Each year, Streptococcus pneumoniae kills hundreds of thousands of children around the world. Its protective capsule is the target of current vaccines, and the capsule is also required for S. pneumoniae to cause severe disease. This work clarifies how the capsule is attached to the cell wall.
By studying double mutants, we found that the putative capsule attaching enzyme CpsA is not required for capsule formation, as other enzymes can also fulfill this role. However, CpsA is also involved in attaching another component of the cell wall, wall teichoic acid (WTA). When CpsA is absent, the regulator WalK becomes essential, probably because WalK senses the defects in the cell wall due to loss of WTA and causes the upregulation of cell wall remodeling enzymes to compensate.
Impact to Field/Science
This work clarifies how the capsule and WTA are attached to the cell wall, and how WalK is involved in this process. Similar processes occur in many related bacteria (besides Streptococcus pneumoniae). Also, because the formation of the cell wall is the target of many antibiotics, these results could someday inform the development of new treatments.
Significance to ENIGMA
This work illustrates how to adapt ENIGMA’s randomly-barcoded transposon sequencing technology (RB-TnSeq) to study two gene disruptions at a time, instead of just one. Specifically, by moving a library of transposon insertions into different genetic backgrounds, interactions between a few genes and all other genes can be measured. Findings from a 2025 study published in Science by Zik et al. led Zik to investigate the capsule further as described in the current paper.
Example of transposon mutagenesis data (RB-TnSeq) in a mutant background, with or without Zn-dependent expression of cpsE (top and bottom panels, respectively). Arrows highlight insertions on the minus strand that increase fitness by driving overexpression of lytR, which can compensate for lack of cpsA (CpsA and LytR are attachment enzymes with overlapping roles). This benefit is only seen when capsule precursors are being made.
Image Credit: Justin Zik
Collaborations
This is a complementary project affiliated with ENIGMA. Most of the work was done in Chris (Lok-To) Sham’s lab at the National University of Singapore.
Funding Acknowledgement
This work was supported by grants from the National Medical Research Council, Singapore (OFIRG23jan-0057 to L.-T.S.); the Ministry of Education, Singapore (MOE-T2EP30220-0012 and MOE-T2EP30224-0012 to L.-T.S.; MOE-T2EP30223-0002 and MOE-T2EP10124-0001 to Y.Q.); the National Research Foundation Singapore (NRF-F-CRP-2024-0002 to L.-T.S.); the National Institutes of Health Grants (5R35GM150669 to J.F.-K.); the Smith Family Foundation (Grant for Excellence in Biomedical Research to J.F.-K.); and the UMass Chan (Startup funds and BMB Excellence Award Grant to J.F.-K.). This material by ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies; http://enigma.lbl.gov), a Science Focus Area Program at Lawrence Berkeley National Laboratory, is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under contract number DE-AC02-05CH11231.
Publication
Zik JJ, Fu Z, Price MN, Li Y, Yuan Q, Arkin AP, Deutschbauer AM, Flores-Kim J, Sham L.2026.The WalRK two-component system in Streptococcus pneumoniae ensures robustness of secondary wall polymer attachment. mBio17:e01075-26.https://doi.org/10.1128/mbio.01075-26. OSTI:17138085
Contact
Morgan Price