The eSTKs regulate streptococcal peptidoglycan synthesis with distinct subcellular localizations and coordination with Pbps in S. suis and S. pneumoniae

Abstract

Compared with traditional drugs targeting penicillin-binding proteins (Pbps), targeting the upstream coordinators of bacterial peptidoglycan (PG) synthesis is a highly promising strategy for overcoming the frequent occurrence of resistance to β-lactam drugs. Eukaryotic-like serine/threonine kinases (eSTKs) are core coordinators of bacterial cell division and PG synthesis in many pathogenic bacteria, and their activity is exemplified in the well-studied ovoid bacterium Streptococcus pneumoniae. Here, we demonstrate that eSTK activity in Streptococcus suis (STKss), another ovoid bacterium, is crucial for the normal cell division cycle and morphology, especially in septum formation. The abnormalities observed during the cell division cycle in S. suis mutants were similar in both the S. pneumoniae STK (STKsp) mutant and the kinase activity-deficient mutant. However, orthologous eSTKs in these two species failed to functionally replace each other in reciprocal complementation assays. STKss and STKsp localization experiments in wild-type and mutant strains, including the replacement of different domains of the two eSTKs, revealed that incompatibility stemmed from their divergent localization patterns during the cell cycle, which were primarily dictated by their extracellular PASTA4 domains. During septal PG synthesis, STKss and STKsp consistently localized to the inner and outer edges of the septum, respectively. Additionally, both STKss and STKsp interact with similar PG synthesis and remodeling proteins, with differences in their spatial coordination with the core PG synthases Pbp2B (peripheral synthesis) and Pbp2X (septal synthesis). Collectively, these findings indicate that diverse eSTK localizations are directed by the PASTA4 domain and coordination with Pbps during the regulation of cell division among closely related ovoid bacteria. Given the essentiality of eSTKs in governing the growth, metabolism and virulence of various species of pathogenic bacteria, eSTKs have become promising drug targets for the development of novel antibacterial agents.

Keywords

Streptococcus pneumoniae / Streptococcus suis / Cell division / eSTKs / Peptidoglycan synthesis / Antibacterial target

References

[1] Antimicrobial Resistance CollaboratorsGlobal burden of bacterial antimicrobial resistance in 2019: A systematic analysis. Lancet, 2022, 39910325629655.

[2] Beilharz K, Nováková L, Fadda D, Branny P, Massidda O, Veening J-W. Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP. Proceedings of the National Academy of Sciences of the United States of America, 2012, 10915E905913.

[3] Blair JMA, Webber MA, Baylay AJ, Ogbolu DO, Piddock LJV. Molecular mechanisms of antibiotic resistance. Nature Reviews Microbiology, 2015, 1314251.

[4] Boutte CC, Baer CE, Papavinasasundaram K, Liu W, Chase MR, Meniche X, Fortune SM, Sassetti CM, Ioerger TR, Rubin EJ. A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis. eLife, 2016, 5. e14590

[5] Cui L, Yang R, Huo D, Li L, Qu X, Wang J, Wang X, Liu H, Chen H, Wang X. Streptococcus pneumoniae extracellular vesicles aggravate alveolar epithelial barrier disruption via autophagic degradation of OCLN (occludin). Autophagy, 2024, 20715771596.

[6] Daniel RA, Errington J. Control of cell morphogenesis in bacteria: Two distinct ways to make a rod-shaped cell. Cell, 2003, 1136767776.

[7] Eberhardt A, Wu LJ, Errington J, Vollmer W, Veening J-W. Cellular localization of choline-utilization proteins in Streptococcus pneumoniae using novel fluorescent reporter systems. Molecular Microbiology, 2009, 742395408.

[8] Fenton AK, Manuse S, Flores-Kim J, Garcia PS, Mercy C, Grangeasse C, Bernhardt TG, Rudner DZ. Phosphorylation-dependent activation of the cell wall synthase PBP2a in Streptococcus pneumoniae by MacP. Proceedings of the National Academy of Sciences of the United States of America, 2018, 1151128122817.

[9] Fleurie A, Cluzel C, Guiral S, Freton C, Galisson F, Zanella-Cleon I, Di Guilmi A-M, Grangeasse C. Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae. Molecular Microbiology, 2012, 834746758.

[10] Fleurie A, Lesterlin C, Manuse S, Zhao C, Cluzel C, Lavergne J-P, Franz-Wachtel M, Macek B, Combet C, Kuru E, et al. MapZ marks the division sites and positions FtsZ rings in Streptococcus pneumoniae. Nature, 2014, 5167530259262.

[11] Fleurie A, Manuse S, Zhao C, Campo N, Cluzel C, Lavergne J-P, Freton C, Combet C, Guiral S, Soufi B, et al. Interplay of the serine/threonine-kinase StkP and the paralogs DivIVA and GpsB in pneumococcal cell elongation and division. PLoS Genetics, 2014, 104e1004275

[12] Foulquier E, Pompeo F, Freton C, Cordier B, Grangeasse C, Galinier A. PrkC-mediated phosphorylation of overexpressed YvcK protein regulates PBP1 protein localization in Bacillus subtilis mreB mutant cells. Journal of Biological Chemistry, 2014, 289342366223669.

[13] Hamidi M, Nagarajan SN, Ravikumar V, Gueguen-Chaignon V, Laguri C, Freton C, Mijakovic I, Simorre J-P, Ravaud S, Grangeasse C. The juxtamembrane domain of StkP is phosphorylated and influences cell division in Streptococcus pneumoniae. Mbio, 2025, 165e0379924

[14] Hanks SK, Hunter T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J, 1995, 98576596.

[15] Hardt P, Engels I, Rausch M, Gajdiss M, Ulm H, Sass P, Ohlsen K, Sahl H-G, Bierbaum G, Schneider T, et al. The cell wall precursor lipid II acts as a molecular signal for the Ser/Thr kinase PknB of Staphylococcus aureus. International Journal of Medical Microbiology, 2017, 3071110.

[16] Hu Q, Yao L, Liao X, Zhang L-S, Li H-T, Li T-T, Jiang Q-G, Tan M-F, Li L, Draheim RR, et al. Comparative phenotypic, proteomic, and phosphoproteomic analysis reveals different roles of serine/threonine phosphatase and kinase in the growth, cell division, and pathogenicity of Streptococcus suis. Microorganisms, 2021, 9122442

[17] Iannelli F, Pearce BJ, Pozzi G. The type 2 capsule locus of Streptococcus pneumoniae. Journal of Bacteriology, 1999, 181826522654.

[18] Jani C, Eoh H, Lee JJ, Hamasha K, Sahana MB, Han J-S, Nyayapathy S, Lee J-Y, Suh J-W, Lee SH, et al. Regulation of polar peptidoglycan biosynthesis by Wag31 phosphorylation in Mycobacteria. BMC Microbiology, 2010, 10. 327

[19] Jarick M, Bertsche U, Stahl M, Schultz D, Methling K, Lalk M, Stigloher C, Steger M, Schlosser A, Ohlsen K. The serine/threonine kinase Stk and the phosphatase Stp regulate cell wall synthesis in Staphylococcus aureus. Scientific Reports, 2018, 8113693

[20] Jiang Q, Li B, Zhang L, Li T, Hu Q, Li H, Zou W, Hu Z, Huang Q, Zhou R. DivIVA interacts with the cell wall hydrolase MltG to regulate peptidoglycan synthesis in Streptococcus suis. Microbiology Spectrum, 2023, 113e04750-22

[21] Kang C-M, Abbott DW, Park ST, Dascher CC, Cantley LC, Husson RN. The Mycobacterium tuberculosis serine/threonine kinases PknA and PknB: Substrate identification and regulation of cell shape. Genes & Development, 2005, 191416921704.

[22] Kant S, Asthana S, Missiakas D, Pancholi V. A novel STK1-targeted small-molecule as an ‘antibiotic resistance breaker’ against multidrug-resistant Staphylococcus aureus. Scientific Reports, 2017, 715067

[23] Kant S, Sun Y, Pancholi V. StkP- and PhpP-mediated posttranslational modifications modulate the S. pneumoniae metabolism, polysaccharide capsule, and virulence. Infection and Immunity, 2023, 914e0029622

[24] Kaspy I, Rotem E, Weiss N, Ronin I, Balaban NQ, Glaser G. HipA-mediated antibiotic persistence via phosphorylation of the glutamyl-tRNA-synthetase. Nature Communications, 2013, 4. 3001

[25] Kieser KJ, Boutte CC, Kester JC, Baer CE, Barczak AK, Meniche X, Chao MC, Rego EH, Sassetti CM, Fortune SM, et al. Phosphorylation of the peptidoglycan synthase PonA1 governs the rate of polar elongation in mycobacteria. PLoS Pathogens, 2015, 116e1005010

[26] Kwon A, Scott S, Taujale R, Yeung W, Kochut KJ, Eyers PA, Kannan N. Tracing the origin and evolution of pseudokinases across the tree of life. Science Signaling, 2019, 12578eaav3810

[27] Lacks S, Hotchkiss RD. A study of the genetic material determining an enzyme in Pneumococcus. Biochimica Et Biophysica Acta, 1960, 39: 508-518.

[28] Li W, Liu L, Chen H, Zhou R. Identification of Streptococcus suis genes preferentially expressed under iron starvation by selective capture of transcribed sequences. FEMS Microbiology Letters, 2009, 2921123133.

[29] Li H, Li T, Hu Q, Yao Z, Li L, Huang Q, Zhou R. Inhibitors targeting the autophosphorylation of serine/threonine kinase of Streptococcus suis show potent antimicrobial activity. Frontiers in Microbiology, 2022, 13. 990091

[30] Li S, Ma Z, Lin H, Pan F, Zhou H, Tang J, Fan H. STK-mediated FadR phosphorylation regulates the acid resistance and virulence of Streptococcus suis. PLoS Pathogens, 2025, 219e1013534

[31] Li T, Liu X, Zhang L, Li H, Ni M, Zou W, Liang M, Gong R, Hu Q, Zhao L, et al. EzrA promotes Z-ring formation through interaction of its QNR motif with FtsA. Journal of Bacteriology, 2025, 2077e0012525

[32] Libby EA, Goss LA, Dworkin J. The eukaryotic-like Ser/Thr kinase PrkC regulates the essential WalRK two-component system in Bacillus subtilis. PLoS Genetics, 2015, 116e1005275

[33] Manuse S, Fleurie A, Zucchini L, Lesterlin C, Grangeasse C. Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis. FEMS Microbiology Reviews, 2016, 4014156.

[34] Martin B, Prudhomme M, Alloing G, Granadel C, Claverys JP. Cross-regulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae. Molecular Microbiology, 2000, 384867878.

[35] Martínez-Caballero S, Freton C, Molina R, Bartual SG, Gueguen-Chaignon V, Mercy C, Gago F, Mahasenan KV, Muñoz IG, Lee M, et al. Molecular basis of the final step of cell division in Streptococcus pneumoniae. Cell Reports, 2023, 427112756

[36] Massidda O, Nováková L, Vollmer W. From models to pathogens: How much have we learned about Streptococcus pneumoniae cell division?. Environmental Microbiology, 2013, 151231333157.

[37] Morlot C, Bayle L, Jacq M, Fleurie A, Tourcier G, Galisson F, Vernet T, Grangeasse C, Di Guilmi AM. Interaction of penicillin-binding protein 2x and Ser/Thr protein kinase StkP, two key players in Streptococcus pneumoniae R6 morphogenesis. Molecular Microbiology, 2013, 90188102.

[38] Nováková L, Sasková L, Pallová P, Janecek J, Novotná J, Ulrych A, Echenique J, Trombe M-C, Branny P. Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates. The FEBS Journal, 2005, 272512431254.

[39] Pereira SFF, Goss L, Dworkin J. Eukaryote-like serine/threonine kinases and phosphatases in bacteria. Microbiology and Molecular Biology Reviews, 2011, 751192212.

[40] Pereira SFF, Gonzalez RL, Dworkin J. Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor. Proceedings of the National Academy of Sciences of the United States of America, 2015, 11225E3274E3281.

[41] Perez AJ, Cesbron Y, Shaw SL, Bazan Villicana J, Tsui H-CT, Boersma MJ, Ye ZA, Tovpeko Y, Dekker C, Holden S, et al. Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae. Proceedings of the National Academy of Sciences, 2019, 116832113220.

[42] Perez AJ, Boersma MJ, Bruce KE, Lamanna MM, Shaw SL, Tsui HT, Taguchi A, Carlson EE, VanNieuwenhze MS, Winkler ME. Organization of peptidoglycan synthesis in nodes and separate rings at different stages of cell division of Streptococcus pneumoniae. Molecular Microbiology, 2021, 115611521169.

[43] Perez AJ, Lamanna MM, Bruce KE, Touraev MA, Page JE, Shaw SL, Tsui H-CT, Winkler ME. Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell of Streptococcus pneumoniae. Proceedings of the National Academy of Sciences of the United States of America, 2024, 12125e2401831121

[44] Pinho MG, Kjos M, Veening J-W. How to get (a)round: Mechanisms controlling growth and division of coccoid bacteria. Nature Reviews. Microbiology, 2013, 119601614.

[45] Pompeo F, Foulquier E, Serrano B, Grangeasse C, Galinier A. Phosphorylation of the cell division protein GpsB regulates PrkC kinase activity through a negative feedback loop in Bacillus subtilis. Molecular Microbiology, 2015, 971139150.

[46] Pompeo F, Byrne D, Mengin-Lecreulx D, Galinier A. Dual regulation of activity and intracellular localization of the PASTA kinase PrkC during Bacillus subtilis growth. Science and Reports, 2018, 8: 1660.

[47] Pozzi G, Masala L, Iannelli F, Manganelli R, Havarstein LS, Piccoli L, Simon D, Morrison DA. Competence for genetic transformation in encapsulated strains of Streptococcus pneumoniae: Two allelic variants of the peptide pheromone. Journal of Bacteriology, 1996, 1782060876090.

[48] Rajpurohit YS, Bihani SC, Waldor MK, Misra HS. Phosphorylation of Deinococcus radiodurans RecA regulates its activity and may contribute to radioresistance. Journal of Biological Chemistry, 2016, 291321667216685.

[49] Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P. The penicillin-binding proteins: Structure and role in peptidoglycan biosynthesis. FEMS Microbiology Reviews, 2008, 322234258.

[50] Sisley TA, Park Y, Santiago AG, Beroual W, Sobolewski IA, Lee W, Paulo JA, Walker S. AuxB interacts directly with GpsB and PknB to coordinate cell envelope processes that contribute to intrinsic antibiotic resistance in Staphylococcus aureus. Mbio, 2025, 1610e0185825

[51] Stauberová V, Kubeša B, Joseph M, Benedet M, Furlan B, Buriánková K, Ulrych A, Kupčík R, Vomastek T, Massidda O, et al. GpsB coordinates StkP signaling as a PASTA kinase adaptor in Streptococcus pneumoniae cell division. Journal of Molecular Biology, 2024, 43622168797

[52] Sung CK, Li H, Claverys JP, Morrison DA. An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae. Applied and Environmental Microbiology, 2001, 671151905196.

[53] Takamatsu D, Osaki M, Sekizaki T. Thermosensitive suicide vectors for gene replacement in Streptococcus suis. Plasmid, 2001, 462140148.

[54] Tang J, Guo M, Chen M, Xu B, Ran T, Wang W, Ma Z, Lin H, Fan H. A link between STK signalling and capsular polysaccharide synthesis in Streptococcus suis. Nature Communications, 2023, 1412480

[55] Thakur M, Chakraborti PK. GTPase activity of mycobacterial FtsZ is impaired due to its transphosphorylation by the eukaryotic-type Ser/Thr kinase, PknA. Journal of Biological Chemistry, 2006, 281524010740113.

[56] Tsui H-CT, Boersma MJ, Vella SA, Kocaoglu O, Kuru E, Peceny JK, Carlson EE, VanNieuwenhze MS, Brun YV, Shaw SL, et al. Pbp2x localizes separately from Pbp2b and other peptidoglycan synthesis proteins during later stages of cell division of Streptococcus pneumoniae D39. Molecular Microbiology, 2014, 9412140.

[57] Tsui H-CT, Joseph M, Zheng JJ, Perez AJ, Manzoor I, Rued BE, Richardson JD, Branny P, Doubravová L, Massidda O, et al. Negative regulation of MurZ and MurA underlies the essentiality of GpsB- and StkP-mediated protein phosphorylation in Streptococcus pneumoniae D39. Molecular Microbiology, 2023, 1203351383.

[58] Turapov O, Forti F, Kadhim B, Ghisotti D, Sassine J, Straatman-Iwanowska A, Bottrill AR, Moynihan PJ, Wallis R, Barthe P, et al. Two faces of CwlM, an essential PknB substrate, in Mycobacterium tuberculosis. Cell Reports, 2018, 2515767.e5.

[59] Ulrych A, Holečková N, Goldová J, Doubravová L, Benada O, Kofroňová O, Halada P, Branny P. Characterization of pneumococcal Ser/Thr protein phosphatase phpP mutant and identification of a novel PhpP substrate, putative RNA binding protein Jag. BMC Microbiology, 2016, 161247.

[60] Wang J, Ge P, Lei Z, Lu Z, Qiang L, Chai Q, Zhang Y, Zhao D, Li B, Su J, et al. Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity. EMBO Reports, 2021, 226e52175

[61] Yin H, Tian Y, Zhou Z, Lang S, Li Y, Peng L, Li J, Fang R. Phosphorylation of EF-P aggravates Streptococcus suis-induced blood-brain barrier damage by enhancing serine protease production. Veterinary Research, 2025, 561215.

[62] Young TA, Delagoutte B, Endrizzi JA, Falick AM, Alber T. Structure of Mycobacterium tuberculosis PknB supports a universal activation mechanism for Ser/Thr protein kinases. Nature Structural & Molecular Biology, 2003, 103168174.

[63] Zapun A, Contreras-Martel C, Vernet T. Penicillin-binding proteins and β-lactam resistance. FEMS Microbiology Reviews, 2008, 322361385.

[64] Zhang C, Sun W, Tan M, Dong M, Liu W, Gao T, Li L, Xu Z, Zhou R. The eukaryote-like serine/threonine kinase STK regulates the growth and metabolism of zoonotic Streptococcus suis. Frontiers in Cellular and Infection Microbiology, 2017, 7: 66.

[65] Zhang L, Zou W, Ni M, Hu Q, Zhao L, Liao X, Huang Q, Zhou R. Development and application of two inducible expression systems for Streptococcus suis. Microbiology Spectrum, 2022, 104e0036322

[66] Zucchini L, Mercy C, Garcia PS, Cluzel C, Gueguen-Chaignon V, Galisson F, Freton C, Guiral S, Brochier-Armanet C, Gouet P, et al. PASTA repeats of the protein kinase StkP interconnect cell constriction and separation of Streptococcus pneumoniae. Nature Microbiology, 2018, 32197209.

Funding

National Natural Science Foundation of China(32573361)

Key Science and Technology Projects of Wuhan City(No. 2025021102020384)

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