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Pneumococcal S protein coordinates cell wall modification and repair to resist host antimicrobials

Nature Microbiology volume 11, pages 282–300 (2026)Cite this article

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Abstract

S protein is conserved among streptococci and contributes to group A Streptococcus virulence, but the mechanisms involved are unclear. Here we used genetic, biochemical, single-molecule, in vitro and in vivo analyses to show that S protein is crucial for resistance against host-derived antimicrobials by coordinating cell wall modification and repair. We observed that S protein was localized to the streptococcal septum dependent on its transmembrane domain, while S protein function was dependent on its peptidoglycan (PG)-binding LysM domain. Direct interactions between the pneumococcal S protein and the PG synthase PBP1a as well as the PG deacetylase PgdA were detected. Loss of S protein reduced the proportion of circumferentially moving PBP1a molecules, altered streptococcal morphology and increased susceptibility to cell-wall-targeting antibiotics, suggesting that S protein activates PBP1a. Streptococcus pneumoniae ess mutants lacking the gene encoding S protein were more susceptible to human antimicrobial peptide LL-37 and lysozyme, while their virulence was decreased compared with wild-type bacteria in zebrafish and mice. These data suggest that S protein activates the PG repair and modification complex, providing defence against host-derived and environmental antimicrobials.

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Fig. 1: The S protein is localized at midcell through its transmembrane-spanning domain.
Fig. 2: Pneumococcal S protein directly interacts with PBP1a and PgdA.
Fig. 3: S protein is involved in cell wall homeostasis.
Fig. 4: essSp is genetically linked to pbp1a, pgdA, mpgA and rodZ.
Fig. 5: S protein protects streptococcal cells from host innate immune system damages, and PBP1a contributes to ΔessSp phenotypes.
Fig. 6: Streptococcal S protein coordinates the PBP1a–PgdA sentinel cell wall repair–modification complex required for protection from host antimicrobial challenges.

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Data availability

The data that support the findings of this study are incorporated in the paper and its supporting information. Genome sequences, assemblies and sequencing reads are available at NCBI (BioProject accession number PRJNA1198892). All raw MS data together with raw output tables are available via the Proteomexchange data repository (www.proteomexchange.org) under the accession PXD055534. Source data are provided with this paper.

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Acknowledgements

We thank the members of the Veening group for valuable discussions; F. Patrick Bock for helping with protein structure prediction and V. de Bakker for helping with CRISPRi-seq data analysis; J. Dénéréaz and P. Gibson for technical support; the UNIL GTF for sequencing, PAF for proteomics and EMF for TEM; N. Vastenhouw for access to the zebrafish facility and continued support; D. van Swaay (Wünderlichips) for design and production of the micropillars chip; D. Gonzalez (UC Berkeley School of Public Health, California, USA) for strains and insightful discussions. Work in the lab of J.-W.V. was supported by SNSF grants 310030_192517, 310030_200792 and NCCR 51NF40_180541. Work in the lab of M.E.W. was supported by NIH grant R35GM131767 and NIH equipment grant S10OD024988 to the Indiana University Bloomington Light Microscopy Imaging Center. W.V. was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC; BB/W013630/1).

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Author notes

  1. These authors contributed equally: Jessica Burnier, Clement Gallay.

Authors and Affiliations

  1. Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland

    Jessica Burnier, Clement Gallay, Louise Martin, Kin Ki Jim, Amelieke J. H. Cremers, Johann Mignolet & Jan-Willem Veening

  2. Department of Biology, Indiana University Bloomington, Bloomington, IN, USA

    Kevin E. Bruce, Ho-Ching Tiffany Tsui & Malcolm E. Winkler

  3. Department of Pediatrics, University of California San Diego, La Jolla, CA, USA

    Elisabet Bjånes & Victor Nizet

  4. Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK

    Daniela Vollmer, Jacob Biboy & Waldemar Vollmer

  5. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA

    Victor Nizet

  6. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

    Waldemar Vollmer

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Contributions

J. Burnier and J.-W.V. wrote the paper with input from all authors. J. Burnier, C.G., K.E.B., E.B, L.M., K.K.J., H.-C.T.T., A.J.H.C., J.M., D.V. and J. Biboy performed the experiments. J. Burnier, C.G., K.E.B., E.B., K.K.J., H.-C.T.T., A.J.H.C., J.M., D.V., J. Biboy, V.N., W.V., M.E.W. and J.-W.V. designed, analysed and interpreted the data.

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Correspondence to Jan-Willem Veening.

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J.-W.V. is a scientific advisory board member at i-Seq Biotechnology. The other authors declare no competing interests.

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Nature Microbiology thanks Silvia Cardona, Elaine Tuomanen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 S protein conservation in Bacilli.

(A) Multiple sequence alignment of Streptococci S protein sequences. Sequence similarities are highlighted in dark blue. (B) Percentages indicate the percent identity for each species compared to S. pneumoniae S protein. L. monocytogenes S protein identity score compared to S. pneumoniae S protein is 21% using Clustal Omega. Gene co-occurrence in several bacilli genomes (data obtained from genome annotation in NCBI: see Methods). Multiple sequence alignment of possible S protein sequences of several bacilli. Sequence similarities are highlighted in dark blue. (C) AlphaFold model of the S protein of different bacilli bacteria.

Extended Data Fig. 2 PBP1a and PgdA conservation in Streptococci.

(A) PBP1a and PgdA conservation in Streptococci. Lineage tree based on 16S rRNA sequence of each specie (see methods) was constructed using MEGA. Mycobacterium tuberculosis was used as an outgroup and numbers represent bootstrap. Percentages indicate the percent identity of PBP1a and PgdA for each species compared to S. pneumoniae PBP1a and PgdA respectively. (B) Multiple sequence alignment of Streptococci PBP1a or PgdA sequences. Sequence similarities are highlighted in dark blue.

Extended Data Fig. 3 S protein localization, susceptibility to divers antibiotics and co-deletions.

(A) Deconvolved epifluorescence microscopy of live S. pneumoniae cells containing truncation fragments of S protein tagged with msfGFP (GFP). Numbers represent amino acids. Transmembrane (TM) domain is necessary for S protein septal localization. Scale bar = 2 μm. (B-H) Growth curve at 37°C. Data are represented as the mean of n ≥ 3 replicates and shading represents SEM. (B) Growth curve of pneumococcal cells deleted with either ΔlytA or ΔessSp, or both. (C) Growth curves of WT or ΔessSp cells to DNA targeting antibiotic ciprofloxacin (CIP). ΔessSp mutant has the same susceptibility to DNA targeting antibiotic compared to the WT. (D) Growth curves of WT or ΔessSp cells subject to diverse cell-wall targeting antibiotics; penicillin (PEN), piperacillin (PIP) or ceftriaxone (CTR). ΔessSp mutant is more susceptible to cell wall targeting antibiotics (PEN, PIP, CTR) than the WT. (E) Confirmation of CRISPRi-seq synthetic lethal screen. Growth curve of pneumococcal cells deleted with ΔessSp, and/or ΔdivIB (left), as well as ΔessSp, and/or mreD−/+ (right). (F) Growth curves of pneumococcal cells subject to cefotaxime (CTX) show that the ΔpgdA mutants is not more susceptible compared to the WT. (G) Growth curves of S. salivarius HSISS4 cells subject to penicillin (PEN) at 37°C show that the Δess mutants is more susceptible compared to the wild type. (H) Growth curve of pneumococcal cells deleted with either Δpbp2a or ΔessSp, or both.

Source data

Extended Data Fig. 4 Split-luciferase assay.

(A) All proteins tested by split-luciferase assay. High relative luminescence unit (RLU, normalized log) indicate close proximity (0 represent no proximity, 3 represent close proximity), see material and methods for details of normalization. HlpA-LgBit HlpA-SmBit (HlpA-HlpA) is used as a positive control. Protein representations are not drawn to scale, and when relevant, C and N indicate the sub-cellular localization of the C- and N-terminal SmBit fusion respectively. (B) Fusion of the LgBit to the C-ter of MpgA and RodZ confirmed their close proximity to S protein.

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Extended Data Fig. 5 Pneumococcal PBP1a and PgdA are co-localizing and the removal of the S protein does not impact their localization.

Double-labeled cells were grown in C + Y medium at 37°C and mid-exponentially growing cells were collected for fluorescence microscopy. Fluorescent proteins were expressed from their native locus as only copy in the cell. (A) Deconvolved epifluorescence microscopy. Scale bar = 2 µm. (B) Fluorescent signal of one replicate from >1,000 cells per strain are ordered by cell length and represented by demographs plot performed using MicrobeJ40 (see Methods). (C-D) same as A and B but in an ess deleted background.

Source data

Extended Data Fig. 6 S protein is an integral component of a cell wall repair complex comprising PBP1a, PgdA, and possibly MpgA and RodZ.

(A) AlphaFold prediction of all five proteins generated separately using AlphaFold 3. (B) AlphaFold prediction of the potential complex formed by the S protein, PBP1a, PgdA, MpgA and RodZ. For clarity purposes, the amino-acids 651 to 719 of PBP1a and amino acid 1 to 173 of MpgA are not shown.

Extended Data Fig. 7 Time course experiment.

(A) Growth curves of ΔessSp deleted pneumococcal cells shows increased cell lysis compared to D39V wild type at 37°C. Dotted lines represent the times at which cells were harvested for microscopy. Peaks are due to the plate reader being opened to take samples for microscopy. (B) Phase contrast microscopy of ΔessSp deleted pneumococcal cells at different time point. Scale bar = 2 μm.

Source data

Extended Data Fig. 8 Removal of the S protein specifically decreases the frequency of circumferentially moving PBP1a.

n = the total number of molecules analyzed from two biological replicates and ns (not significant), P > 0.05. (A) Deleting ess does not affect the velocity or duration of circumferentially moving iHT-PBP1a. All strains express a functional fusion of the HaloTag (iHT) domain fused to PBP1a. (left) Velocities and (right) durations of circumferentially moving HT-labeled single molecules of PBP1a are shown. Dots represent individual measurements, black horizontal line shows median, error bars denote interquartile range. Mean, standard deviation (±SD) and n = circumferential molecules. (B-C) Deleting ess decreases the frequency of circumferentially moving iHT-aPBP1a molecules, but does not affect their velocity. Both strains are merodiploids that express a functional fusion of the HaloTag (iHT) domain fused to PBP1a from the native locus of pbp1a as well as from an ectopic site under the control of a zinc-inducible promoter. (B) Deleting ess decreases the frequency of circumferentially moving iHT-aPBP1a in unencapsulated D39W. Merodiploid strains expressed a functional fusion of the HaloTag (iHT) domain to PBP1a from both the native locus of pbp1a as well as from an ectopic site under the control of a zinc-inducible promoter. Movement of >400 iHT-aPBP1a single molecules were recorded and analyzed as described for Fig. 5a. Similar frequency distributions were obtained in independent experiments using comparable single-copy strains (see Fig. 5a). See Methods (Single-molecule dynamics) for details of statistical tests. * P ≤ 0.0254; ** P ≤ 0.0098; all other comparisons (not shown) were not significant, P > 0.05. (C) (left) Velocities and (right) durations of circumferentially moving HT-labeled single molecules of PBP1a are shown and plotted as described above. (D-E) Deleting ess does not affect the frequency or velocity of circumferentially moving molecules of iHT-PBP2b, but reduces their duration. Merodiploid strains of iHT-bPBP2b were constructed similarly to the iHT-aPBP1a merodiploids described in panel B. (D) Movement of >220 iHT-bPBP2b single molecules were recorded and analyzed as described for Fig. 5a (E) Velocities and durations of circumferentially moving HT-labeled single molecules of PBP2b are shown. Mean, standard deviation (±SD) and n = circumferential molecules; ****, P = 0.0001. See Methods (Single-molecule dynamics) for details of statistical tests.

Source data

Extended Data Fig. 9 Deacetylated muropeptides are reduced in a S protein mutant.

(A) Muropeptide profiles of exponentially growing cells in C + Y medium at 37°C were obtained by reversed-phase HPLC55. The area under each peak (numbers) were calculated for each strain (Supplementary Table 5). Peak number 2, 17 and 18 correspond to Tri[deAc], TetraTri[deAc]‡, and TetraTri[deAc]‡, respectively55,94, as reported before by (Bui et al. 2011). Experiments were performed in duplicates and a single profile is shown for each strain. (B) Relative amount of deacetylated peaks of muropeptide profile done in duplicates. Also see (Supplementary Table 4, 5). (C) Pneumococcal susceptibility to host-induced damage in the absence of the S protein can be complemented and is also seen in pneumococcal cells lacking the capsule. Heatmap of the area under the curve (AUC) of the growth curves in liquid media. Lysozyme and LL-37 act synergistically on the ΔessSp mutant. Empirical AUC was plotted between 0-7 hours.

Supplementary information

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Supplementary Tables 1–7

Compilation of the tables, with legend for each table (sheet) found at the top.

Supplementary Data 1 and 2

Statistical source data for Supplementary Figs. 1 and 2.

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Burnier, J., Gallay, C., Bruce, K.E. et al. Pneumococcal S protein coordinates cell wall modification and repair to resist host antimicrobials. Nat Microbiol 11, 282–300 (2026). https://doi.org/10.1038/s41564-025-02184-4

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