WhyD tailors surface polymers to prevent premature bacteriolysis and direct cell elongation in Streptococcus pneumoniae
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This study explores autolysis regulation in the gram positive pathogen Streptococcus Pneumoniae and finds that the turnover of wall teichoic acids at the septum controls cell elongation. Specifically, a possible new enzyme WhyD regulates the level of wall teichoic acids (WTA) at the bacterial septum, limiting the number of WTA-associated autolysins such as LytA at division sites. The study provides new insights into the molecular interactions between enzymes that regulate PG-associated polymers and enzymes that hydrolyze to regulate cell morphogenesis. In principle, such regulation will also apply to other monoderm bacteria. This article will be of interest to microbiologists who are studying bacterial cell envelope dynamics. The experimental data support the conclusions drawn and the work provides tools, concepts and remaining questions to address that will be instrumental to understand the mechanism underpinning autolytic control.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Microbiology and Infectious Disease (eLife)
Abstract
Penicillin and related antibiotics disrupt cell wall synthesis in bacteria causing the downstream misactivation of cell wall hydrolases called autolysins to induce cell lysis. Despite the clinical importance of this phenomenon, little is known about the factors that control autolysins and how penicillins subvert this regulation to kill cells. In the pathogen Streptococcus pneumoniae ( Sp ), LytA is the major autolysin responsible for penicillin-induced bacteriolysis. We recently discovered that penicillin treatment of Sp causes a dramatic shift in surface polymer biogenesis in which cell wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked teichoic acids (LTAs). Because LytA binds to both species of teichoic acids, this change recruits the enzyme to its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA misactivation and lysis during exponential growth . We show that WhyD is a WTA hydrolase that restricts the WTA content of the wall to areas adjacent to active peptidoglycan (PG) synthesis. Our results support a model in which the WTA tailoring activity of WhyD during exponential growth directs PG remodeling activity required for proper cell elongation in addition to preventing autolysis by LytA.
Article activity feed
-
-
Author Response
Reviewer #2 (Public Review):
Weaknesses:
Although this is certainly a technically difficult goal, the paper does not show a direct interaction between WhyD (or its GlpQ sub-domain) with WTAs. While the effect of WhyD over WTA levels showed here is undeniable, and the proposed interaction is the simplest explanation, it's not possible to assert whether this is the case without a crosslink co-purification using an inactive mutant of WhyD.
We show in Figure 3 that the purified GlpQ domain of WhyD hydrolyzes WTAs from purified cell wall sacculi. It’s hard to imagine how the enzyme could accomplish this without making direct contact with WTAs. We therefore think this result along with the finding that the ∆whyD mutant has high levels of WTAs provides strong support for our conclusion that WhyD acts on WTAs.
Another …
Author Response
Reviewer #2 (Public Review):
Weaknesses:
Although this is certainly a technically difficult goal, the paper does not show a direct interaction between WhyD (or its GlpQ sub-domain) with WTAs. While the effect of WhyD over WTA levels showed here is undeniable, and the proposed interaction is the simplest explanation, it's not possible to assert whether this is the case without a crosslink co-purification using an inactive mutant of WhyD.
We show in Figure 3 that the purified GlpQ domain of WhyD hydrolyzes WTAs from purified cell wall sacculi. It’s hard to imagine how the enzyme could accomplish this without making direct contact with WTAs. We therefore think this result along with the finding that the ∆whyD mutant has high levels of WTAs provides strong support for our conclusion that WhyD acts on WTAs.
Another aspect the paper could improve is the explanation of the labeled cell-wall analogs, very well established in the cell-wall field but likely obscure to other biologists. Especially on figures that nothing at all is said about the data (Figures 4 and 5). The microscopy data, despite evidently being well-performed, begs for better quantitation and visualization. For example, it's not clear whether there were replicates, the sample size (informing that at least 300 cells were used is not enough information to inform on sample size effects). Sub-panels where no signal is apparently detected (e.g. Figure 7 and supplements) should be clarified and the background should be displayed.
We thank the reviewer for requesting more information for the general reader. We have included more information about the probes in the Materials and Methods section.
-
Evaluation Summary:
This study explores autolysis regulation in the gram positive pathogen Streptococcus Pneumoniae and finds that the turnover of wall teichoic acids at the septum controls cell elongation. Specifically, a possible new enzyme WhyD regulates the level of wall teichoic acids (WTA) at the bacterial septum, limiting the number of WTA-associated autolysins such as LytA at division sites. The study provides new insights into the molecular interactions between enzymes that regulate PG-associated polymers and enzymes that hydrolyze to regulate cell morphogenesis. In principle, such regulation will also apply to other monoderm bacteria. This article will be of interest to microbiologists who are studying bacterial cell envelope dynamics. The experimental data support the conclusions drawn and the work provides tools, concepts …
Evaluation Summary:
This study explores autolysis regulation in the gram positive pathogen Streptococcus Pneumoniae and finds that the turnover of wall teichoic acids at the septum controls cell elongation. Specifically, a possible new enzyme WhyD regulates the level of wall teichoic acids (WTA) at the bacterial septum, limiting the number of WTA-associated autolysins such as LytA at division sites. The study provides new insights into the molecular interactions between enzymes that regulate PG-associated polymers and enzymes that hydrolyze to regulate cell morphogenesis. In principle, such regulation will also apply to other monoderm bacteria. This article will be of interest to microbiologists who are studying bacterial cell envelope dynamics. The experimental data support the conclusions drawn and the work provides tools, concepts and remaining questions to address that will be instrumental to understand the mechanism underpinning autolytic control.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
The work by Flores-Kim et al. reports the identification and characterisation of WhyD, an enzyme involved in the hydrolysis of wall teichoic acids (WTAs) in S. pneumoniae. They explore the role of WhyD in autolysis control and cell growth and division. This study reveals that WhyD controls the abundance and localization of WTAs, which in turn inhibits autolytic activities.
The methods used convincingly address the questions asked by the authors and overall, the data are robust and support the conclusions drawn, providing experimental evidence for a mechanism proposed a long time ago but that remained poorly characterised. This work provides a convincing model to explain how the enzymatic activity of WhyD contributes to control peptidoglycan hydrolysis in the context of actively growing and dividing S. …
Reviewer #1 (Public Review):
The work by Flores-Kim et al. reports the identification and characterisation of WhyD, an enzyme involved in the hydrolysis of wall teichoic acids (WTAs) in S. pneumoniae. They explore the role of WhyD in autolysis control and cell growth and division. This study reveals that WhyD controls the abundance and localization of WTAs, which in turn inhibits autolytic activities.
The methods used convincingly address the questions asked by the authors and overall, the data are robust and support the conclusions drawn, providing experimental evidence for a mechanism proposed a long time ago but that remained poorly characterised. This work provides a convincing model to explain how the enzymatic activity of WhyD contributes to control peptidoglycan hydrolysis in the context of actively growing and dividing S. pneumoniae cells. It also highlights that WhyD activity is not sufficient to prevent autolysis and cell death during late stationary phase, indicating that this process awaits further analysis.
-
Reviewer #2 (Public Review):
Authors identified the gene whyD, an essential factor for the bacterial human pathogen Streptococcus pneumoniae (Sp) survival. Surprisingly, WhyD importance is reversed in the absence of LytA, making cells' survival dependent on the simultaneous deletion of both genes. The authors investigated the relationship between these 2 proteins and the production and localization of peptidoglycan (PG), teichoic acids (WTAs), and lipoteichoic acids (LTAs) in the cell wall. Combining genetically engineered strains, in vivo tagging of proteins, chemical probes, and purification of recombinant proteins, authors concluded that more than regulating levels of WTA and LTA, WhyD acts as a topological factor, supporting the spatio-temporal synthesis and degradation of cell wall necessary for cell elongation.
Strengths:
Working …
Reviewer #2 (Public Review):
Authors identified the gene whyD, an essential factor for the bacterial human pathogen Streptococcus pneumoniae (Sp) survival. Surprisingly, WhyD importance is reversed in the absence of LytA, making cells' survival dependent on the simultaneous deletion of both genes. The authors investigated the relationship between these 2 proteins and the production and localization of peptidoglycan (PG), teichoic acids (WTAs), and lipoteichoic acids (LTAs) in the cell wall. Combining genetically engineered strains, in vivo tagging of proteins, chemical probes, and purification of recombinant proteins, authors concluded that more than regulating levels of WTA and LTA, WhyD acts as a topological factor, supporting the spatio-temporal synthesis and degradation of cell wall necessary for cell elongation.
Strengths:
Working with Streptococcus, as with any less-studied bacterial model compared to B. subtilis and E. coli, is challenging but increasingly important to understand human pathogens and their interactions with antibiotic drugs. This work represents a tour de force and joins a relatively small collection of state-of-the-art studies by combining genetics, biochemistry, and cell biology approach to solve a specific problem. The questions asked in each experiment are clear and the performed experiments were, in the majority, well designed with proper controls. The paper is well-written and accessible to the general scientific community.
One of the highlights of this study is the development of a good proxy for WTA localization - something not trivial - providing the field with endless possibilities to study the immediate and lasting effects of antibiotic resistance and other genetic pathways in this model. A second important development - not so obvious as the first one - is the successful use of purified hydrolases from one species (in this case, LytA from S. pneumoniae) in orthogonal organisms (here, B. subtilis). This is an elegant assay that can be useful to study the function of proteins from challenging model systems.
Weaknesses:
Although this is certainly a technically difficult goal, the paper does not show a direct interaction between WhyD (or its GlpQ sub-domain) with WTAs. While the effect of WhyD over WTA levels showed here is undeniable, and the proposed interaction is the simplest explanation, it's not possible to assert whether this is the case without a crosslink co-purification using an inactive mutant of WhyD.
Another aspect the paper could improve is the explanation of the labeled cell-wall analogs, very well established in the cell-wall field but likely obscure to other biologists. Especially on figures that nothing at all is said about the data (Figures 4 and 5). The microscopy data, despite evidently being well-performed, begs for better quantitation and visualization. For example, it's not clear whether there were replicates, the sample size (informing that at least 300 cells were used is not enough information to inform on sample size effects). Sub-panels where no signal is apparently detected (e.g. Figure 7 and supplements) should be clarified and the background should be displayed.
-
Reviewer #3 (Public Review):
In this study, the authors aim at identifying new factors regulating the activity of bacterial cell wall hydrolases using Streptococcus pneumoniae as a model. Based on previous Tn-Seq screens, they showed that the gene whyD becomes non essential in a ∆lytA genetic background and that this protein restrains the activity of LytA. To explain this observation, they provide in vivo and in vitro evidence showing that WhyD specifically hydrolyses wall teichoic acids. A series of experiments is then achieved to demonstrate that WhyD regulates the activity of the cell wall hydrolase LytA to prevent cell lysis and that of other cell wall hydrolases to allow the insertion of new peptidoglycan in the preexisting polymer, promoting thus cell elongation. Notably, it is shown that WhyD localizes at mid-cell and together …
Reviewer #3 (Public Review):
In this study, the authors aim at identifying new factors regulating the activity of bacterial cell wall hydrolases using Streptococcus pneumoniae as a model. Based on previous Tn-Seq screens, they showed that the gene whyD becomes non essential in a ∆lytA genetic background and that this protein restrains the activity of LytA. To explain this observation, they provide in vivo and in vitro evidence showing that WhyD specifically hydrolyses wall teichoic acids. A series of experiments is then achieved to demonstrate that WhyD regulates the activity of the cell wall hydrolase LytA to prevent cell lysis and that of other cell wall hydrolases to allow the insertion of new peptidoglycan in the preexisting polymer, promoting thus cell elongation. Notably, it is shown that WhyD localizes at mid-cell and together with FDAA labelling and the use of an inactive form of LytA as a WTA localization probe, it is further shown that WhyD co-localizes with nascent peptidoglycan, while WTA are more abundant in the peripheral cell wall, during cell elongation. By contrast, WTA localization changes in cells nearing cell constriction, co-localizing with WhyD and nascent peptidoglycan. It is concluded that WhyD modulates the presence of wall teichoic acids in the PG layer in the course of the cell cycle, modulating in time and space their availability for choline-binding hydrolases in order to prevent aberrant cell lysis, proper cell elongation and final separation. The methodology used is logical, appropriate and well-executed. The data are clear and the interpretations are reasonable. This work represents a nice contribution to our understanding of the regulation of cell wall hydrolysis in bacterial growth and morphogenesis.
-
