Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as indicated by sCRilecs-seq

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1. 

   Version published to 10.7554/elife.75607 on eLife

   Jun 24, 2022
2. 

   eLife

   Jun 14, 2022

   Author Response

   Reviewer #1 (Public Review):

   This well-written, convincing paper ties together three major topics. The authors first detail a general strategy to combine CRISPRi approaches previously reported by the authors in S. pneumoniae with FACS to identify larger or smaller cells. The goal is to carry out a CRISPRi screen coupled to FACS to identify genes whose decreased expression distorts pneumococcal cell shape, with focus here on increased cell size. A strength of this strategy, which the authors call SCRilecs-seq, is the availability of a robust CRISPRi system and fluorescent-protein labeling methods in S. pneumoniae that have been developed and reported in several previous publications from the Veening lab. Sorting based on increased forward light scattering (FSC) indicative of increased cell size led to the …

   Author Response

   Reviewer #1 (Public Review):

   This well-written, convincing paper ties together three major topics. The authors first detail a general strategy to combine CRISPRi approaches previously reported by the authors in S. pneumoniae with FACS to identify larger or smaller cells. The goal is to carry out a CRISPRi screen coupled to FACS to identify genes whose decreased expression distorts pneumococcal cell shape, with focus here on increased cell size. A strength of this strategy, which the authors call SCRilecs-seq, is the availability of a robust CRISPRi system and fluorescent-protein labeling methods in S. pneumoniae that have been developed and reported in several previous publications from the Veening lab. Sorting based on increased forward light scattering (FSC) indicative of increased cell size led to the identification of 17 genes, whose decreased expression leads to increased FCS. This set includes genes involved in cell division, peptidoglycan (PG) synthesis, and teichoic acid synthesis, including two operons in the mevalonic acid synthesis pathway. The paper then explores how mevalonic acid synthesis is linked to cell size and PG synthesis, and further, how inhibition of this pathway can be used to potentiate amoxicillin killing of resistant pneumococcal strains.

   The shift to an interesting biological problem means that the SCRilecs-seq method is presented here as a workable pilot method that could be further optimized. The authors point out in the Results and Discussion several classes of known essential genes that mediate cell size that were not detected in this first screen ("false negatives), some of which are in operons with other essential genes. They also point out that the encapsulated D39 used forms chains of cells that must be separated by vigorous vortexing that could potentially lead to loss of sensitive cells. Besides mentioning some potential issues, the authors might consider including additional suggestions of steps that could be taken in future studies to further optimize the SCRilecs-seq method. For example, isogenic unencapsulated D39 mutants do not form chains and often show more severe cell division and PG synthesis phenotypes than encapsulated strains. Therefore, unencapsulated strains would obviate the need for vortexing and perhaps increase the shape phenotypes caused by expression inhibition of some genes. Additional suggestions about ways to optimize the method in the future would add to this paper.

   We have now added possible points of improvement of our screening approach, including the use of an unencapsulated strain, to our discussion.

   The second topic concerns how the mevalonic acid synthesis pathway causes elongation of pneumococcal cells. This section forms a playbook on following up candidates detected in SCRilecsseq screens. The authors construct depletion/complementation merodiploid strains for operon 1 and operon 2 that encode genes in the pathway. They confirm elongation of cells by static and time-lapse phase-contrast microscopy and statistically robust determinations of cell lengths. They demonstrate complementation in strains with and without the fluorescent-protein reporters. As expect, from the pathways, deletion mutants of operon 1 can be fed mevalonic acid for growth. These physiological data are of high quality. One question that is not fully resolved is why depletion of operon 1 or operon 2 expression in culture leads to a drop in culture OD that levels out, which is interpreted as surviving cells, whereas depletion on agarose pads leads to complete lysis by microscopy. Likewise, starvation of an operon 1 mutant for mevalonic acid leads to complete lysis of the culture. Whether the depleted cultures actually contain survivors or debris can be checked by Live/Dead staining. Suppressor accumulation is suggested, but seems not tested. These phenotypes in different growth conditions might be tied together a bit more. Nevertheless, transformation assays and the mevalonic acid starvation experiments show that the pathways are essential in pneumococcus.

   Whereas data on suppressor accumulation was indeed not shown in our original manuscript, we did test this hypothesis and confirmed that the acquisition of suppressor mutations in depletion (inducible ectopic operon copy present) but not deletion (operon completely absent) strains is responsible for the apparent difference in behavior between these strains. Indeed, we find that cultures that manage to grow in absence of IPTG in these complementation strains are either mutated in the Plac promoter or in the lacI repressor. This is now mentioned in the revised manuscript and provided in the supplementary information of our revised manuscript.

   Experiments that localize FtsZ-rings or regions of active PG synthesis with fluorescent-D-amino acids led to a remarkable result. TEM indicates that elongated cells depleted for operon 1 or operon 2 expression start to pinch in a little, but stop. Similarly, depletion of operon 1 or operon 2 expression leads to elongated cells with multiple unconstricted FtsZ-rings and relative faint bands of FDAA labeling with the around cell diameters. Constricted FtsZ-rings and FDAA labeling less than cell diameters are absent. This pattern strongly supports the interpretation that septal ring closure and PG synthesis does not occur, while PG elongation continues at a reduced level. This pattern is highly reminiscent of the cells depleted for the GpsB regulator that is required for septal closure and PG synthesis, but phenotypically acts like a negative regulator of PG elongation. An interesting future question is whether the residual peripheral PG elongation during depletion is carried out by PBP2b:RodA as during normal growth or by other PBPs as part of a stress response.

   Based on the results of our second sCRilecs-seq screen performed on a strain depleted for mevalonate, we conclude that Pbp2b:RodA is at least partly responsible for the observed cell elongation. Results indicate that when pbp2b or rodA expression is halted during mevalonate depletion, cells are not able to become as long as when Pbp2b and RodA are fully active. We have now explicitly mentioned this observation at line 539. Additionally, we have tried to characterize the contribution of RodA more quantitatively towards cell elongation in the mevalonate depletion phenotype. However, this double depletion strain is extremely sensitive to the amount of inducer for both rodA expression and the expression of the mevalonate genes. We were therefore unable to identify conditions in which cells behaved properly that could be used as a starting point to perform this experiment.

   The authors further test whether cell elongation upon mevalonate limitation occurs because of reduced undecaprenol-phosphate amount and whether this correlates with capsule, teichoic acid, or PG synthesis, that all use undecaprenol-phosphate as a carrier. In a well-designed series of physiology experiments, the authors first showed that mutants blocked later in the pathway to undecaprenol synthesis have the same elongation morphology as that caused by mevalonic acid depletion. In addition, only a late block in PG synthesis produced MraY depletion produced cells with the elongation phenotype observed during mevalonic acid synthesis. In contrast, deletion of capsule did not produce a growth defect, and depletion of teichoic acid synthesis led to elongated cells with a completely different cell morphology defect. The inference from the matching phenotypes is that the division defects caused by mevalonic acid limitation are largely caused by a lack of PG precursor, and this limitation is leads to residual PG elongation, but no septal closure. Several reasonable hypotheses for the mechanism of this differential synthesis are presented in the Discussion for future testing. Although the physiological data are high consistent with this interpretation no direct determinations of the final Lipid-II precursor were included in this study.

   The authors did one further series of clever experiments to understand the phenotypes caused by mevalonic acid limitation. They applied the SCRilecs-seq method to cells depleted for mevalonic acid to identify decreased gene expression that would lead to even longer or smaller cells. The results show that depletion of gene expression involved in cell division or septal PG synthesis (e.g., FtsW, PBP2x) further elongated mevalonate-depleted cells, whereas genes involved in blocking protein expression, energy metabolism, or PG elongation (e.g., DivIVA, RodA, PBP2b) led to smaller cells.

   The last topic builds on the idea that synergy between two antibiotics is often greatest when they target the same process or pathway. Since locking mevalonic acid synthesis limits undecaprenol and blocks PG synthesis, then there may be synergy with the beta-lactam antibiotic, amoxicillin that inhibits PBP transpeptidase activity, especially in amoxicillin-resistant clinical strains. To test this idea, the authors built on a previous result from Staphylococcus aureus showing that the FDA-approved drug fertility drug, clomiphene, inhibits undecaprenol synthesis. The authors used cell elongation morphology and mevalonic acid depletion to confirm that clomiphene likely inhibits undecaprenol synthesis in S. pneumoniae, although Lipid II levels were not determined. In addition, synergy between clomiphene and amoxicillin inhibition was observed for the virulent, sensitive D39 strain and for three more recently isolated amoxicillin-resistant clinical strains. This synergy did not occur between clomiphene and antibiotics that do not inhibit PG synthesis. An attempt was made to test the synergy in a murine lung model of infection with one of the clinical strains (19F) that showed an intermediate synergy between clomiphene and amoxicillin. However, no synergy was detected in vivo, perhaps because insufficient clomiphene dosing. Further experiments could try other clinical strains with greater in vitro synergy (e.g., 23F) or improving the clomiphene dosing.

   We are indeed performing additional experiments to try and find conditions where the clomiphene-amoxicillin combination displays potentiation in vivo. Our current strategy is mainly focused at optimizing clomiphene to increase its potency in vivo. For example, clomiphene is a prodrug that is metabolized by the liver but because of clomiphene’s capability to boost amoxicillin action in vitro, this metabolic conversion is undesirable for our applications. Slight changes to the compound that prevent this conversion could therefore boost its activity. We hope that these and other minor adjustments will help to increase the in vivo efficacy of the clomiphene-amoxicillin drug combination. However, this is a work in progress and results will be presented at a later time.

   Together, this paper presents a number of important new findings that will strongly impact the field. A successful pilot screening method has been developed for S. pneumoniae, a major human pathogen. This method will undoubtedly be optimized, refined, and expanded in future screens. The screen pointed to the critical role of mevalonic acid in pneumococcal cell shape determination. While not totally unexpected, this paper is the first to systematically study this pathway in pneumococcus. This line of investigation led to the remarkable observation that mevalonic acid and undecaprenol deficiency preferentially blocks septal closure and septal PG synthesis over weakened PG elongation, not necessarily by the normal peripheral PG elongasome proteins. The basis for this phenotype will be explored in future studies. Finally, once characterized, depletion of undecaprenol synthesis showed synergy with amoxicillin in amoxicillin-resistant clinical strains of pneumococcus for the first time. Thus, this paper goes from unbiased screening, to characterization of a pathway that affects cell shape through modulation of PG synthesis, to manipulation of this pathway for antibiotic potentiation in resistant strains.

   Reviewer #3 (Public Review):

   This manuscript describes a powerful high throughput screening in the bacterial pathogen Streptococcus pneumoniae, that couples genome-wide CRISPR interference depletion with FACS sorting of elongated cells. This approach is therefore not limited to measuring changes in fitness but can be used to screen for mutants with any phenotype that can be detected by flow cytometry.

   The results from the screen uncovered an important role of the mevalonate pathway on cell length, as well as new factors with unknown functions required for proper cell morphology in Streptococcus pneumoniae.

   Upon depletion of mevalonate, overall peptidoglycan (PG) synthesis rate decreased. However, peripheral PG synthesis for cell elongation continued after septal peptidoglycan synthesis for cell division was inhibited. This suggests a form of regulation that directs PG synthesis towards septal or peripheral, depending on the availability of PG precursors. However, this mechanism is not identified.

   Finally, authors use the knowledge gained from the screening to design a combination therapy of amoxicillin with clomiphene that resensitizes amoxicillin-resistant S. pneumoniae strains. This is in agreement with previous data from Eric Brown´s lab showing that clomiphene potentiates the activity of β-lactam antibiotics against methicillin-resistance Staphylococcus aureus strains. The authors then show that clomiphene/amoxicillin combination was not effective in vivo, using a murine pneumonia disease model, possibly because the active concentration of clomiphene in the lung was too low.

   The manuscript is very clear and the experiments were carefully done. There are two major points that should be addressed

   1. Authors mention that depletion of mevalonate operons prevents cell division and leads to cell elongation. This conclusion is based on cell morphology (longer cells) and on microscopy experiments assessing peptidoglycan incorporation (Fig 4D). However, it is not clear from Fig 4D that there is no septal synthesis. For example, in the panel corresponding to depletion of mevalonate operon 1, the top cell is constricted in the middle and the RADA labelling shows a constricted ring, similar to what is seen for the wild type strain. Authors should point clearly to what they consider peripheral and septal synthesis to be; should overlay the green and red signals so that localization of PG synthesis over time can be more easily seen; and should discuss their data in the context of the recent model for peptidoglycan assembly during the cell cycle of S. pneumoniae by the Morlot group (https://doi.org/10.1016/j.cub.2021.04.041) which proposes that "the ovoid-cell morphogenesis (relies) on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct successive phases of peripheral and septal synthesis".

   Indeed, Figure 4D contains a cell that is still able to constrict slightly. We do not mean to claim that there is no constriction possible at all upon depletion of the mevalonate operons. However, constriction is very severely hampered and diminished strongly in comparison to a wild-type strain. We hope this can be appreciated qualitatively from the cells shown in Figure 4D and Figure 5F and from all acquired images that have been deposited on the EMBL-EBI BioImages Archive: https://www.ebi.ac.uk/biostudies/studies/S-BIAD477. Additionally, we have also attempted to quantify this phenotype. We recognize, however, that it was not apparent from our original manuscript how the distinction between septal and peripheral peptidoglycan synthesis was made. We have therefore now explicitly mentioned this at line 362: “Indeed, when defining septal peptidoglycan synthesis as FDAA bands that are less than 70% of the maximal cell width (i.e. sites of constriction, a criterion also used previously), quantitative analysis of hundreds of peptidoglycan synthesis sites demonstrated that the amount of septal synthesis is drastically reduced when transcription of the mevalonate operons is repressed (Figure 4E).”

   Figure 4D and Figure 5F now contain overlays of the sBADA and RADA signal so the progression of peptidoglycan synthesis can be interpreted more easily by the reader.

   Finally, we have included the recently formulated model for peptidoglycan synthesis postulated by the Morlot group in our manuscript. We have included this important work and have interpreted our results in light of this new and highly plausible model. This is part of our discussion starting at line 746. Additionally, we have rephrased some statements throughout our manuscript to allow for this new alternative interpretation of results.

   1. Authors propose that the elongation phenotype due to downregulation of the mevalonate pathway is caused by "insufficient transport of cell wall precursors across the cell membrane due to a limitation in the production of undecaprenyl phosphate (Und-P)". However, this conclusion is based almost exclusively on the similarity of phenotypes of the uppS deletion mutant and the mevalonate mutants. The levels of Und-P were not measured. An alternative to measuring these levels could be adding exogenous Und-P, which should revert the elongation phenotype. In S. aureus addition of exogenous Und-P suppressed the activity of clomiphene, indicating that cells are able to incorporate the compound (doi: 10.1073/pnas.1511751112)

   Many thanks for pointing us to this interesting result. We have referred to this experiment in the revised manuscript at line 572.

   The authors focus only on the elongation phenotype upon mevalonate depletion. However cells are also considerably wider (cell width increases 1.35X). This is similar to what happenes in Bacillus subtilis, where clomiphene was shown to cause swelling of cells (doi: 10.1073/pnas.1511751112). It would be interesting to discuss what may be the cause for this phenotype.

   Unfortunately, we do not know why cell width increases upon mevalonate depletion. However, it has been well established that both chemical and genetic inhibition of PBP2x, the PBP that is dedicated to septal cell wall synthesis, also leads to increases in cell width in S. pneumoniae which are often described as a “lemon-shape”. Why cells adopt this lemon-shape remains unknown and understudied. We can therefore currently not provide any plausible explanation for why cell width increases.

   We have, however, included a description of the Und-P inhibition phenotype of B. subtilis in our discussion section and have compared this phenotype to that of S. pneumoniae starting at line 705.

   Read the original source
3. 

   eLife

   Mar 11, 2022

   Evaluation Summary:

   Three experts in the field reviewed this manuscript from slightly different perspectives. All three reviewers are generally positive about this interesting, well-presented paper and think that it leads to several advances in the field. However, the reviewers also think changes can be made that would considerably strengthen the current version and its impact. Specific modifications have been requested to improve analysis of the screening data, to discuss hits besides the mevalonate pathway that increase Streptococcus pneumoniae cell length and shape, to clarify some issues about how mevalonate depletion changes pneumococcal cell shape and peptidoglycan synthesis, and to provide more context for clomiphene potentiation of amoxicillin killing of Streptococcus pneumoniae in comparison to previously published results in …

   Evaluation Summary:

   Three experts in the field reviewed this manuscript from slightly different perspectives. All three reviewers are generally positive about this interesting, well-presented paper and think that it leads to several advances in the field. However, the reviewers also think changes can be made that would considerably strengthen the current version and its impact. Specific modifications have been requested to improve analysis of the screening data, to discuss hits besides the mevalonate pathway that increase Streptococcus pneumoniae cell length and shape, to clarify some issues about how mevalonate depletion changes pneumococcal cell shape and peptidoglycan synthesis, and to provide more context for clomiphene potentiation of amoxicillin killing of Streptococcus pneumoniae in comparison to previously published results in Staphylococcus aureus.

   (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.)

   Read the original source
4. 

   eLife

   Mar 11, 2022

   Reviewer #1 (Public Review):

   This well-written, convincing paper ties together three major topics. The authors first detail a general strategy to combine CRISPRi approaches previously reported by the authors in S. pneumoniae with FACS to identify larger or smaller cells. The goal is to carry out a CRISPRi screen coupled to FACS to identify genes whose decreased expression distorts pneumococcal cell shape, with focus here on increased cell size. A strength of this strategy, which the authors call SCRilecs-seq, is the availability of a robust CRISPRi system and fluorescent-protein labeling methods in S. pneumoniae that have been developed and reported in several previous publications from the Veening lab. Sorting based on increased forward light scattering (FSC) indicative of increased cell size led to the identification of 17 genes, …

   Reviewer #1 (Public Review):

   This well-written, convincing paper ties together three major topics. The authors first detail a general strategy to combine CRISPRi approaches previously reported by the authors in S. pneumoniae with FACS to identify larger or smaller cells. The goal is to carry out a CRISPRi screen coupled to FACS to identify genes whose decreased expression distorts pneumococcal cell shape, with focus here on increased cell size. A strength of this strategy, which the authors call SCRilecs-seq, is the availability of a robust CRISPRi system and fluorescent-protein labeling methods in S. pneumoniae that have been developed and reported in several previous publications from the Veening lab. Sorting based on increased forward light scattering (FSC) indicative of increased cell size led to the identification of 17 genes, whose decreased expression leads to increased FCS. This set includes genes involved in cell division, peptidoglycan (PG) synthesis, and teichoic acid synthesis, including two operons in the mevalonic acid synthesis pathway. The paper then explores how mevalonic acid synthesis is linked to cell size and PG synthesis, and further, how inhibition of this pathway can be used to potentiate amoxicillin killing of resistant pneumococcal strains.

   The shift to an interesting biological problem means that the SCRilecs-seq method is presented here as a workable pilot method that could be further optimized. The authors point out in the Results and Discussion several classes of known essential genes that mediate cell size that were not detected in this first screen ("false negatives), some of which are in operons with other essential genes. They also point out that the encapsulated D39 used forms chains of cells that must be separated by vigorous vortexing that could potentially lead to loss of sensitive cells. Besides mentioning some potential issues, the authors might consider including additional suggestions of steps that could be taken in future studies to further optimize the SCRilecs-seq method. For example, isogenic unencapsulated D39 mutants do not form chains and often show more severe cell division and PG synthesis phenotypes than encapsulated strains. Therefore, unencapsulated strains would obviate the need for vortexing and perhaps increase the shape phenotypes caused by expression inhibition of some genes. Additional suggestions about ways to optimize the method in the future would add to this paper.

   The second topic concerns how the mevalonic acid synthesis pathway causes elongation of pneumococcal cells. This section forms a playbook on following up candidates detected in SCRilecs-seq screens. The authors construct depletion/complementation merodiploid strains for operon 1 and operon 2 that encode genes in the pathway. They confirm elongation of cells by static and time-lapse phase-contrast microscopy and statistically robust determinations of cell lengths. They demonstrate complementation in strains with and without the fluorescent-protein reporters. As expect, from the pathways, deletion mutants of operon 1 can be fed mevalonic acid for growth. These physiological data are of high quality. One question that is not fully resolved is why depletion of operon 1 or operon 2 expression in culture leads to a drop in culture OD that levels out, which is interpreted as surviving cells, whereas depletion on agarose pads leads to complete lysis by microscopy. Likewise, starvation of an operon 1 mutant for mevalonic acid leads to complete lysis of the culture. Whether the depleted cultures actually contain survivors or debris can be checked by Live/Dead staining. Suppressor accumulation is suggested, but seems not tested. These phenotypes in different growth conditions might be tied together a bit more. Nevertheless, transformation assays and the mevalonic acid starvation experiments show that the pathways are essential in pneumococcus.

   Experiments that localize FtsZ-rings or regions of active PG synthesis with fluorescent-D-amino acids led to a remarkable result. TEM indicates that elongated cells depleted for operon 1 or operon 2 expression start to pinch in a little, but stop. Similarly, depletion of operon 1 or operon 2 expression leads to elongated cells with multiple unconstricted FtsZ-rings and relative faint bands of FDAA labeling with the around cell diameters. Constricted FtsZ-rings and FDAA labeling less than cell diameters are absent. This pattern strongly supports the interpretation that septal ring closure and PG synthesis does not occur, while PG elongation continues at a reduced level. This pattern is highly reminiscent of the cells depleted for the GpsB regulator that is required for septal closure and PG synthesis, but phenotypically acts like a negative regulator of PG elongation. An interesting future question is whether the residual peripheral PG elongation during depletion is carried out by PBP2b:RodA as during normal growth or by other PBPs as part of a stress response.

   The authors further test whether cell elongation upon mevalonate limitation occurs because of reduced undecaprenol-phosphate amount and whether this correlates with capsule, teichoic acid, or PG synthesis, that all use undecaprenol-phosphate as a carrier. In a well-designed series of physiology experiments, the authors first showed that mutants blocked later in the pathway to undecaprenol synthesis have the same elongation morphology as that caused by mevalonic acid depletion. In addition, only a late block in PG synthesis produced MraY depletion produced cells with the elongation phenotype observed during mevalonic acid synthesis. In contrast, deletion of capsule did not produce a growth defect, and depletion of teichoic acid synthesis led to elongated cells with a completely different cell morphology defect. The inference from the matching phenotypes is that the division defects caused by mevalonic acid limitation are largely caused by a lack of PG precursor, and this limitation is leads to residual PG elongation, but no septal closure. Several reasonable hypotheses for the mechanism of this differential synthesis are presented in the Discussion for future testing. Although the physiological data are high consistent with this interpretation no direct determinations of the final Lipid-II precursor were included in this study.

   The authors did one further series of clever experiments to understand the phenotypes caused by mevalonic acid limitation. They applied the SCRilecs-seq method to cells depleted for mevalonic acid to identify decreased gene expression that would lead to even longer or smaller cells. The results show that depletion of gene expression involved in cell division or septal PG synthesis (e.g., FtsW, PBP2x) further elongated mevalonate-depleted cells, whereas genes involved in blocking protein expression, energy metabolism, or PG elongation (e.g., DivIVA, RodA, PBP2b) led to smaller cells.

   The last topic builds on the idea that synergy between two antibiotics is often greatest when they target the same process or pathway. Since locking mevalonic acid synthesis limits undecaprenol and blocks PG synthesis, then there may be synergy with the beta-lactam antibiotic, amoxicillin that inhibits PBP transpeptidase activity, especially in amoxicillin-resistant clinical strains. To test this idea, the authors built on a previous result from Staphylococcus aureus showing that the FDA-approved drug fertility drug, clomiphene, inhibits undecaprenol synthesis. The authors used cell elongation morphology and mevalonic acid depletion to confirm that clomiphene likely inhibits undecaprenol synthesis in S. pneumoniae, although Lipid II levels were not determined. In addition, synergy between clomiphene and amoxicillin inhibition was observed for the virulent, sensitive D39 strain and for three more recently isolated amoxicillin-resistant clinical strains. This synergy did not occur between clomiphene and antibiotics that do not inhibit PG synthesis. An attempt was made to test the synergy in a murine lung model of infection with one of the clinical strains (19F) that showed an intermediate synergy between clomiphene and amoxicillin. However, no synergy was detected in vivo, perhaps because insufficient clomiphene dosing. Further experiments could try other clinical strains with greater in vitro synergy (e.g., 23F) or improving the clomiphene dosing.

   Together, this paper presents a number of important new findings that will strongly impact the field. A successful pilot screening method has been developed for S. pneumoniae, a major human pathogen. This method will undoubtedly be optimized, refined, and expanded in future screens. The screen pointed to the critical role of mevalonic acid in pneumococcal cell shape determination. While not totally unexpected, this paper is the first to systematically study this pathway in pneumococcus. This line of investigation led to the remarkable observation that mevalonic acid and undecaprenol deficiency preferentially blocks septal closure and septal PG synthesis over weakened PG elongation, not necessarily by the normal peripheral PG elongasome proteins. The basis for this phenotype will be explored in future studies. Finally, once characterized, depletion of undecaprenol synthesis showed synergy with amoxicillin in amoxicillin-resistant clinical strains of pneumococcus for the first time. Thus, this paper goes from unbiased screening, to characterization of a pathway that affects cell shape through modulation of PG synthesis, to manipulation of this pathway for antibiotic potentiation in resistant strains.

   Read the original source
5. 

   eLife

   Mar 11, 2022

   Reviewer #2 (Public Review):

   A major aspect of the authors' manuscript is their "sCRilecs-seq" method. This method sorts a CRISPRi library according to cell size and identifies mutants with significant cell size defects. Despite the promise of this novel, fundamentally sound method, much optimization of the experimental and/or analytical methods is required before this method will be of broad utility to the microbiological community. The major issue is regarding the reproducibility of the data, which then causes major issues with hit identification and limits the power of the method. To the extent that the authors wish to emphasize the method as part of the manuscripts' significance, these issued must be addressed head on and resolved.

   The authors use their method to identify 17 operons with significantly larger cell size. Both operons …

   Reviewer #2 (Public Review):

   A major aspect of the authors' manuscript is their "sCRilecs-seq" method. This method sorts a CRISPRi library according to cell size and identifies mutants with significant cell size defects. Despite the promise of this novel, fundamentally sound method, much optimization of the experimental and/or analytical methods is required before this method will be of broad utility to the microbiological community. The major issue is regarding the reproducibility of the data, which then causes major issues with hit identification and limits the power of the method. To the extent that the authors wish to emphasize the method as part of the manuscripts' significance, these issued must be addressed head on and resolved.

   The authors use their method to identify 17 operons with significantly larger cell size. Both operons encoding components of the mevalonate pathway are in this set, which suggests that inhibiting mevalonate synthesis may synergize with amoxicillin, an aminopenicillin antibiotic. In a series of thoughtful and elegant experiments, the authors show that indeed, inhibiting UppS (which synthesizes Und-PP from Farnesyl-PP for which mevalonate is an upstream precursor) using clomiphene sensitizes cells to amoxicillin. Although this finding may be of some clinical relevance, its significance is lessened by the fact that clomiphene has been shown to sensitize both Staphylococcus aureus MRSA and B. subtilis to beta-lactams, including amino-penicillins since at least 2015. Given the conservation of this synergy between MRSA and B. subtills, it is not surprising that it holds for Streptococcus. Moreover, since this synergy has been previously observed, it is not clear that the authors' sCRilecs-seq method was necessary to discover it.

   Considered as a whole, the authors' method and follow-up experiments have the potential to uncover novel aspects of Streptococcus biology, but at the current level of analysis, it falls short of this goal. In my opinion, this paper could be substantially improved if the sCRilecs-seq method and/or analysis were improved, and if the authors' findings were better contextualized to highlight differences and similarities to MRSA and B. subtilis.

   Read the original source
6. 

   eLife

   Mar 11, 2022

   Reviewer #3 (Public Review):

   This manuscript describes a powerful high throughput screening in the bacterial pathogen Streptococcus pneumoniae, that couples genome-wide CRISPR interference depletion with FACS sorting of elongated cells. This approach is therefore not limited to measuring changes in fitness but can be used to screen for mutants with any phenotype that can be detected by flow cytometry.

   The results from the screen uncovered an important role of the mevalonate pathway on cell length, as well as new factors with unknown functions required for proper cell morphology in Streptococcus pneumoniae.

   Upon depletion of mevalonate, overall peptidoglycan (PG) synthesis rate decreased. However, peripheral PG synthesis for cell elongation continued after septal peptidoglycan synthesis for cell division was inhibited. This suggests a …

   Reviewer #3 (Public Review):

   This manuscript describes a powerful high throughput screening in the bacterial pathogen Streptococcus pneumoniae, that couples genome-wide CRISPR interference depletion with FACS sorting of elongated cells. This approach is therefore not limited to measuring changes in fitness but can be used to screen for mutants with any phenotype that can be detected by flow cytometry.

   The results from the screen uncovered an important role of the mevalonate pathway on cell length, as well as new factors with unknown functions required for proper cell morphology in Streptococcus pneumoniae.

   Upon depletion of mevalonate, overall peptidoglycan (PG) synthesis rate decreased. However, peripheral PG synthesis for cell elongation continued after septal peptidoglycan synthesis for cell division was inhibited. This suggests a form of regulation that directs PG synthesis towards septal or peripheral, depending on the availability of PG precursors. However, this mechanism is not identified.

   Finally, authors use the knowledge gained from the screening to design a combination therapy of amoxicillin with clomiphene that resensitizes amoxicillin-resistant S. pneumoniae strains. This is in agreement with previous data from Eric Brown´s lab showing that clomiphene potentiates the activity of β-lactam antibiotics against methicillin-resistance Staphylococcus aureus strains.
   The authors then show that clomiphene/amoxicillin combination was not effective in vivo, using a murine pneumonia disease model, possibly because the active concentration of clomiphene in the lung was too low.

   The manuscript is very clear and the experiments were carefully done. There are two major points that should be addressed

   1.Authors mention that depletion of mevalonate operons prevents cell division and leads to cell elongation. This conclusion is based on cell morphology (longer cells) and on microscopy experiments assessing peptidoglycan incorporation (Fig 4D). However, it is not clear from Fig 4D that there is no septal synthesis. For example, in the panel corresponding to depletion of mevalonate operon 1, the top cell is constricted in the middle and the RADA labelling shows a constricted ring, similar to what is seen for the wild type strain. Authors should point clearly to what they consider peripheral and septal synthesis to be; should overlay the green and red signals so that localization of PG synthesis over time can be more easily seen; and should discuss their data in the context of the recent model for peptidoglycan assembly during the cell cycle of S. pneumoniae by the Morlot group (https://doi.org/10.1016/j.cub.2021.04.041) which proposes that "the ovoid-cell morphogenesis (relies) on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct successive phases of peripheral and septal synthesis".

   2. Authors propose that the elongation phenotype due to downregulation of the mevalonate pathway is caused by "insufficient transport of cell wall precursors across the cell membrane due to a limitation in the production of undecaprenyl phosphate (Und-P)". However, this conclusion is based almost exclusively on the similarity of phenotypes of the uppS deletion mutant and the mevalonate mutants. The levels of Und-P were not measured. An alternative to measuring these levels could be adding exogenous Und-P, which should revert the elongation phenotype. In S. aureus addition of exogenous Und-P suppressed the activity of clomiphene, indicating that cells are able to incorporate the compound (doi: 10.1073/pnas.1511751112)

   The authors focus only on the elongation phenotype upon mevalonate depletion. However cells are also considerably wider (cell width increases 1.35X). This is similar to what happenes in Bacillus subtilis, where clomiphene was shown to cause swelling of cells (doi: 10.1073/pnas.1511751112). It would be interesting to discuss what may be the cause for this phenotype.

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7. 

   Version published to 10.1101/2021.09.13.460059v2 on bioRxiv

   Nov 16, 2021
8. 

   Version published to 10.1101/2021.09.13.460059v1 on bioRxiv

   Sep 13, 2021