-3997.

[] Pericone CD, Bae D, Shchepetov M, McCool T, Weiser JN. Short-sequence tandem and nontandem DNA repeats and endogenous hydrogen peroxide production contribute to genetic instability of Streptococcus pneumoniae. J Bacteriol, 2002, 184: 4392-4399.

[] Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Protoc, 2016, 11: 1650-1667.

[] Ramos-Montanez S, Tsui HC, Wayne KJ, Morris JL, Peters LE, Zhang F, Kazmierczak KM, Sham LT, Winkler ME. Polymorphism and regulation of the spxB (pyruvate oxidase) virulence factor gene by a CBS-HotDog domain protein (SpxR) in serotype 2 Streptococcus pneumoniae. Mol Microbiol, 2008, 67: 729-746.

[] Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P. The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev, 2008, 32: 234-258.

[] Slager J, Aprianto R, Veening JW. Deep genome annotation of the opportunistic human pathogen Streptococcus pneumoniae D39. Nucleic Acids Res, 2018, 46: 9971-9989.

[] Sung W, Ackerman MS, Gout J-F, Miller SF, Williams E, Foster PL, Lynch M. Asymmetric context-dependent mutation patterns revealed through mutation-accumulation experiments. Mol Biol Evol, 2015, 32: 1672-1683.

[] Sung W, Ackerman MS, Miller SF, Doak TG, Lynch M. Drift-barrier hypothesis and mutation-rate evolution. Proc Natl Acad Sci USA, 2012, 109: 18488-18492.

[] Tajiri T, Maki H, Sekiguchi M. Functional cooperation of MutT, MutM and MutY proteins in preventing mutations caused by spontaneous oxidation of guanine nucleotide in Escherichia coli. Mutat Res, 1995, 336: 257-267.

[] Thorvaldsdóttir H, Robinson JT, Mesirov JP. Integrative genomics viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform, 2013, 14: 178-192.

[] Tsui HT, Boersma MJ, Vella SA, Kocaoglu O, Kuru E, Peceny JK, Carlson EE, VanNieuwenhze MS, Brun YV, Shaw SL, Winkler ME. Pbp2x localizes separately from Pbp2b and other peptidoglycan synthesis proteins during later stages of cell division of Streptococcus pneumoniae D39. Mol Microbiol, 2014, 94: 21-40.

[] Tsui HC, Keen SK, Sham LT, Wayne KJ, Winkler ME. Dynamic distribution of the SecA and SecY translocase subunits and septal localization of the HtrA surface chaperone/protease during Streptococcus pneumoniae D39 cell division. mBio, 2011, 2: e00202-00211.

[] Tsui HC, Zheng JJ, Magallon AN, Ryan JD, Yunck R, Rued BE, Bernhardt TG, Winkler ME. Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39. Mol Microbiol, 2016, 100: 1039-1065.

[] Van de Beek D, de Gans J, Tunkel AR, Wijdicks EF. Community-acquired bacterial meningitis in adults. New Engl J Med, 2006, 354: 44-53.

[] Van der Poll T, Opal SM. Pathogenesis, treatment, and prevention of pneumococcal pneumonia. Lancet, 2009, 374: 1543-1556.

[] Wahl LM, Gerrish PJ. The probability that beneficial mutations are lost in populations with periodic bottlenecks. Evolution, 2001, 55: 2606-2610.

[] Watson DA, Musher DM, Jacobson JW, Verhoef J. A brief history of the Pneumococcus in biomedical research: a panoply of scientific discovery. Clin Infect Dis, 1993, 17: 913-924.

[] Waxman DJ, Strominger JL. Penicillin-binding proteins and the mechanism of action of beta-lactam antibiotics. Annu Rev Biochem, 1983, 52: 825-869.

[] Weiser JN, Ferreira DM, Paton JC. Streptococcus pneumoniae: transmission, colonization and invasion. Nat Rev Microbiol, 2018, 16: 355-367.

[] Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comp Biol, 2017, 13.

[] Woodford N, Ellington MJ. The emergence of antibiotic resistance by mutation. Clin Microbiol Infect, 2007, 13: 5-18.

[] Yesilkaya H, Andisi VF, Andrew PW, Bijlsma JJ. Streptococcus pneumoniae and reactive oxygen species: an unusual approach to living with radicals. Trends Microbiol, 2013, 21: 187-195.

[] Yu VL, Chiou CC, Feldman C, Ortqvist A, Rello J, Morris AJ, Baddour LM, Luna CM, Snydman DR, Ip M. An international prospective study of pneumococcal bacteremia: correlation with in vitro resistance, antibiotics administered, and clinical outcome. Clin Infect Dis, 2003, 37: 230-237.

[] Zhang Y, Zhang C, Huo W, Wang X, Zhang M, Palmer K, Chen M. An expectation–maximization algorithm for estimating proportions of deletions among bacterial populations with application to study antibiotic resistance gene transfer in Enterococcus faecalis. Mar Life Sci Technol, 2023, 5: 28-43.

[] Zurek L, Ghosh A. Insects represent a link between food animal farms and the urban environment for antibiotic resistance traits. Appl Environ Microbiol, 2014, 80: 3562-3567.